Virtual Genome Project Blog

2010-07-05T11:14:00.000-07:00

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

John T. Singer, Ryan T. Phennicie, Matthew J. Sullivan, Laura A. Porter, Valerie J. Shaffer, and Carol H. Kim

Fluorescent proteins have been used to visualize different biological processes. One such process is the study of immune response to a bacterial infection in vivo. The organism being studied here is the zebrafish, which has a transparent exoskeleton in its early developmental stages, making visualization of fluorescently labeled pathogens possible. The goal of the study was to develop plasmids producing red-fluorescent-protein (RFP) in a non-toxic manner in a variety of gram negative bacteria. The labeled bacteria could then be introduced into embryonic zebrafish, followed by detection of immune response. So while bacteria were labeled with RFP, macrophages and neutrophils, which are the innate immune cells of the zebrafish, were labeled with green-fluorescent-protein (GFP) helping in detection of any interaction between the two. The plasmid they chose was a broad –host –range mobilizable, IncQ, plasmid called pMMB66EH, that has a tac promoter and a lacI repressor. They used four variants of RFP, which had shorter maturation times and higher brightness. These genes were cloned into pMMB66EH at a site downstream of the tac promoter. Since this plasmid is not self transferable, it had to be mobilized by another plasmid pRK2013 into three pathogens i.e., Edwardsiella tarda, Vibrio anguillarum and Pseudomonas aeruginosa. To be sure that their RFP producing plasmids were stable in the three bacteria, they conducted plasmid stability assays and found that most of their plasmids were stable. Of all plasmid and host combinations tested, they found P. aeruginosa PA14 bearing p67T1 to be the most stable and used this for studying immune response in zebrafish. They injected zebrafish embryos with P. aeruginosa PA14 (p67T1). Red fluorescence was visible using a wide-field epifluorescence microscope at X40 magnification. The bacteria were found to colonize the yolk initially and then spread to other regions such as the pericardium and head. Since the zebrafish was a transgenic variety capable of expressing GFP in macrophages and neutrophils, their movement towards the sites of infection could be seen as well. The most interesting result was that they were able to see phagocytosis of the bacteria by the immune cells. Although their initial plasmid constructs were designed to produce RFP under the regulation of the tac promoter, spontaneous mutations occurring in the lacI repressor resulted in constitutive production of RFP. The relevance of constitutive expression is not clear. In discussion they say that the plasmid that constitutively expresses RFP confers no additional burden on the zebrafish. Not having measured burden of any of the other plasmids that regulate the production of RFP, it is hard to say that constitutively expressed plasmids provide any benefit.
To summarize, the authors aimed to create a set of plasmids that would express RFP in a non-toxic manner in a variety of bacterial hosts that could be used in studying immune response in zebrafish. They were successful in creating plasmids that could be transferred to members of gamma-proteobacteria only. To transfer their plasmids to more unrelated bacteria, use of an IncP plasmid as the helper would be better.

Diya Sen
Graduate student
University of Idaho

Introducing the Chromid

2010-05-31T22:24:00.000-07:00

Harrison, PW, et al. 2010. Introducing the bacterial ‘chromid’: not a chromosome, not a plasmid. Trends in microbiology.18:4.

Although it is common to think that a scientist’s job is to make new discoveries, equally or perhaps more important is a scientist’s ability to communicate. A brilliant discovery does the world no good if it can’t be explained to other scientists or the populous at large. Defining terminology in a useful and biologically meaningful way is therefore an important aspect of biological pursuits. In a recent article Harrison et al. recognize this necessity in proposing a new term: the bacterial “chromid.” Typically bacterial genomes consist of one circular plasmid, but may also contain smaller replicons as well. These replicons may be standard plasmids or (usually) larger entities that contain plasmid-type replication machinery but also core genes that are essential to bacterial growth and survival. Currently these larger replicons are classified as second chromosomes due to their necessity for a functional cell. Here the authors argue that “chromid” rather than second chromosome is a more apt classification, as they are distinct from plasmids and the chromosome in important ways.

Here the authors defined chromids with the following criteria: “i. chromids have plasmid-type maintenance and replication systems; ii. Chromids have a nucleotide composition close to that of the chromosome; iii. Chromids carry core genes that are found on the chromosome in other species.” Chromids therefore share certain commonalities between plasmids and chromosomes, but have combined aspects of each in a consistent-enough manner, with enough differences from each as to form their own class of replicon. This mélange of chromosome and plasmid could have important consequences for bacterial evolution. Indeed, chromids can be differentiated at the genus level by the core genes they encode, indicating specific phylogentic and evolutionary histories.

As about one in ten sequenced bacterial strains have replicons that fit the definition of a chromid, the authors argue that it is important to have this term in order to clearly communicate about these replicons and their importance in bacterial evolution and adaptation. With new sequencing technologies more and more bacteria are being sequenced and so this clarity of communication will also become increasingly important in the future.

Julie Hughes

University of Idaho

Conjugative plasmids: vessels of the communal gene pool.

2010-04-30T17:39:00.000-07:00

Anders Norman, Lars H. Hansen and Soren Sorensen
Phil. Trans. R. Soc. B 2009 364: 2275-2289

Browsing the Internet for a recent review article talking generally about plasmids, I found an article written by the group of Prof. Sorensen from the University of Copenhagen in Denmark. The first surprise was the journal, which full title is Philosophical Transactions of The Royal Society B: Biological Sciences. The “Royal Society” sounds great and the journal is really good with the IF=5.9 (2008), but may be not very popular especially among molecular biologists now days. Philosophical Transactions B is divided into four cluster areas: Cell and Development, Health and Disease, Environment and Evolution, Neuroscience and Cognition. The leading theme of the August 2009 issue, where this review was published, was 'The network of life: genome beginnings and evolution'. Second, the title “…plasmids as vessels of the communal gene pool”: what does this really mean? The explanation can be found in the abstract. The authors point out that evolution of microorganisms is tightly linked to the environment in which they live and the communal (total) pool of genes within that environment. As conjugative plasmids play a major role in horizontal gene transfer (HGT) within and between different bacterial populations, the accessory genes carried by these plasmids belong to the pool of communal genes.
The review consists of seven main chapters. After a short introduction to the current bacterial evolution research based on single genomes or metagenomic DNA sequences, the authors propose some new terms (chapter 2) such as:
“supergenome” – the total pool of genes readily available to a prokaryotic organism within a particular setting;
“private pool” - which consists of the fixed and ‘idiosyncratic’ genes encoded on the chromosome of the prokaryote;
“communal pool” - which consists of genes encoded on mobile genetic elements (MGEs) and that are thus available to all permissive prokaryotes,
and discuss the relations of these new concepts to older terms like: core genome, which define the genes present in all strains of a prokaryotic species; dispensable genome (or flexible genome), which are genes present in some, but not all, strains of the same species; and pan genome—the sum of the former two.
The difference between these new and old terms is that the latter are related to a single species, while the new ones are more related to the population of microorganisms. Actually, I like the idea because it really reflects the natural state. As new DNA sequencing technologies enable us to analyze whole populations generating gigabytes/bases of information, it is better to treat this as a “supergenome-gene pool” than pan genome, especially since assembling single genomes out of this population is not an easy task and can generate many errors. On the other hand, the authors confine the communal pool to genes present on mobile genetics elements, which in my opinion is not really good as we know that the structure of these elements can be very unstable with almost continuous exchange between different genome parts (chapter 3).
I think that in ‘population genetics’/metagenomic kinds of studies based on current technology in DNA sequencing and analysis one really could concentrate on two things:
1) identification of species within the population based on 16SrDNA sequence;
2) supergenome analysis – presentation of all genes available within a population with their relative abundance – which will reflect the physiology of the analyzed population.

But back to the article; in chapter 4 and 5 –‘The tools of genetic mobility’ and ‘Mechanisms of, and barriers to, horizontal gene transfer’, the authors briefly describe different mobile elements and mechanisms that drive HGT. This leads us to the main part describing ‘The world of conjugative plasmids’, where in a few subchapters the authors describe origin of plasmids and its organization (with a description of plasmid modular structure) and discuss the role of conjugative plasmids in the cell. Finally, in the last part they talk about some methods used to study the communal gene pool and how these studies reflect on our understanding of bacterial evolution.
I found this article very well written and really interesting. It is a very current review article talking about horizontal gene transfer and conjugative plasmids with up-to-date references. Simple and relatively broad presentation of HGT and all the processes that lead to genetic exchange within microbial populations as well as a simple description of conjugative plasmids and their role in HGT make this review an ideal article as an introduction for students and researchers new to this field.

Jarek Krol PhD
UofI

Survival of the Fittest

2010-04-23T11:31:00.000-07:00

Vriezen JAC, Valliere M, Riley MA. 2009. The evolution of reduced microbial killing. Genome. Biol. Evol. 2009:400-8.

One interesting question in the plasmid world is how to classify plasmids. They are commonly compared to parasites in that they require the use of host machinery for replication and protein production, and can “infect” bacterial hosts even if this reduces host fitness (i.e. the bacteria often have no say in whether a plasmid is admitted into its cell or not). Unlike parasites, however, plasmids can be beneficial to their hosts, depending on what genes they code for and the current environment. For instance, some plasmids code for colicin production, which can kill bacteria that are closely related to the host bacteria(1). This gives plasmid-bearing bacteria an edge on competing strains, but at a slight cost due to plasmid maintenance and colicin production. In this article the authors found that after 253 generations of growth in the absence of competing strains, the killing ability of E. coli was reduced in an attempt to reduce the cost of plasmid maintenance in an environment wherein colicin production is unnecessary.

So far, these results aren’t terribly surprising: if colicin production comes with a cost the bacterial host then any bacterium that can reduce this cost will be at a selective advantage. This will allow bacteria with reduced killing affects to become more dominate in the population over time(c.f. 2,3). What is more interesting is that, even though it is the plasmid that codes for colicin production it is the bacteria’s genes that change to reduce colicin production. After 253 generations the plasmid’s sequence remained completely unaltered, whereas the expression of host genes including those for DNA repair, Mg ion uptake, and late prophage genes displayed changes in their regulation. The authors commented that this was also a wider variety of genes that were involved in this evolutionary response to colicin production pressures than expected.

The interactions between plasmids and their host bacteria appear very complex. The fate of each is closely related to the fate of the other, and there are a variety of ways that the plasmid, the host, or both could change to increase the chances of survival of both together. Changes on one partner, in this case the bacteria, can regulate the expression of the other without altering the other at all. The interactions and coevolution of plasmids and bacteria are dynamic, with countless possibilities that have yet to be explored. What seems ever more clear with each such study is that when faced with a problem, in the words quoted in Jurassic Park, “Nature will find a way.”

Julie Hughes
University of Idaho

References\Further Reading:

1. Cascales E, et al. 2007. Colicin biology. Microbiol Mol Biol Rev. 71:158–229.

2. Lenski RE, Winkworth CL, Riley MA. 2003. Rates of DNA sequence evolution in experimental populations of Escherichia coli during 20,000 generations. J Mol Evol. 56:498–508.

3. Modi RI, Adams J. 1991. Coevolution of bacterial-plasmid populations. Evolution. 45:656–667.

4. Walker D, et al. 2004. Transcriptional profiling of colicin-induced cell death of Escherichia coli MG1655 identifies potential mechanisms by which bacteriocins promote bacterial diversity. J Bacteriol. 186:866–869.

2010-04-16T16:38:00.000-07:00

An efficient stress-free strategy to displace stable bacterial plasmids

Lisa Hale, Orestis Lazos, Anthony S. Haines, and Christopher M. Thomas

Plasmid curing is the process of displacing a plasmid from a plasmid-bearing strain. This is useful for studying phenotypic effects of plasmids on their bacterial hosts. Curing is easy to do when plasmids are unstable and easily lost, but more challenging when dealing with stable plasmids, which can be maintained for a long time in the host even in the absence of selection. Traditionally, plasmid curing is achieved by growing the bacterial host under stressors such as high temperature, detergents or mutagens [1]. This process has the disadvantage of inducing mutations in the bacterial host, which is undesirable. The authors propose a method of plasmid curing based on plasmid incompatibility that can avoid chromosomal mutations. Plasmid incompatibility arises when two plasmids having related replication functions find themselves in the same bacterial cell. This results in the displacement of one by the other that has a second, unrelated replicon [2]. However some plasmids have post-seggregational-killing (psk) genes [3], which produce toxins that kill plasmid-free bacteria in the absence of the anti-toxin, which is another hindrance in plasmid curing. To overcome this, the authors included an anti-toxin gene in their displacing plasmid, which can counter the toxin produced in plasmid-free cells and prevent host killing during plasmid curing. They constructed three vectors having similar replication and stability functions to the plasmids that were being displaced. Plasmid pCURE1, which was constructed to cure plasmid pO157 from E. coli O157:H7, had a pO157-related replicon repF1B and an unrelated replicon from pMB1. To prevent psk, anti-toxin genes related to corresponding genes of pO157 were cloned into pCURE1. Plasmid pCURE1 was introduced into the pO157-bearing strain and successfully cured E. coli O157:H7 of plasmid pO157. Thus, while repF1B replicon disrupted replication of pO157, the anti-toxin produced by pCURE1 prevented host lysis. This produced E. coli O157:H7 bearing only pCURE1, which being unstable was lost rapidly in the absence of selection. Similarly two more vectors (pCURE2 and pCURE11) were constructed for displacing an F plasmid and an IncP-1 plasmid. This approach was thus successful in displacing two different kinds of F plasmids as well as those of the IncP-1 family. The authors also ruled out the possibility of chromosomal integration of pO157 through PCR using pO157-specific primers.
In summary, although plasmid curing through incompatibility has been used before [4], this paper presents a new method, which overcomes psk during plasmid curing. The only drawback is that the authors do not mention how frequent psk is and if the inefficiency of the previously used method was shown to be linked to a plasmid-encoded psk system. To show a direct link between plasmid curing and psk, they could have compared the displacing capacity of a vector with a psk system and another without a psk system. Also, while the authors suggest their method to be more efficient than the previously used method, they do not quantitatively compare the two.

References:
1. Stanisich, V.A. 1984. Identification and analysis of plasmids at the genetic level, pp. 5-32. In P.M. Bennett and J. Grinsted (Eds.), Plasmid Technology. Academic Press, London.
2. Novick, R.P. 1987. Plasmid Incompatibility. Microbiol. Rev. 51:381-395.
3. Gerdes, K., S. Ayora, I. Canosa, P. Ceglowski, R. Diaz-Orejas, T. Franch,
A.P. Gultyaev, R. Bugge Jensen, et al. 2000. Plasmid maintenance systems, pp. 49-85. In
C.M. Thomas (Ed.), The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread.
Harwoord Academic Press, Amsterdam.
4. Tatsuno, I., M. Horie, H. Abe, T. Miki, K. Makino, H. Shinagawa, H. Taguchi,
S. Kamiya, et al. 2001. toxB gene on pO157 of enterohemorrhagic Escherichia coli O157: H7 is required for full epithelial cell adherence phenotype. Infect. Immun. 69:6660-6669

Diya Sen
Graduate Student
University of Idaho

Compensatory gene amplification restores fitness after inter-species gene replacement.

2010-03-25T12:07:00.000-07:00

Lind P.A., C. Tobin, O.G. Berg, C.G. Kurland, and D.I. Andersson (2010) Mol. Microbiol. 75: 1078-1089.

Transfer of genes to organisms can occur in various ways especially in microorganisms. Introduction of foreign genes follows one of the three fates: insertion to replacement of an existing homologous gene locus, uncertain locations on chromosome, or inactivation. If the transferred genes are neutral or deleterious to bacteria, they are likely to be lost over time (Berg and Kurland, 2002). Because of this reason, there have been few examples of experimental evidence for evolution of horizontally transferred genes.

To examine whether and how horizontally transferred genes evolve, the authors replaced the ribosomal protein genes of Salmonella typhimuriums with homologous genes of foreign origin and evolved the strain by repeating serial batch culture transfer. Since the ribosomal protein gene is essential in bacteria, the transferred gene will not be lost and the gene needs to adapt to the new host to allow the cell to grow more efficiently (to increase fitness). Low fitness of the six constructed strains was observed as expected, because the transferred gene and its product do not initially fit the new host for many reasons; for example, difference in codon usage causes translational problem. However, within 25-200 generation of growth, adaptive mutations did overcome the fitness defects of the strains.

An interesting finding was that all genetic changes observed in the six evolved strains were not directly related to the changes in the replaced protein coding sequence, but resulted in increased expression of the introduced gene product. It is known that protein concentration imbalance can cause fitness problems for several reasons (Papp and Pai et al. 2003). The increase in protein expression was required because the introduced alien protein was inefficiently expressed due to the differences in codon usage, or because the alien protein did not have sufficient affinities to the partner molecules (RNA and proteins) to reconstitute an effective ribosome complex. All observed mutations were duplication of DNA segments containing the introduced ribosomal protein gene. This indicates that the rate of beneficial mutations in the protein coding sequence, which can change codon usage or the function of the alien protein, is much lower than the rate of recombination event that results in increase in protein expression level. The former mutation can happen, and could eventually be fixed in the population if you kept evolving the strains for long time, but such beneficial mutations were not fixed in the population within 250 generations of growth of this bacterium.

This study support the hypothesis that gene paralogs and orthologs arise upon horizontal gene transfer in the presence of selection for the gene. Although gene duplication under selection is not a rare event (Reams and Neidle, 2004; Kugelberg and Kofoid et al, 2006), the hypothesis is attractive because bioinformatics analysis revealed that duplication is more common in laterally transferred genes than in indigenous genes (Hooper and Berg, 2003).
By the way, how long does it take for the changes in the coding sequence to be fixed? It depends on selection and population size. Ask mathematicians!

References
Lind P.A., C. Tobin, O.G. Berg, C.G. Kurland, and D.I. Andersson (2010) Compensatory gene amplification restores fitness after inter-species gene replacement. Mol. Microbiol. 75: 1078-1089

Papp B., C. Pai, and L.D. Hurst (2003) Dosage sensitivity and the evolution of gene families in yeast. Nature 424: 194-197.

Berg O.D. and C.G. Kurland. (2002) Evolution of microbial genomes: Sequence acquisition and loss. Mol. Biol. Evol. 19:2265–2276

Hooper S.D. and O.D. Berg. (2003) Duplication is more common among laterally transferred genes than among indigenous genes. Genome Biol. 4: R48

Reams A.B. and E.L. Neidle (2004) Gene amplification involves site-specific short homology-independent illegitimate recombination in Acinetobacter sp. strain ADP1. J. Mol. Biol. 338:643-656

Kugelberg E, E. Kofoid, A.B. Reams, D.I. Andersson, J.R. Roth (2006) Multiple pathways of selected gene amplification during adaptive mutation. Proc. Natl. Acad. Sci. USA 103:17319-17324

H. Yano, University of Idaho

2010-03-15T13:35:00.000-07:00

Exploring the evolutionary dynamics of plasmids: the Acinetobacter pan-plasmidome

Marco Fondi, Giovanni Bacci, Matteo Brilli, Cristiana M Papaleo, Alessio Mengoni, Mario Vaneechoutte, Lenie Dijkshoorn, Renato Fani

Bacteria belonging to the genus Acinetobacter are found in diverse ecosystems such as, soil, water and even animals. Some like A. baumannii are well-known opportunistic human pathogens while others can be useful in bioremediation because of their ability to degrade toxic hydrocarbons. Many of these bacteria have been found to have plasmids of different sizes that probably encode genes that help Acinetobacter sp. survive in the different ecosystems. The goal of this study was two-fold: i) reconstruct the evolutionary dynamics of plasmids of Acinetobacter sp. and ii) investigate the evolutionary cross-talk between plasmid and chromosome. A total of 29 plasmids and seven Acinetobacter genomes from NCBI were included in this study. A computation tool called Blast2Network was used for visualzing plasmid and chromosome relationships. For goal one, the authors retrieved 493 protein sequences form all 29 plasmids and used them as input for the Blast2Network program. This resulted in a network where each plasmid is represented by a ring of balls, each ball being a single protein. In addition there are lines connecting homologous proteins. Changing the degree of identity between proteins generates different networks, such that at 50% sequence identity more proteins are linked together than at 100% sequence identity. Overall the pattern remains the same with three distinct clusters. The first represents a group of plasmids called the pKLH-group that were isolated from different species/strains of Acinetobacter. The second cluster includes plasmids form A. baumannii strains and cluster three has plasmids form other Acinetobacter species. This shows that the pKLH plasmids are the most closely related since they have interlinks among all members even at 100% protein sequence identity, while interlinks decrease for the other two clusters. Thus, although the pKLH plasmids were isolated from different hosts, they have a high relatedness. This is an interesting result, since many of these plasmids are tra- and mob- making conjugation an impossible mechanism of gene transfer between bacteria. Another interesting observation is that plasmids from the same strain often have no connections at 100% identity meaning that no recent genetic exchange probably took place between them. This is surprising because transposition and recombination are common means of gene exchange between plasmids residing in the same host. On the other hand, some proteins, were found to have 100% identity between homologs on plasmids from clusters two and three, meaning that some gene exchange did take place between plasmids from different hosts. Thus, overall these networks are an easy way to visualize complex data. Next they analyzed the functional classes of proteins with the most interlinks. Not surprisingly they found transposition related proteins and mercury resistance related proteins to have high connectivity. It is also interesting that out of 493 proteins, 280 did not have any connections, suggesting that plasmids from Acinetobacter encode a high number of unknown functions. For the second goal, the authors included the genomes of seven completely sequenced Acinetobacter sp. and generated a network of plasmid and chromosome encoded proteins, similar to the network generated previously. The networks show the existence of interconnections between all chromosomes and most of the plasmids. The only exception to this is, four plasmids belonging to cluster three which have no connections to any of the chromosomes. The pKLH group on the other hand was found to be strongly interconnected to two A. baumannii strains. These connections were mostly with mercury resistance related proteins and transposases. Interestingly enough, some plasmids belonging to the same strain such as p1ABAYE, p2ABAYE and p4ABAYE were not found to have any connections to proteins of their host chromosome. This means that gene exchange did not take place between plasmid and chromosome in this case. There are many plasmid-encoded proteins that do not have sufficient identity to chromosome-encoded proteins, suggesting that these may have been acquired from other species/strains of Acinetobacter or other genera. Thus, this study provides an interesting visualization of plasmids of Acinetobacter and their relationships to each other and to some Acinetobacter genomes. I think the most surprising and interesting fact I learned is that tra- and mob- plasmids are promiscuous too and their evolutionary history is often independent of their hosts. There were a couple of areas that were unclear to me, such as, their identity thresholds, which seem to be absolute values instead of a range of values. Also, they do not say why they did not use all twenty-nine Acinetobacter genome sequences and restricted their study to only seven genomes. An interesting feature from figure 4, that they seemed to have overlooked is the fact that single plasmid-bearing proteins have numerous, multiple hits on the same chromosome (A. baumannii SDF) at the 90% threshold. To summarise, their illustrations are really pretty and show the different types of plasmid-chromosome relationships. With the availability of more sequences, such figures may get more difficult to create.

Diya Sen
Graduate student
University of Idaho

Monitoring the Dissemination of the Broad-Host-Range Plasmid pB10 in Sediment Microcosms by Quantitative PCR

2010-02-19T12:14:00.000-08:00

Sebastien Bonot and Christophe Merlin
Applied and Environmental Microbiology Vol. 76 No. 1 2010

There have been many discoveries about the process of horizontal gene transfer and our understanding of this subject has increased greatly in recent years. However, the work that can be done on this process is hindered by limitations inherent in laboratory settings. The results of current experiments are difficult to apply to real-world situations with diverse microbial communities. For example, it is believed that we are only able to cultivate approximately 1% of environmental bacteria under laboratory conditions. Also, genetic information transferred under laboratory conditions may show narrow host expression. Quantifications using culture-based techniques may lead to an underrepresentation of the extent of gene transfer that has occurred, especially in complex environments. Recent work involving green fluorescent protein allows us to see a more realistic representation of the gene transfer that would occur in these complex environments. However, the authors suggest that these studies are still not completely representative of natural environments because the elements under study still require genetic manipulation. PCR and other molecular techniques can get around these problems, but are not typically used due to the fact that so many DNA markers are shared among different genomes. In this paper the authors show that with carefully designed, specific primers they were able to use quantitative PCR (qPCR) to monitor the dissemination of the broad-host-range plasmid pB10 in sediment microcosms. E. coli DH5α was chosen to be the donor strain because it had poor chances of survival since the sediment microcosms under study were not its natural habitat. Also, the high numbers of genetic alterations that this lab strain has undergone were likely to further reduce the donor’s chances of survival.

To follow the fate of plasmid pB10 with qPCR the researchers needed to develop primers specific to the plasmid, as well as to the donor bacteria, E. coli DH5α. The researchers were unable to develop traditional primers specific to pB10 due to the fact that the plasmid has many similarities to other genetic elements. The researchers came up with a very interesting solution to this problem. They used the fact that bacterial genomes have a unique structure based on a combination of DNA blocks instead of just specific DNA sequences. The researchers developed unique primers for pB10 that prime on both sides of a junction of these building blocks. They used the same technique to design primers for DH5α. qPCR tests were performed on total environmental DNA to determine whether or not these primers were truly specific. Amplification was only achieved when DNA was present from pB10 or DH5α.

Tests were run to determine the ability of qPCR to quantify DNA from pB10 and DH5α when in complex environments. Samples were inoculated with known amounts of DH5α/pB10, followed by immediate total DNA extraction and qPCR quantification. 20% of the expected pB10 DNA was recovered, and 0.25% of DH5α DNA was recovered. The researchers postulate that this discrepancy occurred because plasmid DNA is much easier to recover than chromosomal DNA. They also determined that these results mean they must analyze qPCR results based on appearance/disappearance of DNA instead of absolute quantities.

The researchers then performed an experiment to monitor the fate of pB10 in sediment microcosms. The microcosms used were blended river and sediment samples. These were inoculated with pB10/DH5α. Microcosms were maintained for 5 days and total community DNA was sampled at intervals. The researchers found that pB10 concentrations remained stable, while DH5α DNA was lost completely after 48 hours. This suggests that pB10 invaded the microcosm relatively early, before DH5α was lost. Controls that were inoculated with naked pB10 DNA showed a rapid loss of plasmid DNA that disappeared completely after 48 hours. This shows that the plasmid could not have been in the environment extracellularly during the experiment.

This experiment is noteworthy because it monitors the dissemination of a broad-host-range plasmid in a complex environment using molecular tools. I found the use of DNA block primers over conventional sequence primers quite interesting. Perhaps this will lead to new methods of studying horizontal gene transfer in complex environments. The use of qPCR to do this would allow researchers to study horizontal gene transfer in these environments with minimal disturbances to the microbial communities. The researchers plan to expand on their current work by including factors such as spatial structure in future analyses.

Ryan Simmons
University of Idaho

Acquisition of prokaryotic genes by fungal genomes

2010-02-09T13:26:00.000-08:00

Marina Marcet-Houben and Toni Gabaldon

Trends in Genetics Vol. 26 No. 1 2010

Horizontal gene transfer between bacteria is widely studied for its numerous consequences including increased antibiotic resistance, virulence and transfer of metabolic pathways. What is less often considered is horizontal gene transfer from a prokaryotic donor to a eukaryotic recipient. Although this is known to happen, its mechanisms and effects are poorly understood. This study searches for genes of bacterial origin in the fungal kingdom to attempt to gain a broader understanding of the frequency of gene transfer from prokaryotes to fungus and what its evolutionary implications might be.

This study was conducted using whole genomes of over 60 different fungi and over 600 genomes from prokaryotic and other eukaryotic organisms. Using a conservative detection method they found 713 genes over 53 genomes that were acquired from prokaryotic sources. This is an indication of the frequency of horizontal gene transfer to eukaryotes, but also represents a novel way to search for horizontal gene transfer events using whole genomes. However, the number of transfer events is difficult to estimate due to gene loss and multiple genes being transferred in a single event.

The distribution of horizontal gene transfer events over the different fungal clades is not even and suggests factors that could aid or hinder transfer. Identification of traits in eukaryotic species that make them good candidates for horizontal gene transfer could have far reaching implications. Several of the genes that were observed to transfer were analyzed and their possible evolutionary advantages discussed. The first to be discussed was the arsenic detoxification pathway. It appears that the specific types of yeast mentioned have the machinery to reduce arsenate to arsenite but a bacterial reductase successfully transferred and replaced the standard yeast reductase. In another example, to convert between optical isomers of amino acids a racemase is necessary. Several different types were found to be transferred into two different members of the yeast family and a rotifer from a bacterial species. This could lead to the ability to use new sources of amino acids. Bacterial catalases were also found to be transferred to pathogenic fungal species. These catalases help protect pathogens from host reactive oxygen defense mechanisms. Finally, the transfer of a functional bacterial metabolic pathway was found in Aspergillus species. What is remarkable about this transfer is that the three genes that make up this pathway appear to have moved as two units, with two of the fused into a single gene, instead of three separate genes as they are found in the donor.

This study has a well thought out approach to searching for horizontal gene transfer events between prokaryotes and eukaryotes that could allow insight into the evolutionary history of many different species with unique abilities. It will be interesting to see if this type of horizontal gene transfer has played a larger role in the evolution of eukaryotes than previously thought.

Brian Lohman

University of Idaho

Mobilization and prevalence of a fusobacterial plasmid

2010-01-23T22:18:00.000-08:00

Mobilization and prevalence of a fusobacterial plasmid.
Brianna M. Claypool, Sean C. Yoder, Diane M. Citron, Sydney M. Finegold, Ellie J.C. Goldstein and Susan Kinder Haake
Plasmid 2010 63:11-19

Fusobacterium nucleatum is a Gram-negative anaerobic rod found in dental plaque biofilms, and is an opportunistic pathogen implicated in periodontitis as well as a wide range of systemic abscesses and infections.
Studies indicate considerable phenotypic variability between F. nucleatum strains, and biochemical analyses have suggested that F. nucleatum represents a “species complex”. Comparative genomic analyses revealed that 25% of the genes encoded by F. nucleatum ATCC 10953 ssp. polymorphum are not found in either of the sequenced genomes of F. nucleatum ssp. nucleatum and vincentii). In addition, 21% of these unique ORFs mapped to clusters of five or more genes, suggesting that they may have been introduced into the genome by horizontal gene transfer. Several plasmids were found in Fusobacterium but they are too small to be self transmissible.
In this paper authors defined the minimal “mobilon” of plasmid pFN1 that included the relaxase, and DNA directly upstream containing ORF4 and ORF7 with the oriT region located upstream of ORF4. The pFN1 mobilon is related by sequence to the mobilons of the staphylococcal plasmids pC221 and pC223. Authors screened 94 clinical and 4 laboratory isolates of F. nucleatum for plasmids and plasmid encoded relaxase gene and they found that 11.5% of isolates.
As the main discovery authors demonstrated for the first time that the fusobacterial plasmid pFN1 encodes genes enabling efficient mobilization between strains of E. coli by the broad-host range plasmid RP4. I found this experiment really interesting. It is true that IncP plasmid RP4 can mobilize plasmid pFN1 between E. coli strains but if we look closer we can find that plasmid pFN1 cannot replicate in E. coli as oriP15A was used for stable replication. On the other hand there is no evidence that pRP4 can transfer to and/or replicate in F. nucleatum. This can raise a question, how is it possible for pRP4 to mobilize the pFN1 plasmid in vivo. Unfortunately, authors did not discuss that point. On the other hand they realize that the main aim to really prove the role of Fusobacterium plasmids in horizontal gene transfer is to show that plasmid transfer occurs in vivo and find what can support the missing conjugation functions.
Our dual reporter system for detection plasmid transfer in situ in anaerobic conditions can help in this research.

Jarek Krol
UofI

Into the Woods

2010-01-12T08:52:00.000-08:00

The fundamental units, processes and patterns of evolution, and the Tree of Life conundrum

Eugene V Koonin and Yuri I.Wolf

Biology Direct (2009), 4:33

Human communication and thought is largely shaped by metaphors. Metaphors allow man to think about concepts and facts in new ways and to make connections that otherwise might have been left undiscovered. However, if one becomes too committed to a particular metaphor there is the danger of the opposite-of stifling creativity and having a distorted view of individual facts or even the world at large.

"The affinities of all the beings of the same class have sometimes been represented by a great tree. I believe this simile largely speaks the truth. The green and budding twigs may represent existing species; and those produced during each former year may represent the long succession of extinct species. .... The limbs divided into great branches, and these into lesser and lesser branches, were themselves once, when the tree was small, budding twigs; and this connexion of the former and present buds by ramifying branches may well represent the classification of all extinct and living species in groups subordinate to groups."

The authors of this article begin with this quote from Charles Darwin(1) in order to assert that the very roots of evolutionary theory are grounded in the metaphor of a “Tree of Life.” In this metaphor all living organisms are the budding tips of living branches in the Tree of Life (TOL), whose trunk was the original organism from which all life derived. While this metaphor has served scientists well for centuries, our increased understanding of horizontal gene transfer has led to a “crisis of the TOL.”

The authors argue that individual viruses, plasmids, transposons, individual genes, and the like, rather than organisms or species, should be considered as the true fundamental units of evolution (FUEs). The evolutionary history of each individual FUE is still accurately represented by a tree in that an ancestral gene can be altered such that a new version “branches off,” with this process continuing until a full tree is formed. An organism, therefore, is made up of many such FUE trees, and so is more properly a “forest of life.”

The more we learn about the importance of horizontal gene transfer among organisms, species, or even kingdoms, the more clear it becomes that the metaphor of a Tree of Life ignores much of the complexity of true evolutionary histories, especially among prokaryotes. We therefore might benefit from reevaluating our metaphor and perhaps, as the authors suggest, acknowledge the true complexity of the Forest of Life.

References\Further Reading:

1. Darwin C: On the Origin of Species. 1st edition. London: Murray; 1859.

2. Hilario E, Gogarten JP: Horizontal transfer of ATPase genes--the tree of life becomes a net of life. Biosystems31(2-3):111-119. 1993,

3. Doolittle WF: Uprooting the tree of life. Sci Am 2000, 282(2):90-95

4. Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Nelson WC, Ketchum KA, et al.: Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 1999, 399(6734):323-329.

Julie M. Hughes

University of Idaho

2009-10-07T22:12:00.000-07:00

Antimicrobial Resistance-Conferring Plasmids with Similarity to Virulence Plasmids from Avian Pathogenic Escherichia coli Strains in Salmonella enterica Serovar Kentucky Isolates from Poultry.

W. Florian Fricke, Patrick F. McDermott, Mark K. Mammel, Shaohua Zhao, Timothy J. Johnson, David A. Rasko, Paula J. Fedorka-Cray, Adriana Pedroso, Jean M. Whichard,
J. Eugene LeClerc, David G. White, Thomas A. Cebula, and Jacques Ravel

Salmonella enterica is a common cause of food –borne gastroenteritis. This combined with the rise of multidrug resistant S. enterica isolates is a grave medical concern. The S enterica subsp. enterica serovar Kentucky is the most common serotype found in chickens [1,2]. Moreover this serotype is often found to be resistant to antibiotics such as tetracycline and streptomycin [2]. The goal of the study was to find clues to the development of multi-drug resistance in S. Kentucky. The authors analyzed the complete sequences of the 3 large plasmids (pCVM29188_146, pCVM29188_101, pCVM29188_46) that they isolated from S. Kentucky CVM29188. Only the two large plasmids (pCVM29188_146 and pCVM29188_101) were found to carry antibiotic resistance genes. Thus, genes coding for resistance to aminoglycosides (strAB) and tetracyclins (tetRA) were found on pCVM29188_146 and those coding for resistance to cephalosporins (bla CMY-2) were found on pCVM29188_101. Both resistance plasmids (pCVM29188_101 and pCVM29188_146) in this study were found to have intact transfer regions, while the smaller plasmid pCVM29188_46 (46kb) did not have any transfer genes. Sequence similarity of the replication and transfer genes to other plasmids suggest that plasmid pCVM29188_101 may belong to the IncI1 group while plasmid pCVM29188_46 may belong to the IncFII group. The backbone of plasmid pCVM29188_146 is very similar to two plasmids isolated from avian pathogenic E. coli strains and also have the same virulence factors. The plasmid pCVM29188_46 has little similarity to other plasmids and has a lot of hypothetical proteins. They next conducted mating experiments with plasmids pCVM29188_146 and pCVM29188_101 and showed their transfer to two strains of Salmonella and a strain of E. coli. Next they wanted to test the abundance of the virulence genes on other isolates of S. Kentucky from meat, clinical and agricultural sources. So they screened 287 S. Kentucky isolates for the presence of virulence genes by PCR with primers specific for the 5 loci of pCVM29188_146 that were responsible for encoding virulence factors. They found that 64% of all S. Kentucky strains tested positive for the presence of at least one locus associated with virulence. The association was even stronger among the S. Kentucky strains that were isolated from chicken. This however was not the case for the 6 other Salmonella serovars that were isolated from chicken Moreover, all S. Kentucky strains that had at least one virulence locus of Pcvm29188_146 also had resistance to tetracycline and a subgroup of these had resistance to streptomycin. This suggests that a strong association may exist between S. Kentucky and plasmids like pCVM29188_146 that encode both virulence factors and resistance to antibiotics such as tetracycline and streptomycin. One explanation that the authors offer for this association is that pathogenic E. coli strains bearing virulence plasmids may have encountered resistance plasmids, leading to integration of the resistance genes into the virulence plasmid. This new plasmid could have transferred into a Salmonella strain such as the S. Kentucky commonly found in chickens. They do acknowledge that this does not explain why other Salmonella strains isolated from chicken do not have this plasmid type. The second explanation is that virulence plasmids may help S. Kentucky in coping with stress or other enterobacteria and hence, the association. This again does not explain why the association is only seen in S. Kentucky isolated from chicken.
This is an interesting study of plasmids from a strain of bacterium that has medical relevance to us. It is indeed surprising that there is such a clear association of the virulence and antibiotic resistance encoding plasmid with the S. Kentucky strain isolated from chicken. Their explanations for the association seem a little weak.

References:

1. FDA. 2008. National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS). Retail meat annual report, 2006. FDA, Bethesda MD. http://www.fda.gov/downloads/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/UCM073302.pdf
2. USDA. 2008. National Antimicrobial Resistance Monitoring System for Enteric
Bacteria (NARMS). Veterinary isolates final report, slaughter isolates,
2006. USDA, Washington, DC. http://www.ars.usda.gov/sp2UserFiles/Place/66120508/NARMS/narms_2006/NARMS2006.pdf.

Diya Sen
University of Idaho

Mobile Microenvironments

2009-10-05T16:24:00.000-07:00

Horizontal Transfer of the Tetracycline Resistance Gene tetM Mediated by pCF10 Among Enteroccus faecalis in the House Fly Alimentary Canal
Mastura Akhtar, Helmut Hirt, Ludek Zurek
Environmental Microbiology

Vectors have largely aided the spread of microorganisms. Vectors move bacteria from one place to another as they themselves go about their life cycle. The movements of a house fly would be a prime example of this type of vector. However, a role of the vector not considered as often is its role as a habitat for a bacterium itself. During transport, or as a permanent environment, bacteria encounter a unique combination of other bacteria and nutrients that only the vector could assemble. This provides for a microenvironment that can play a key role in the evolution of and dissemination of traits beneficial to bacterial species. While inside the fly these traits can be transferred horizontally through conjugation, transduction and transformation. This study considers how plasmid mediated horizontal gene transfer in the gut of a house fly can mediate tetracycline resistance to transfer between bacterial species.
Two strains of enterocci, bacteria normally residing in the gastrointestinal tract, were selected for donor and recipient. The donor contained the tetM gene to identify transformants using selective media. Flies were separated into two groups, one that received the donor first, via infected food supply, and the other received the recipient strain first. After twelve hours flies were given food source containing the opposite strain for one hour. Each group was then subdivided so that while flies were checked for the presence of donor, recipient and transformants over the next five days and half would have their eating appendage sterilized and half would not.
Results showed that regardless of whether the donor or recipient was introduced first, both groups established concentrations of donor and recipient cells that were similar. The was also no statistical difference in rate of gene transfer between the two groups. Concentration of donor and recipient cells in the digestive tract was similar to that of the surface sterilized, suggesting that observed donor, recipient and transformants were localized to the gut.
Transformants began to be detected 24 hours after both stains were combined. Their presence was screened for using selective media. Groups of flies were sterilized at the surface to eliminate the possibility of surface contamination and transformation outside the vector. Portions of the food supplied to the flies were periodically screened for transformants with very little occurrence, suggesting this did not play a key role. However, it is possible that transfer is taking place on the eating appendage. The conditions in which intestinal gene transfer are best suited are not well understood. The final possible explanation could be that transformants could be the product of high plasmid transfer rate and subsequent rapid clonal expansion of transformants.
Horizontal transfer in a vector could lead to the spread of various genes and provide a unique microenvironment for evolution. The house fly’s unique combination of contact with decaying organic matter and food provides ample opportunity for transfer of traits between bacteria evolved to live in harsh environments to those that are common in food. This potential introduces a need to better understand horizontal gene transfer and evolution in microenvironments that can directly affect humans.

Brian Lohman
University of Idaho

Interkingdom Horizontal Gene Transfer—a hot topic in recent years

2009-09-25T13:45:00.000-07:00

Ancient Horizontal Gene Transfer from Bacteria Enhances Biosynthetic Capabilities of Fungi
Schmitt I, Lumbsch HT (2009) PLoS ONE 4(2): e4437. doi:10.1371/journal.pone.0004437

Until recently, the studies of gene transfer have been mostly focused on prokaryotes, and the process of gene transfer is assumed to be of limited significance to eukaryotes. The availability of diverse eukaryotic genome sequence data is dramatically changing our views on the important role gene transfer can play in eukaryotic evolution. The rapid increase in fungal sequence data has promoted this kingdom to the forefront of comparative genomics. As a result, interkingdom HGT became a hot topic in recent years. Whereas there is very few documented evidence for interkingdom HGT, but they did happen. Here, we will present an ancient interkingdom HGT event between bacteria and fungi.

The targeted gene discussed here is the polyketide synthase (PKSs) genes, which involved in antibiotic and mycotoxin production. Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms.

Bacteria and fungi commonly harbor a group of PKSs that consists of a single protein complex carrying all catalytic sites (typeI PKS). In this paper, the authors are focusing on a clade of fungal type I PKSs gene which is closely related to bacterial PKSs. Since 6-methylsalicylic acid synthase (6-MSAS) was the first PKS in this group to be characterized, this clade is also termed as‘‘6-MSAS-type PKS’’. The lichenized fungi, which are characterized by a sophisticated vegetative morphology and a rich polyketide metabolism, were selected as the research materials in this study. The total genomic DNA of the lichenized fungi, which collected from 12 different countries, were extracted, and then the KS domain of fungal 6MSAS-type PKS genes were amplified by a degenerate primer pair, LC3 and LC5c. The amplified fragments were cloned and sequenced, and then all sequences were subjected to BLAST searches. The alignment was analyzed in a Bayesian phylogenetic framework using MrBayes 3.1. The tree resulting from this analysis was used to determine the PKS clades most closely related to the fungal 6-MSAS group. To evaluate potential problems with outgroup selection, three alignments including different outgroups were compared.

As a result, 24 6-MSA synthase sequence tags from lichen-forming fungi were generated. The results from comparative phylogenetics support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication. In the Discussion, the authors inferred that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase. Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi.

This study revealed the phylogenetic origin of the enigmatic fungal 6-MSAS-type PKS biosynthetic gene using comparative analysis. The results provide statistical support to the hypothesis that this PKS was transferred from an actinobacterial source into ascomycete fungi during an ancient HGT event. They also report the finding of 6-MSAS-type PKS genes in a variety of lichen-forming fungi, and speculate about the possible role of lichen symbionts in the evolution of this gene. Overall, this paper added solid evidence to the fact of interkingdom HGT.

Hui Li Ph.D University of Idaho

Plasmid-mediated multiple antibiotic resistance of Escherichia coli in crude and treated wastewater used in agriculture.

2009-09-18T10:56:00.000-07:00

Plasmid-mediated multiple antibiotic resistance of Escherichia coli in crude and treated wastewater used in agriculture.

S. Pignato, M. A. Coniglio, G. Faro, F. X. Weill and G. Giammanco
Journal of Water and Health Vol 07 No 2 pp 251–258

Antibiotic resistant bacteria strains are permanent threat to human populations. Genes encoding antibiotic resistance are commonly located on mobile genetic elements like bacterial plasmids. Horizontal gene transfer (HGT) occurs in the environmental condition. The main mechanism of HGT seems to be bacterial conjugation. This process requires direct contact between plasmid bearing, donor strain and plasmid free recipients. The frequency of conjugation depends on a number of different factors. One of them is the cell density. As we could imagine possibility of meeting of two bacterial cells living in 1ml of water is much lower than if there are millions of different cells occupying the same volume. Density of bacterial populations in environmental samples varies markedly depending on sampling sites. Usually is not very high ~106 cfu/ml. One of the places where bacterial population reaches high densities are wastewater treatment plants. So study of spread antibiotic resistance encoding bacterial plasmids in the waste water is very important.
In presented paper authors pointed out that the guidelines for cleaning water used for irrigation requires treatments to remove pathogens that can cause enteric infections for crop consumers, producers and handlers. According to the microbiological guidelines for safe use of wastewater in agriculture developed by the World Health Organization(WHO) less than 0.1 intestinal nematode eggs must be detected in 1 litre, while up to 1,000 faecal coliform bacteria per 100 ml can be tolerated for unrestricted irrigation. In the United States, much stricter wastewater quality standards for irrigation are recommended by the Environmental Protection Agency but, lacking federal standards for the quality of reclaimed water, individual states have developed guidelines mainly based on the daily monitoring of faecal coliform bacteria on a single, 100-ml sample, assuming a predictive relationship between indicator microorganisms and pathogen presence.
Although wastewater treatments proved to be effective in eliminating Salmonella spp. and in reaching WHO microbiological standards for safe use of wastewater in agriculture, they were ineffective in reducing significantly the frequency of plasmid-mediated multiple antibiotic resistance in surviving E. coli.
It was shown that 22.71%, 19.41%, 16.84% and 14.28% out of 273 isolates were resistant respectively to ampicillin, tetracycline, sulfamethoxazole, and streptomycin. Some other antibiotic resistant strains were detected at low frequency (trimetoprim – 9.15%; nalidixic acid – 8%; chloramphenicol – 5.12% and kanamycin – 2.93%). Also multiple antibiotic resistance was present in 24.17% of the isolates. Antibiotic resistance was detected to be transferred by conjugation from 54% resistant strains. Three different plasmids with the sizes of 125kb, 54kb and 60kb were isolated from those strains. Also some other mobile elements like class 1 integrons were detected in resistant strains.

Since multiple antibiotic-resistant bacteria carrying integrons and conjugative R plasmids can constitute a reservoir of antibiotic-resistance genes in wastewater reclaimed for irrigation, risks for public health should be considered. Bacterial strains carrying R plasmids and integrons could contaminate crops irrigated with reclaimed wastewater and transfer their resistances to the consumers’ intestinal bacteria. So we should remember to wash every vegetables and fruits before we will eat them…

Jarek Krol
UofI

Competition favors reduced cost of plasmids to host bacteria

2009-09-04T16:46:00.000-07:00

Rembrandt J. F. Haft, John E. Mittler, and Beth Traxler

The ISME Journal (2009) 3, 761-769

When it comes to their relationship with their hosts it can sometimes be difficult to define what plasmids are. They often encode beneficial traits that can be useful, or even vitally necessary, for their host bacteria. A bacterium that finds itself in the gut of a patient taking antibiotics, for instance, may require plasmid-encoded resistance in order to survive. However, despite the potential usefulness of a given plasmid, plasmid carriage also comes with certain costs. In certain environments the same plasmid that used to be essential for survival can become a burden to its host due to the energy required of the host for plasmid maintenance and upkeep. In such circumstances plasmids can be viewed as parasitic; they need the host to survive but only confer costs, not benefits, to the host. Many plasmids have therefore developed clever ways with which to ensure their survival in bacterial populations, even in the absence of external selective pressures for plasmid maintenance (e.g. the presence of antibiotics).

It’s easy to imagine that in the absence of such selective pressures plasmid-free bacteria would out-compete plasmid-bearing cells. Through vertical inheritance alone this would ensure the eventual loss of plasmids from a mixed population of plasmid-bearing and free cells. One tool that most plasmids have to combat this loss is the ability to pass copies of themselves to neighboring cells via the horizontal gene transfer mechanism of conjugation. Conjugation allows plasmids to infect new hosts such that even in the absence of selection plasmids can survive in a population, even as they reduce the fitness of their hosts. Yet many plasmids have developed systems that inhibit their own horizontal transfer. The authors of this paper used a combination of mathematical models and laboratory experiments to determine when and why a plasmid might benefit in repressing, rather than promoting, their own conjugation.

What the authors predicted in their models (and confirmed in their experiments) is that by reducing transfer frequency plasmids can also reduce the cost that they’re imposing on their hosts. While plasmids that don’t repress conjugation will spread through a bacterial population more quickly on their own than plasmids with functioning repression systems, when both types of plasmids are present the latter will eventually take become dominant. This is because in limiting their horizontal transfer they give the competitive edge to their hosts, which will be more fit and grow faster than the bacteria harboring the plasmids that transfer more frequently, and consequently these transfer-limited plasmids are spread mainly through vertical transfer.

This paper not only elucidates the mystery of the persistence of transfer repression systems in plasmids, but also has broader applications to parasitic strategies in general. The authors point out that for many parasites being slightly less virulent gives some parasites a competitive advantage over those that kill their host before they are able to spread to a new one. The mathematical model that they developed could therefore be applied to a much large scope of systems, of which plasmid transfer is just one.

Additional Reading:

Bahl MI, Hansen LH, Sorensen SJ. (2007). Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5 in bacterial populations. FEMS Microbiol Lett 266: 250-6.

De Gelder L, Ponciano JM, Joyce P, Top EM. (2007). Stability of a promiscuous plasmid in

different hosts: no guarantee for a long-term relationship. Microbiology 153: 452-63.

Dionisio F. (2005). Plasmids survive despite their cost and male-specific-phages due to

heterogeneity of bacterial populations. Evolution Ecol Res 7: 1-19.

Freter R, Freter RR, Brickner H. (1983). Experimental and mathematical models of

Escherichia coli plasmid transfer in vitro and in vivo. Infect Immun 39: 60-84.

Kerr B, Neuhauser C, Bohannan BJ, Dean AM. (2006). Local migration promotes competitive

restraint in a host-pathogen 'tragedy of the commons'. Nature 442: 75-8.

Kover PX, Clay K. (1998). Trade-off between virulence and vertical transmission and the

maintenance of a virulent plant pathogen. Am Nat 152: 165-175.

Turner PE, Cooper VS, Lenski RE. (1998). Tradeoff between horizontal and vertical modes of transmission in bacterial plasmids. Evolution 52: 315-329.

Julie Hughes

University of Idaho

Need we optimize the mating system when deal with every different genus？

2009-08-21T15:07:00.000-07:00

Assessment of horizontal gene transfer in Lactic acid bacteria – A comparison of mating techniques with a view to optimising conjugation conditions
Niamh Toomey, Áine Monaghan, Séamus Fanning, Declan J. Bolton
Journal of Microbiological Methods, 2009, 77: 23–28

The most common laboratory techniques used to assess HGT in vitro include: plate, filter and broth mating protocols. While there is a general acceptance that higher transfer frequencies occur when using solid-phase mating mediums (since conjugation requires mating cells to be in close contact with each other), few studies have experimentally compared HGT techniques, with a view to standardizing these approaches, thereby providing meaningful direct comparisons.
In this study, plate, filter and broth mating techniques were assessed and compared by using a taxonomically diverse group, Lactic acid bacteria (LAB). LAB is a Gram-positive, catalase-negative group, which share the capacity to ferment sugars into lactic acid. Due to their aerotolerant anaerobic nature they are found in a variety of different environments. LAB also has the potential to act as resistance gene reservoirs with the ability to transfer these genes in a range of different environments; including transfer to pathogenic species. Genes conferring resistance to antimicrobials such as tetracycline, erythromycin, streptomycin, chloramphenicol, and vancomycin, have been found in LAB isolated from foods. These resistance genes were found to be located on transferable elements including plasmids and conjugative transposons. In present study, one L. lactis strain with the broad-host range plasmid pAMβ-1 [containing an erythromycin resistance marker, erm(B)], and two L. lactis strains with the conjugative transposon Tn916 [expressing a tetracycline resistance gene, tet(M)] were used as the donor strains, along with a marked (Strr, Rifr) L. lactis strain as recipient.
The plate mating technique used in this study is almost the same with that used in TopLab, except that following overnight incubation and scraping of the bacterial spots, additional medium was used to wash the plate for accurately calculating the transfer frequency. The filter mating technique is exactly the same with that we used. The broth mating method is just to add equal volume donor culture and recipient culture into one tube, and then following the overnight incubation, directly dilute the mixed culture and spread onto selective plates. Transconjugants were confirmed as the true transconjugants but not the reverted mutants by using antibiotic selection, E-tests to determine MICs, PCR assays to detect the corresponding marker genes, DNA fingerprinting by pulsed-field gel electrophoresis (PFGE), and Southern blotting.
Based on the results of transfer frequency, the general trend is plate > filter > broth. In addition, in most cases, there is no significant difference between plate mating and filter mating. Effects of different pH, varying from 6.0 to 8.0, on the transfer rate were also detected. The pH of medium between 6.0 and 7.0 had no significant effect on transfer rate, while at pH 8.0 the conjugation was completely inhibited.
As the author said, this paper is the first study to examine the influence of mating protocols on both plasmid and transposon conjugal transfer between lactococcal strains. The results provide the detailed information about which system should be recommended in future LAB HGT studies.
However, some of the other research works are not in agreement with the results from this study. Both Lampkowska et al. (2008) and Langella et al. (1996) suggested that the filter facilitates a greater degree of donor-recipient contact than the plate method. Therefore, a question rises up. Need we optimize the mating system (including the mating techniques and environmental factors, such as temperature, pH) when we deal with every different genus, such as Pseudomonas, Agrobacterium, and Cupriavidus, which currently used in our lab? If it does, then a great amount of work we should do. Do you think it is necessary? Feel free to take part in the discussion. It is a Blog, and there should be some discussion here.

Additional reference:

Lampkowska, J., Feld, L., Monaghan, A., Toomey, N., Schjørring, S., Jacobsen, B., van der Voet, H., Andersen, S.R., Bolton, D., Aarts, H., Krogfelt, K.A., Wilcks, A., Bardowski, J., 2008. A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species. Int. J. Food Microbiol. 127, 172–175.

Langella, P., Zagorec, M., Ehrlich, S.D., Morel-deville, F., 1996. Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake. FEMS Microbiol. Lett. 139, 51–56.

Hui Li, PhD
University of Idaho

The defective prophage pool of Escherichia coli O157: Prophage-prophage interactions potentiate horizontal transfer of virulence determinants

2009-08-16T17:14:00.000-07:00

Asadulghani M. and Ogura Y. et al, (2009) PLOS Pathogens 5: 1-15.

Bacteriophage is one of the major genetic elements promoting horizontal gene transfer between bacteria. Enterohemorrhagic Escherichia coli O157:H7 (Sakai isolate) contains eighteen prophages in its genome (Ohnishi et al. 1999). Two of the eighteen prophages carry Shiga toxin gene clusters; stx1AB and stx2AB; these gene products kill eukaryotic cells by inhibiting protein synthesis. Interestingly, all prophages in O157 have mutations in genes that encode basic phage functions such as site-specific recombination, replication, cell lysis, or structural proteins constituting the head and tail. Evidence obtained by genome analysis suggests that each prophage is defective. Thus, one might think this strain is not very problematic. But, it may be not true.

To assess the role of defective prophages in horizontal gene transfer of virulence determinants, the authors analyzed each basic function of the prophages in O157: excision from chromosome, phage DNA amplification in response to DNA damage, ability to be released as phage particles, and infection of new recipient cells.

Among eighteen prophages, eight prophages including the stx gene-containing phages were found to be released as phage particles, and among them four were able to infect both or either of the two E. coli recipient strains tested. These results strongly suggest that prophages in O157 are complementing defective functions with each other to make practically transferable phages.

Importantly, the phage particle produced often contains a chimeric DNA fragment made of two different prophages' fragments, indicating the recombination of prophages in host cells. Although the newly generated phage DNA were not extensively sequenced in this study, it is possible that some phage fragments reconstituted intact, functional phage through recombination.

This paper nicely explains the way bacteriophages live and propagate in a host. I think this paper is a good example of a post-genomic sequence study. In this era, we can understand how mobile genetic elements are acting in a population by combining sequence analysis and standard genetic experiments. I suspect that plasmids and transposons are also repeating cycles of inactivation and activation as shown for these phages. They will never be extinguished as long as similar mobile genetic elements exist in the world.

Additional reference:
Ohnishi M. and Tanaka C. et al., (1991) DNA Res. 6: 361-368

Posted by H. Yano, Univerisity of Idaho

Plasmid Conjugation in an Activated Sludge Microbial Community

2009-07-31T17:22:00.000-07:00

By Ruoting Pei and Claudia K. Gunsch

ENVIRONMENTAL ENGINEERING SCIENCE,

Vol 26, No 4, 2009, p. 825

This is jet another paper describing plasmid transfer and their role in so called bioaugmentation in natural kind of environments.

Bioaugmentation involves the addition of exogenous micro-organisms that have the ability of degrading the compound of interest. Using this process, new biodegradation pathways can be added, which inherently improve the metabolic conversion of the contaminants. The main problem of this method is that the laboratory strains used to introduce new genetic traits usually cannot grow in natural environmental conditions. This means that bioaugmentation can fail due to the poor establishment and=or survival of new strains under field environmental conditions. The second process used for enhancing natural biodegradation capabilities is biostimulation which consists of adding nutrients (e.g., carbon, nitrogen, electron acceptor, etc.) to promote the growth of indigenous microorganisms. This method is preferred over bioaugmentation because no additional bacterial strains are introduced but it requires the presence of indigenous microorganisms, which are capable of breaking down the contaminant of interest. To overcome the limitations associated with biostimulation and bioaugmentation, it might be possible to combine these two strategies into a technique called genetic bioaugmentation. This technique would consist of adding bacteria with the needed genes and inducing their horizontal gene transfer (HGT) to indigenous bacterial species utilizing natural prokaryotic adaptation mechanisms.

In this paper authors described pWWO plasmid transfer from Pseudomonas putida strain to bacterial population present in activated sludge. The pWWO plasmid is well known for its toluene degradation capability. The plasmid DNA was tagged with transposone containing gfp gene driven by lac promoter. In the donor strain which overproduce LacI repressor gfp is not expressed. In other bacterial hosts gfp is expressed and gives bright green fluorescence which can be measured. Using flow cytometry authors showed that plasmid was actually transferred into bacterial community of activated sludge with number of bacteria expressing GFP protein up to 6%. They showed that the highest number of transconjugants is reached at the third day of experiment and it depends on the donor to recipient ratio with the best results at the 1:20 ratio. Authors tried to connect the plasmid transfer with the actual capability of activated sludge to toluene degradation. This experiment did not really work as the level of toluene degradation remained unchanged during the time of the experiment.

The paper stays in a main stream of microbial ecology and engineering facing the problem of global pollutions and waists utilization. It is very important to use new methods for detecting plasmid transfer in natural environment. Authors were facing the same problem as in previously published papers that there are some limitations in using fluorescent proteins as a plasmid infection marker. The true plasmid bearing cells are not known until gfp gene is expressed and protein is properly matured to give strong fluorescence, which can be detected by flow cytometry. Authors cannot also link the presence of TOL phenotype (toluene degradation) with the number of plasmid bearing cells as the sludge microbial community shows already high toluene degradation activity.

Perhaps, it is not a very “big” paper, but it is a small step toward understanding how the plasmid transfer occurs in natural environment and how can we use this to save our polluted world.

Jaroslaw Krol, PhD

UofI

The SOS Response Controls Integron Recombination

2009-07-16T14:24:00.000-07:00

Émilie Guerin, Guillaume Cambray, Neus Sanchez-Alberola, Susana Campoy, Ivan Erill, Sandra Da Re, Bruno Gonzalez-Zorn, Jordi Barbé, Marie-Cécile Ploy, and Didier Mazel

Science, May 2009 p 1034, Vol. 324, No. 5930.

An ever-growing concern in the medical world is the rise of antibiotic-resistant pathogens. One source of comfort that we have had in this fight against the spread of resistance is that the expression of resistance genes confers a cost to bacteria. In the absence selection for antibiotics it is thought that this cost will give a selective advantage to non-resistant bacteria, and so bacterial populations will loose resistance over time in the absence of antibiotics. The authors of this paper suggest that this is unfortunately not always the case.

In the words of Jurassic Park, “Nature will find a way.” For bacteria, one of the weapons in their arsenal against antibiotics is the SOS response. Under normal conditions the protein LexA binds to the operator region controlling SOS genes to repress their expression. In the event of DNA damage LexA is cleaved from this region and a series of DNA repair mechanisms are activated, some of which are low- fidelity and therefore lead to an increase of mutations, some of which may be beneficial in resisting further DNA damage.

In terms of antibiotic resistance, this paper explains that LexA repression and SOS expression also influences recombination of genes cassettes (which often code for antibiotic resistance. During SOS expression cassette recombination is induced. This can result in either silencing or reactivation of cassettes. In other words, a cassette carrying resistance genes can be reactivated through recombination under times of stress and then revert to a “dormant” form that provides no cost to the bacterium once the SOS response is again repressed.

Additional Reading:
Erill, S. Campoy, J. Barbe, FEMS Microbiol. Rev. 31, 637 (2007).
C. M. Collis, R. M. Hall, Antimicrob. Agents Chemother. 39, 155 (1995).
A. Aertsen, C. W. Michiels, Trends Microbiol. 14, 421 (2006).
D. I. Andersson, Curr. Opin. Microbiol. 9, 461 (2006).

Julie Hughes,
Graduate Student, University of Idaho

A rapid method to construct gene cassette libraries enriched with first gene cassettes and an associated screening method for the clone selection

2009-06-26T10:12:00.000-07:00

First Gene Cassettes of Integrons as Targets in Finding Adaptive Genes in Metagenomes
Lionel Huang, Christine Cagnon, Pierre Caumette, and Robert Duran
Applied and Environmental Microbiology, 2009, 75（11）：3823–3825

Here, I will introduce a short publication focusing on a very narrow specific problem. This paper propose a rapid method for the selection of clones carrying an integron first gene cassette that is useful for finding adaptive genes in environmental metagenomic libraries.

Integrons are genetic structures capable of capturing and excising gene cassettes, which usually encode adaptive proteins in different environmental contexts, such as genes for degradation of pollutants, and resistance to antibiotics or heavy metals. Thus, environmental pressures may favor the propagation of cassettes conferring a selective advantage.

Integrons are not a new and hot topic, but there is an increasing interest in finding of adaptive genes associated with integrons among the huge metagenomes. The integration of a new gene cassette, catalyzed by the integrase, occurs by recombination between the attC site and the attI site of the integron. The first gene cassette of an integron is, therefore, the last one integrated. In previous studies, the determinations of gene cassette collection from environmental metagenomes did not target first gene cassettes, since they were performed by PCR methods targeting attC sites. As the first gene cassette is the closest gene to the promoter, its expression level is the highest in the integron. Thus, it is a good target to find new adaptive genes in metagenomes.

The method was developed by using a pure strain isolate, Xanthomonas campestris ATCC 33913T, which carry an integron possessing 23 gene cassettes. One environmental sample, coastal sediment, was used to validate the method. There are two key points in developing this method, one is the construction of first gene cassettes libraries. To amplify the first gene cassettes, a forward primer targeting the intI gene or attI site must be used. Forward primer AJH72 was used for PCR of X. campestris DNA, and primer ICC48 (intB-inverted primer), targeting the class 1 integron intI, was used for PCR of sediment metagenome. A less-degenerated primer ICC21, was designed to target the attC sites from class 1 and 2 integrons. The other key point is the trick for clone selection. Due to the particular structure of the attC site with inverted repeat sequences, the reverse primer was also used in the forward direction. As a result, several amplified fragments were obtained, and the sequence analyses revealed that most of them were gene cassettes other than the first one. Here, the authors develop a triplex PCR method by labeling the forward primer with HEX (6-carboxyhexafluorescein). The fluorescent PCR fragments were selected for further sequencing.

This method was then applied to coastal mud metagenomes, and 23 fluorescent fragments were detected and sequenced. A total of 29 open reading frames (ORF) were characterized as potentially transcribed by an integron promoter. The first-gene cassettes of integrons appear to be good candidates to find gene cassettes, which aid bacteria in effecting a rapid adaptive response. We are now able to reveal integron last gene acquisitions of environmental bacterial communities submitted to stressful conditions. The PCR method combined with the screening method leads to 100% of clones carrying a first gene cassette. Thus, this new method allows the focus to be on spreading first gene cassettes in metagenomes after a specific stress.

In this paper, the authors propose a good idea to construct gene cassette libraries enriched with first gene cassettes and an associated screening method for the clone selection. However, the only one drawback in this paper is that the relationship between the function of ORF and the oil degradation should be discussed further. Since the first gene cassettes was enriched by adding oil into the coastal sediment, it should be served as the environmental pressure for selecting adaptive genes. I have checked the information from EMBL, where the author deposited the sequences, but still no related information in the database.

Hui Li, PhD
University of Idaho

DNA transfer proteins of broad-host-range plasmid can mediate chromosomal DNA transfer

2009-06-19T17:41:00.000-07:00

The R1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome. Richard Meyer (2009) J. Bacteriol. 191: 1574-1580

It is well-known that plasmids mediate horizontal gene transfer among bacteria and play major roles in the rapid spread of antibiotic resistance. Generally, plasmid-mediated horizontal gene transfer from one bacterial chromosome to another requires the help of transposons as follows; in the first step, a transposon that has captured chromosomal genes moves into the plasmid; in the second step, the plasmid moves into a recipient cell by conjugative transfer; and in last step the transposon on the plasmid jumps into the chromosome of the recipient cell.

This paper showed that the above-described scheme is not the only way that plasmids can participate in the horizontal transfer of chromosomal genes. The author found that a plasmid can directly transfer donor chromosomal DNA into the recipient chromosome.

In a canonical model, conjugative DNA transfer starts with the DNA cleavage at the origin of transfer "oriT", a unique site on the plasmid, which is mediated by a protein called "relaxase". Previously, the author found that relaxase of the broad-host-range plasmid R1162 (also called RSF1010) can initiate DNA transfer at several sequence variants of oriTR1161. The observed promiscuous activity of the relaxase raised the possibility that plasmids can directly mediate the transfer of chromosomal DNA from oriT-like sequences in a chromosome (Jandal S. and Meyer R., 2006).

Draper et al. (2005) showed that relaxase can mediate recombination between two directly repeated oriTs on the transferred DNA in the recipient cell. This nature of the relaxase was used in the experiment to test the hypothesis that relaxase can initiate DNA transfer from cryptic oriT in a chromosome. A plasmid that has the original oriT and a selectable drug-resistance gene marker was artificially integrated into the downstream region of one of the candidate oriT sites in the chromosome of donor strain Pectobacterium atrosepticum to make directly repeated oriT sites. If single-strand DNA is branched out from the candidate oriT and moves into recipient cells, the transferred DNA would become a plasmid that carries a hybrid oriT comprised of the candidate oriT and the original oriT due to the recombination activity of the relaxase. As the author expected, the plasmid that has a part of the donor chromosome and a hybrid oriT was obtained in the recipient E. coli. This result indicates that the initiation of transfer does happen at the candidate oriT in P. atrosepticum.

The next questions are "What length of DNA is it possible to transfer?" and "Is the transferred chromosomal DNA integrated into chromosome in the recipient cell?" To answer these questions, the author used an E. coli strain as donor strains, that has a drug-resistance gene marker at a particular location in the chromosome. In the presence of helper plasmids that express relaxase and pillus proteins, the drug resistance gene marker was transferred and integrated into the chromosome of recipient E. coli. The candidate oriT closest to the resistance gene was 40 kbp away from the resistance gene in the donor chromosome. It suggests that a chromosomal DNA fragment of at least 40 kbp was transferred in the mating process. Surprisingly, even when the closest oriT was eliminated from the donor chromosome, the transfer of the drug resistance maker was observed with almost the same frequency as it was in the presence of the closest candidate oriT. This result suggests that chromosome transfer can be initiated at multiple cryptic oriT sites in the donor chromosome. Given that the second closest candidate oriT is 708 kbp away from the resistance gene, a DNA fragment of at least 708 kbp was indicated to be transferable in this experiment.

The authors estimated that there are 10 candidate oriT sites in the P. atrosepticum chromosome and 8 in the E. coli chromosome, which could be active in the presence of R1162 relaxase. Although it is still not clear how many plasmids have a potential to mobilize fragments of the chromosome, this article clearly showed that there is a novel manner of horizontal gene transfer.

Bacteriophage are also known to mediate the transfer of host's DNA in the manner called "general transduction", where host's DNA are accidentally packed in phage's capsid and are introduced into new host cells. Relaxase-mediated chromosomal DNA transfer resembles phages' general transduction, but different in that the length of transferable DNA is not limited in the relaxase-mediated chromosomal DNA transfer; the size of transferable DNA is limited in general transduction due to the limited size of phages' capsid. Given the size of transferable DNA, it seems that plasmids play much more important roles in bacterial evolution than bacteriophages.

References:

Meyer R. (2009) The R1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome. J. Bacteriol. 191: 1574-1580

Jandle S. and Meyer R. (2006) Stringent and relaxed recognition of oriT by related systems for plasmid mobilization; implications for horizontal gene transfer. J. Bacteriol. 188: 499-506

Draper O., César C. E., Machón C., de la Cruz F., and Llosa M. (2005) Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells. Proc. Natl. Acad. Sci. USA 102: 16385–16390.

H.Yano. University of Idaho

Plasmid Capture by the Bacillus thuringiensis Conjugative Plasmid pXO16

2009-05-27T08:46:00.000-07:00

Plasmid Capture by the Bacillus thuringiensis Conjugative Plasmid pXO16
Sophie Timmery, Pauline Modrie, Olivier Minet, and Jacques Mahillon
Journal of Bacteriology, April 2009, p. 2197-2205, Vol. 191, No. 7

In this article authors describe the ability of Bacillus thuringiensis plasmid pXO16 to transfer as well as mobilize three other plasmids. It is very important issue to study plasmid transfer in Gram positive bacteria as this process is not as well known as plasmid transfer in Gram negative bacteria. Gram positive bacteria are very important as they can be as deadly as Bacillus anthracis – the causative agent of anthrax, but also can be opportunistic pathogens associated with food, like Bacillus cereus. The widely used B. thuringiensis plays very important role as the source of insecticidal toxins. Other Gram positive bacteria like Streptococcus sp. and Staphylococcus sp. are also pathogenic for human. The presence and horizontal transfer of mobile genetic elements that can play a role in spreading an antibiotic resistance as well as pathogenic determinants is very important issue.
So this paper gives us some information about pXO16 plasmid transfer and its ability to mobilize other plasmids. I would like to point out some parts of this paper. First, I was intrigued by the title of this paper. The title could suggest that pXO16 can capture other plasmids DNA molecules and incorporate into its own DNA. From the second sentence we can figured out what authors have on mind talking about capture of other plasmids by pXO16, and that it is the mobilization and retromobilization of other plasmids to the host cells by this Bacillus thuringiensis plasmid.
In the introduction section authors presented very briefly the “state of art” in the plasmid transfer and mobilization field. The special interest is put on retromobilization which is very interesting event. Retromobilization occurs when recipient DNA, either plasmid or chromosomal markers are transferred to the donor during conjugation. This reciprocal DNA transfer is very interesting and it was discussed as it is against the unidirectional DNA transfer rule. The schematic representation of retromobilization models on Fig. 1. is very easy and easy understandable. In the results section we have a very nice story about pXO16 plasmid transfer, and mobilization of “mob” plasmids pUB110, and pE194, as well as no mobilizable plasmid pC194. One thing that bothers me is the way to present conjugation efficiency as the transconjugant to recipient ratio. I know that it is the way, but it does not show overall number of donor, recipient and transconjugant cells in the conjugation mixture and I personally do not like it. In the triparental matings with the plasmid free cells as recipients only two plasmids were used, pUB110 and pC194. Generally lower frequencies of plasmid transfer were observed in triparental than in biparental matings. Authors described also the ability of pXO16 to mobilize pUB110 in three different “media” cow, soy and rice milk and showed the influence of media on mobilizable plasmid transfer. It was also shown that plasmid pXO16 can be used to capture the pUB110 plasmid from other bacteria in all 3 kinds of milk. This is quite interesting that food products can be used as media in this kind of experiments and that biological events can occur in such an environment. We should consider this and look on the food quality especially on expiration date in “milk related” products.
Very interesting results are presented in the section describing plasmid transfer kinetics where it is shown that the plasmid transfer occurs only in a short period of time reaching plateau after certain period of time. It is consistent with other results and proves that plasmid transfer is controlled by specific mechanisms in the cell and these functions are not the only plasmid related.
In discussion authors focused on the mode of retrotransfer and showed that retrotransfer in the case of B. thuringiensis pXO16, and plasmids used in the paper support the two step model. It is interesting as we always thought that we need two kinds of cells, donor and recipient for conjugation to occur. And now we have two kinds of cells but both contain the same conjugative plasmid. Mobilizable plasmid can be transfer only if two cells form mating pair. Mobilizable plasmid encode no functions related to mating pair formation so it means that two “donor” cells can form mating pair and maybe reciprocally transfer DNA molecules. That is really exciting…
To summarize I found this paper quite interesting but some weak points, like poorly written materials and methods, as well as graphical representation of results depreciate the quality of this work and should be corrected before publication.

Jarek Krol, PhD
UofI

A new Family of Gram-positive bacterial plasmids

2009-05-17T14:05:00.000-07:00

Weaver, K. E., Kwong, S. M., Firth, N. & Francia, M. V. (2009).The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids. Plasmid 61, 94–109.

Considering the rate at which sequence databases, and therefore our knowledge of existing plasmids, are growing, it is becoming increasingly important to organize and categorize plasmids into relevant groups. Proper organization of known plasmids would allow us to unravel evolutionary histories and make more efficient the process of integrating our current knowledge. In this article, Weaver et. al. propose to create a family of plasmids characterized by RepA_N, a highly conserved domain in the initiator protein.

Unlike most other plasmid classification systems, this family is not based upon the incompatibility of two plasmids due to similar replication machineryc. Indeed, many of the plasmids within the RepA_N family can stably coexist within a single host bacterium (Kwong et. al. 2008). Rather, the determining characteristic of this plasmid is this conserved initiator protein domain. Phylogenies based on RepA_N matched those of each plasmid’s hosts. What’s more, members of this family are narrow host range plasmids but are found in a diverse range of low G+C gram-positive bacteria (Firth et al., 2000). This suggests that RepA_N family plasmids were present in ancestral gram-positive bacteria of low G+C content and then proceeded to diverge with individual hosts at an early split in the host evolution.

When compared to phylogenies based on other protein domains the modular nature of plasmid evolution becomes apparent. Phylogenies based on RepB, for instance, do not match with host phylogenies or those of RepA_N. RepB is just one among many examples of how plasmids can acquire complete, functional units of DNA from various sources throughout their evolutionary histories. The authors site the specific examples of the replication, partition, and conjugative components of RepA_N plasmids as evolving by “shuffling” between various other plasmids that are found within the same host. Again, RepA_N serves as a good classification marker in that it is conserved within each host and matches its host’s phylogeny.

This article touched on several points that bear particular attention. To begin, the authors point out that this sort of a study cannot be effective without a certain volume of raw data in sequence databases, which was not feasible even a few years ago but is now available and growing. Secondly, with such data evolutionary histories of plasmids and their coevolution with their bacterial hosts can be elucidated and that such information is vital to our understanding of why plasmids are distributed as they are today (e.g., how a family of plasmids can be both broadly distributed and only stably transferred to and maintained in a narrow host range). Finally, this study provides several excellent examples of the modular nature of plasmid evolution. Hopefully in the future available information on previously uncharacterized plasmids will continue to grow and will continue to be organized such that more such evolutionary insights may be made in the future.

Citations:

Kwong, S.M., Lim, R., LeBard, R.J., Skurray, R.A., Firth, N., 2008. Analysis of the pSK1 replicon, a prototype from the staphylococcal multiresistance plasmid family. Microbiology 154, 3084–3094.

Firth, N., Apisiridej, S., Berg, T., O’Rourke, B.A., Curnock, S., Dyke, K.G.H., Skurray, R.A., 2000. Replication of staphylococcal multiresistance plasmids. J. Bacteriol. 182, 2170–2178.

Julie Hughes
Graduate Student
Department of Biological Sciences
University of Idaho

2009-05-11T10:19:00.000-07:00

Bacterial Toxin–Antitoxin Systems: More Than Selfish Entities?
Laurence Van Melderen and Manuel Saavedra De Bast

Bacterial Toxin-Antitoxin systems are diverse and widespread in the prokaryotic world. They are composed of two components, a toxin that can harm the host and its corresponding antitoxin that is needed by the host to prevent cell death. TA systems that are found on chromosomes are hypothesized to have been acquired by horizontal gene transfer. Some bacteria are known to have around 50 putative TA systems such as Nitrosomonas europeae, and Sinorhizobium meliloti. Others have none or a few TA systems. Plasmid encoded TA systems act as addiction modules and help in maintaining plasmid-containing cells. Thus, while the function of plasmid encoded TA systems is well known, those found on chromosomes are not as well understood. There are several theories on the physiological roles of chromosomal TA systems. Following are some of these proposed models:
1) The programmed cell death model: This model is based on the chromosomally located mazEF system of E. coli [1]. Under conditions of stress such as amino acid starvation, high temperature or presence of antibiotics, transcription of mazEF is affected. This is followed by degradation of MazE (antitoxin) by an ATP-dependent protease and subsequent toxification by previously produced MazF (toxin). This in turn leads to cell death.
2) The growth modulation model: This model is based on the relBE system of E coli [2,3]. The primary difference between this model and the previous one is that this model proposes cell growth inhibition under conditions of amino acid starvation and not cell death.
3) The developmental model: This model was proposed for the toxin gene in Myxococcus xanthus [4] an organism that forms fruiting bodies under nutrient starved conditions. The genome of this organism has a homologue of the mazF toxin gene. During fruiting body formation, MazF protein is produced which induces cell death. In fact, nearly 80% of the cells that undergo fruiting body formation die by lysis. However, MazF has also been found to be essential for fruiting body formation.
4) The stabilization model: The model proposes that TA systems could help stabilize some regions of the genome that are unstable and prone to being lost [5]. Such TA systems are often found on structures called super integrons that carry many essential and non-essential genes. The TA systems stabilize the super integrons as well as unstable plasmids or genomic regions.
5) The anti-addiction model: This model proposes that chromosomal TA systems can benefit their hosts during post seggregational killing [6]. The chromosomal TA system of Erwinia chrysanthemi 3937 was found to prevent post seggregational killing of bacterial cell after loss of plasmid.
The above models show the different ways in which TA systems can confer a selective advantage to their hosts.
Thus TA systems on chromosomes are diverse and have evolved multiple roles from being simple addiction modules to more complex systems involved in cell physiology.
References:
[1] Engelberg-Kulka H, Amitai S, Kolodkin-Gal I, Hazan R. Bacterial programmed cell death and multicellular behavior in bacteria. PLoS Genet. 2006;2:e135. doi:10.1371/journal.pgen.0020135.
[2] Christensen SK, Mikkelsen M, Pedersen K, Gerdes K. RelE, a global inhibitor of translation, is activated during nutritional stress. Proc Natl Acad Sci U S A. 2001;98: 14328–14333.
[3] Christensen SK, Pedersen K, Hansen FG, Gerdes K. Toxin-antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA. J Mol Biol. 2003; 332:809–819.
[4] Nariya H, Inouye M. MazF, an mRNA interferase, mediates programmed cell death during multicellular Myxococcus development. Cell. 2008;132:55–66.
[5] Rowe-Magnus DA, Guerout AM, Biskri L, Bouige P, Mazel D. Comparative analysis of superintegrons: engineering extensive genetic diversity in the Vibrionaceae. Genome Res. 2003;13:428–442.
[6] Saavedra De Bast M, Mine N, Van Melderen L. Chromosomal toxin-antitoxin systems may act as antiaddiction modules. J Bacteriol. 2008;190:4603–4609.

DIYA SEN
GRADUATE STUDENT
BIOLOGICAL SCIENCES
UNIVERSITY OF IDAHO

A novel gene module protecting bacteria from phage infection

2009-03-31T17:20:00.000-07:00

The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Fineran PC, Blower TR, Foulds IJ, Humphreys DP, Lilley KS, and Salmond GP. (2009) Proc Natl Acad Sci U S A. 106:894-899.

Recently it has become easy to determine the complete sequence of 100 kb long plasmids. However, the ensuing annotation process, assigning a function to a DNA sequence, is still a frustrating process for people who are working on sequence analysis; often, the newly sequenced DNA segment does not show homology to well-known genes which makes difficult to judge whether or not there is a gene in the segment.

Let's hope that no homology is a good sign for a big discovery. Here, I introduce a discovery of the novel gene module on Erwinia carotovora plasmid pECA1039, which protect host bacterium from phage infection.

Erwinia carotovora is a plant pathogenic bacterium that causes rot in diverse vegetables. The author's group has been studying virulence mechanisms of E. carotovora (Barnard A.M. and Bowden S.D. et al., 2007). They identified a probable protein coding sequence, named toxN, through the complete sequence analysis of E. carotovora's 5,620-bp cryptic plasmid., whose product shows 31% amino-acid identity to a protein associated with phage abortive infection (Abi) in gram-positive bacteria (Emond E. and Shirley E.D. et al., 1998). Genes related to Abi generally exert a cellular process that shuts down the phage lytic cycle or that kills phage-infected cells to prevent the phage particle production (Chopin M.C. and Chopin A. et al., 2005). The toxN gene on pECA1039 is adjacent to a potential coding sequence toxI that includes five copies of a 36-bp sequence tandem repeat, followed by an inverted repeat sequence. The potential gene product ToxI exhibits no similarity to proteins in databases. Since the Abi system was not so common among gram-negative bacteria, the authors focused the study on the toxI-toxN region and experimentally showed that the toxI-toxN region confers phage-resistance to the host.

The authors have two major subject to be addressed: one is the mechanism of phage resistance, and another is whether or not toxI encodes a protein.

The authors showed that toxN encodes a toxic protein that inhibits the host's growth, which means that the mechanism of phage resistance might be a growth inhibition induced by phage infection. The authors also found that the co-expression of toxI with toxN can counteract the toxic activity of ToxN. But, the hypothetical protein ToxI seemed to not be produced from the toxI gene region according to Western blotting analysis, using the modified toxI gene fused with a sequence coding for hexa-histidine tag.

It is possible that toxI RNA itself has an activity to counteract ToxN. To test this hypothesis, the authors introduced a translational stop codon into the toxI coding sequence and found that the ToxN-counteracting activity was still retained in the mutant toxI region. Furthermore, point mutations that do not change the amino-acid sequence of ToxI but do change the transcript sequence resulted in the loss of ToxN-counteracting activity. These results suggest that the toxI RNA is responsible for the antitoxin function. They also pointed out that similar gene modules that are comprised of the tandem repeat region and the toxN homologous gene are present in diverse Eubacteria and Archea.

Detailed mechanisms of toxin (ToxN) induction and the interaction between the toxI RNA and the ToxN protein are still unclear. However, it is obvious that the authors identified a novel type of functional RNA. The authors' work proved that we can still discover new biological concepts from plasmid sequences.

References:
Fineran PC, Blower TR, Foulds IJ, Humphreys DP, Lilley KS, Salmond GP. (2009) The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Proc Natl Acad Sci U S A. 106:894-899.

Barnard AM, Bowden SD, Burr T, Coulthurst SJ, Monson RE, Salmond GP. (2007) Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria. Philos Trans R Soc Lond B Biol Sci. 362:1165-1183.

Chopin MC, Chopin A, Bidnenko E.　(2005) Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8:473-479.

Emond E, Dion E, Walker SA, Vedamuthu ER, Kondo JK, Moineau S. (1998) AbiQ, an abortive infection mechanism from Lactococcus lactis. Appl Environ Microbiol. 64:4748-4756.

posted by H. Yano (University of Idaho)

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

2009-03-25T16:02:00.000-07:00

Scott A. Lujan, Laura M. Guogas, Heather Ragonese, Steven W. Matson, Matthew R. Redinbo,
PNAS 2007 vol. 104 no. 30 12282-12287

Since mid sixties when so-called “R factors” were found to spread antibiotic resistance between different bacterial strains, lots of effort has been put on finding the simplest and universal method to stop horizontal plasmid transfer. Many papers described the effect of various substances on conjugation. Many substrates have a detrimental effect on plasmid transfer just by influencing the bacterial propagation and general life functions. Sometimes the results could be quite surprising and unexpected, but some ordinary everyday products like green tea (epigallocatechin gallate), coffee (caffeine), and papaya seed macerate can inhibit conjugation and ipso facto spreading of an antibiotic resistance. The most common approach to this kind of study is just to add the material under investigation to the conjugation mixture and evaluate plasmid transfer efficiency over a time. Conjugation is a very complicated, multistep process. The functions necessary for conjugation are encoded by plasmids and do not depend on the host. Many studies have been done to establish the function of particular genes. One of the most important proteins involved in plasmid transfer is a DNA relaxase. The conjugative relaxase initiates DNA transfer with a site- and strand-specific ssDNA nick in the transferred strand (T-strand) at the origin of transfer (oriT), forming a covalent 5′-phosphotyrosine intermediate. The nicked T-strand moves from the donor cell to the recipient cell via an intercellular junction mediated by a type IV secretion system. The relaxase completes DNA transfer by reversing the covalent phosphotyrosine linkage and releasing the T-strand. In the F plasmid, this relaxase is located in the N-terminal domain of a large multifunctional protein, TraI (DNA helicase I). The relaxase active site contains one or several tyrosine residues: F-like relaxases (found in IncF, IncN, IncP9 and IncW plasmids) contain 2 to 5 tyrosines while relaxases of IncQ, IncP, IncI plasmids and Ti plasmid of Agrobacterium sp. possess only one. F-like relaxases encoded by the traI gene shares significant sequence identity with relaxases of many R plasmids (e.g., 98% with R100 TraI); thus, the F plasmid serves as a model system for examining conjugative plasmids and the inhibition of conjugative transfer.
In this study the authors first describe the role that the relaxase enzyme plays in the initiation and termination of DNA conjugation and then use that information to identify potent relaxase-specific inhibitors. This is the first paper which described a bottom-up approach to identify the first small molecule inhibitors of conjugative DNA transfer.
The authors determined the 2.4-Å crystal structure of the 300-residue N-terminal relaxase domain of F plasmid TraI and found it similar to other, previously described relaxase domains. They found that the tyrosine at the active site is responsible for binding the oriT thymidine. Based on electron density they found presence of a divalent cation in the active site and identified it as Mg2+. A survey of magnesium-binding proteins in the Protein Data Bank revealed that the chelation of Mg2+ by neutral amino acid residues is diagnostic of a site that simultaneously binds to multiple phosphate groups. Mutation of the metal-chelating residue histidine-159 to glutamic acid eliminated relaxase activity. These data indicated that the 2+ charge on the bound metal ion is critical to relaxase function. The proposed models for relaxase role in binding DNA strands during conjugation suggested that relaxase binds two phosphate groups. To prove this theory authors used a simple and relatively stable bisphosphonate – imidobisphosphate (PNP) molecule and found that at nanomolar concentration PNP inhibited relaxase activity in vitro. Further studies established that relaxase can be effectively inhibited by substrates where two phosphonate residues are separated by three or fewer atoms and have no additional negative charge at pH 7.4. Five additional inhibitors were found: methylenediphosphonic acid (PCP), iminobis(methylphosphonic acid) (PCNCP), etidronic acid (ETIDRO), clodronic acid (CLODRO), and 1,2-bis(dimethoxyphosphoryl)benzene (PBENP). ETIDRO and CLODRO are bisphosphonates clinically approved as drugs used to treat bone loss by inhibiting farnesyl diphosphate synthase Two other inhibitors identified, PCP and PNP, have been used as radioisotope carriers in humans. The simplest inhibitors, PCP, ETIDRO, and CLODRO, were then characterized further by using a kinetic assay and exhibited purely competitive inhibition, with Kic,app values ranging from 3 to 145 nM. Taken together with the PNP results, these data validate the prediction that F-like conjugative relaxases can accommodate two phosphotyrosine intermediates simultaneously within their active sites. Significantly, these data also establish that bisphosphonates (including clinically approved compounds) potently inhibit the in vitro relaxase activity of F TraI with Ki values in the nanomolar range.
The in vivo tests confirmed the results obtained in vitro. In addition to conjugation inhibition, micromolar concentrations of PNP caused death of plasmid-containing, but not plasmid free cells by blocking relaxase activity.
The presented results show that the clinically approved bisphosphonates etidronate (Didronel) and clodronate (Bonefos) are potently effective at killing F+ cells and preventing conjugative DNA transfer. These particular compounds could also be combined with existing antibiotics to create potent antimicrobial cocktails. Etidronate and clodronate exhibit low absorption and can be administered at high oral doses. According to the authors, extrapolating from the results, approved doses of etidronate and clodronate would be expected kill >90% of plasmid + cells and to stop >80% of conjugative transfer within the gastrointestinal tract. Such results are relatively mild, given the large bacterial populations present in the gastrointestinal tract or at wound sites, but may be enough shift the balance toward success in a variety of recalcitrant clinical infections, especially given the prevalence of conjugative plasmids within multidrug-resistant bacterial strains. The treatment of skin infections, primary sites of nosocomial antibiotic resistance transfer, using topical applications of bisphosphonates may also be effective. In summary, this study establishes conjugative relaxases as a unique antimicrobial target. The results suggest that approved therapeutics could have an immediate impact, alone or in combination with existing antibiotics, in the prevention of resistance propagation during clinical treatment of bacterial infections, thus extending the lifetime of our antibiotic arsenal.
In conclusion this paper shows us a very important thing: basic research on the conjugation process in plasmid transfer that showed the crucial role of relaxase protein led to more detailed application studies that give us a potential weapon to fight bacterial conjugation and the spread of antibiotic resistance in the world of microorganisms.

Additional papers.

Zhao, W.-H. , Z.-Q. Hu, Y. Hara and T. Shimamura 2001 Inhibition by epigallocatechin gallate (EGCg) of conjugative R plasmid transfer in Escherichia coli. J.Infect. Chemotherapy 7: 195-197

Tiagunenko IuV, Glatman LI, Antsiferova NG., 1975. Caffeine as an inhibitor of the conjugation transfer of R-factors. A study of certain aspects of the mechanism of action of caffeine on the conjugation transfer of R-factors], Antibiotiki 20: 253-257.

Leite A.A.M., Nardi R.M.D., Nicoli J.R., Chartone-Souza E. and Nascimento A.M.A., 2005. Carica papaya seed macerate as inhibitor of conjugative R plasmid transfer from Salmonella typhimurium to Escherichia coli in vitro and in the digestive tract of gnotobiotic mice. Gen. Appl. Microbiol. 51: 21-26

Fernandez-Lopez R., Machón C., Longshaw C.M., Martin S., Molin S., Zechner E.L., Espinosa M., Lanka E. and de la Cruz F. 2005. Unsaturated fatty acids are inhibitors of bacterial conjugation. Microbiology 151: 3517–3526

dr Jaroslaw E. Krol
UofI

Genome's barcodes

2009-03-13T19:26:00.000-07:00

It is important to assign short sequence fragments generated by metagenomic studies to original sources (genomes). Zhou et al. (2008) investigated the oligonucleotide (k-mer) frequencies termed 'barcode' for this purpose.

From the barcodes (Figure 1), 4-mer frequency distrubitions are stable along the chromosomes, and phylogenetically closely related species have more similar barcodes than distantly related species. This is completely agreement with previous studies by Karlin and colleagues, using 2-mer frequeinces (dinucleotide relative abundance, termed 'genomic signature').

Concerning plasmids, the author stated that 'The barcodes of all plasmid genomes also tend to have similar characteristics among themselves, possibly due to being under similar selection pressure caused by their frequent transferring among cell cultures.' It isn't clear what the selection pressure is.

One interesting observation is that different classes of genomes (prokaryotes, eukaryotes, plastids, plasmids, and mitochondria) were separated by two features derived from their barcodes (Figure 4). One feature (x-axis) is the average variation of 4-mer frequencies (across a whole genome across all 4-mers), and the other (y-axis) is the overall similarity (in 4-mer frequencies) among all fragments of the genome. Note that the neighboring genomes in this feature space do not necessarily have similar barcodes. Although the feature space clearly separated these five different classes of genomes, biological implications of the separations were not described.

This has inspired us to investigate factors contributing variations in barcodes (oligonucleotide frequencies) among different genomes. The possible factors include restriction site (Abe et al., 2003), synonymous codon usage, amino acid usage, G+C content (Sandberg et al., 2003), and mosaic structure of the genome.

PRIMARY ARTICLE:
Zhou F, Olman V, Xu Y. BMC Bioinformatics. (2008) 9:546. Barcodes for genomes and applications.

ADDITIONAL REFERENCES:
Karlin S, Campbell AM, Mrázek J. Annu Rev Genet. 1998;32:185-225. Comparative DNA analysis across diverse genomes.

Abe T, Kanaya S, Kinouchi M, Ichiba Y, Kozuki T, Ikemura T. Genome Res. 2003 Apr;13(4):693-702. Informatics for unveiling hidden genome signatures.

Sandberg R, Bränden CI, Ernberg I, Cöster J. Gene. 2003 Jun 5;311:35-42. Quantifying the species-specificity in genomic signatures, synonymous codon choice, amino acid usage and G+C content.

Dr. Haruo Suzuki
University of Idaho

The Diversity of Nature

2009-03-10T13:43:00.000-07:00

With the advent of next-generation sequencing capabilities, many questions that were previously beyond our capabilities to explore are now well within reach. Metagenomic approaches in particular have recently experienced a burst of recognition. One such question that can no be addressed is the extent to which isolated cultures used in laboratory settings represent the true genetic diversity apparent in nature (Tettelin 2005). Pyrosequencing is especially important for such studies in that it is culture-independent. That is, genome sequences can be obtained from a sample even if the species in that sample cannot typically be grown in a laboratory setting. The authors of this paper applied 454 pyrosequencing to marine and coastal samples of cyanobacteria of the genus Synechococcus. This allowed them to identify the levels of diversity present in natural Synechococcus populations and acknowledge the importance of horizontal gene transfer in marine and coastal populations of cyanobacteria.

The authors were able to enrich their samples with Synechococcus by capitalizing on the genus’ natural fluorescence and thereby sorting out the bacteria in the samples using a high-speed flow cytometer. After this enrichment, 454 pyrosequencing was conducted on the two coastal and one open-ocean cyanobacterial populations. These sequences were then compared to the four complete Synechococcus genome sequences available (including representatives from the two most abundant clades-I and IV- as well as clades II and III)( Fuller 2003). Up to 25% of the reads could be mapped to the previously sequenced genomes when the authors used lenient cutoffs (70% identity, 70% sequence length). Of the gene models that had no hits, ca. 48% had atypical trinucleotide content, suggesting that they may have been recently acquired through horizontal gene transfer. Indeed, at least three plasmid families were found in the samples sequenced, again pointing to the potential contribution of horizontal gene transfer to the diversity within marine Synechoccoccus. This is the first reported incidence of plasmid detection in marine, rather than freshwater, cyanobacteria.

In summary, the authors provided this evidence that plasmids play a part in marine cyanobacterial diversity and indicated that, while backbone genes were highly conserved across the metagenome sample, accessory genes were widely varied. Therefore, nature is much more diverse than would be indicated if we only took into account those few cultured representatives that we use in the lab.

Primary article:
B. Palenik, Q. Ren, V. Tai and I. T. Paulsen. (2009) Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity. Environmental Microbiology 11(2):349-359.

Additional Reading:
Fuller, N.J., Marie, D., Partensky, F., Vaulot, D., Post, A.F., and Scanlan, D.J. (2003) Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea. Appl Environ Microbiol 69: 2430–2443.

Glover, H.E. (1985) The physiology and ecology of the marine cyanobacterial genus Synechococcus. Adv Aquat Microbiol 3: 49–107.

Tettelin, H., Masignan, I.V., Cieslewicz, M.J., Donati, C., Medini, D., Ward, N.L., et al. (2005) Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial ‘pan-genome’. Proc Natl Aca Sci USA 102: 13950–13955.

By Julie Hughes
Graduate Student
University of Idaho

2009-03-03T10:48:00.000-08:00

Prevalence of tetracycline resistance genes in Greek seawater habitats

Theodora L. Nikolakopoulou, Eleni P. Giannoutsou, Adamandia A. Karabatsou, and Amalia D. Karagouni

Tetracyclines are antibiotics that have been used for over 40 years as a therapeutic agent in human and veterinary science and also as growth promoters in animal husbandry. Tetracyclines inhibit bacterial growth by interfering with protein synthesis. Overuse of such antibiotics has lead to the rapid spread of antibiotic resistance genes among bacteria. The most common mechanisms of resistance are tetracycline efflux, ribosome protection and tetracycline modification. Since these resistance genes are often found on mobile genetic elements such as transposons, they can be spread rapidly across bacterial species (Schmidt et al., 2001; Roberts, 2005).

The goal of the research presented in this paper was to analyze the presence of 12 tetracycline resistance (tet A,B,C,D,E,G,H,K,L,M,O,T) genes in seawater sampled from different locations in Greece. The broader goal of this research is to study a complex ecosystem such as the marine environment, which acts like a reservoir of antimicrobial compounds and resistant bacteria (Aoki, 1992; Chee-Sanford et al., 2001).

Water was sampled from 4 different habitats: a) seawater near a wastewater treatment facility b) seawater near a fish farm c) sea water near a tourist spot and d) sea water from an uninhabited location. These water samples were plated onto a nutrient medium and then incubated. Colonies were picked and grown individually. Dilutions were made of these cultures and plated onto Agar containing tetracycline for 3-7 days at 20° C. Distinct colonies having different morphologies from each sample were isolated in pure culture. A total of 89 TcR colonies were picked: 36 from the fish farm, 23 from wastewater, 14 from the tourist place and 16 from the uninhabited location. These 89 colonies were analyzed for the presence of genes conferring TcR by polymerase chain reaction (PCR) using primers specific for the 12 kinds of TcR genes and then southern blotted. This showed that 60 colonies had more than one TcR (tet) gene and the remaining 29 had only tetK which encodes a tetracycline efflux pump. Thus, tetK was clearly the most dominant of the 12 genes. Plasmid extraction from the 60 colonies that had more than one tet gene followed by gel electrophoresis revealed the presence of plasmids (around 40 kb in size) in 37 of the colonies. Ten of these 60 colonies had IncP-type plasmids as revealed by PCR using primers specific to sequences of IncP, IncQ, IncW and IncN plasmids. Thus, it is possible that the TcR genes were carried by these large plasmids. To confirm the presence of plasmids in all seawater samples, exogenous plasmid isolations were performed (Hills et al., 1996; Smalla et al., 2000) using tetracycline as a selective agent. TcR plasmids ranging in size between 40 and 80 kb were isolated from 80 transconjugants. 59 of 80 plasmids possessed one or more tet gene. The tetA gene was dominant as it was found in 36 plasmids as the only tet gene and was found with tetK and tetC on a smaller number of plasmids. PCR showed that 27 of the 59 plasmids belonged to the IncP group of broad host range plasmids.

To summarize, this study showed the presence of TcR bacteria in all seawater 4 samples. However, when the community composition of each sample was analyzed, the samples were found to vary from each other in the content of bacterial species. Thus despite the fact that TcR genes were found in these samples they were probably found on different bacteria. Of the 12 kinds of tet genes, only 4 genes (tet K,A,M,C) were found in these samples while others (such as tet B, D, E, G, H, L, O, and T) were not found at all. This is the first study that reported the presence of tetK in the bacterial strains identified from the seawater samples. Moreover many of the tet genes were found on IncP- type broad host range plasmids. This suggests that the spread of TcR genes in marine environments could be because of their association with IncP-type broad host range plasmids.

References:

Schmidt, A.S., M.S. Bruun, I. Dalsgaard, and J.L. Larsen. 2001.Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment.Appl. Environ. Microbiol. 67, 5675-5682.

Roberts, M.C. 2005. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 245, 195-203.

Aoki, T. 1992. Present and future problems concerning the development of antibiotic resistance in aquaculture, p. 254-262. In C. Michael and D.J. Alderman (eds.), Chemotherapy in aquaculture: from theory to reality-1992. Office International des Epizooties, Paris, France.

Chee-Sanford, J.C., R.I. Aminov, I.J. Krapac, N. Garrigues-Jeanjean, and R.I. Mackie. 2001. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol. 67, 1494-1502.

Hill, K.E., J.R. Marchesi, and J.C. Fry. 1996. Conjugation and mobilization in the epilithon, p. 5.2.2/1-5.2.2/28. In D.L. Akkermans, J.D. Van Elsas, and F.J. De Bruijn (eds.). Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Smalla, K., H. Heuer, A. Götz, D. Niemeyer, E. Krögerrecklenfort, and E. Tietze. 2000a. Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-Like plasmids. Appl. Environ. Microbiol. 66, 4854-4862.

Diya Sen
Graduate student, University of Idaho

Stabilization of pSW100 from Pantoea stewartii by the F conjugation system

2009-02-12T16:41:00.000-08:00

Mei-Hui Lin and Shih-Tung Liu (2008) J. Bacteriol. 190: 3681-3689

Plasmids are selfish parasite? It must be partially true as long as plasmids confer no selective advantages on host bacteria. This article tells us one of such aspects of a small plasmid.

When host bacteria divide to produce daughter cells, plasmids are subjected to the risk of segregation from cells. To reduce the risk of segregational loss, most naturally occurring plasmids possess an active partitioning system (Funnell B. E. and Slavcev R. A., 2004; Graumann P.L. 2007) and/or post-segregational killing systems (VGP Blog Dec 28, 2008). If there was a plasmid that did not seem to have such systems, it could be just because we are not aware of the strategy of the tricky plasmid.

A plant pathogen Pantoea stewartii strain SW2 harbors more than 11 plasmids whose size are ranging from 4 kb to 320 kb. One of the plasmids pSW100 is a 4-kb ColE1-like plasmid that encodes two proteins related to DNA-transfer function. Despite that pSW100 does not seem to possess any genes related to the function to avoid segregational loss, pSW100 is stably maintained in the original host SW2 and E. coli strain HB101. Interestingly, pSW100 is unstable in another E. coli stain DH5α.

The question is why pSW100 is stable in HB101?.

The authors thus posit that pSW100 use a novel maintenance system, which is highly dependent on strain-specific factors. To find chromosomal factors responsible for the stable maintenance of pSW100, the authors created a mutant library of HB101 using transposon mutagenesis. From the mutant library, clones that became unable to support the stable maintenance of pSW100 were obtained. When transposon-insertion sites were sequenced, it turned out that most clones had an insertion of transposon in the sex pillus protein gene on the chromosome, which is considered to be remnant of F-plasmid. The authors also found that the 38-bp sequence of pSW100 was responsible for its stable maintenance in HB101, and they showed that the TraC protein provided from the F-plasmid remnant actually binds to the DNA segment containing the 38-bp sequence. This result leads us to speculate that pSW100 is stabilizing itself by attaching to the cytoplasmic component of the sex pillus of F-plasmid. The authors also showed that plasmid pSW1200 that coexists with pSW100 in strain SW2 carries traC homologue. This result suggests that pSW100 is parasitic on pSW100 in strain SW2.

This article raised novel and important ideas that (i) sex pillus components can stabilize plasmids by directly binding to plasmid DNA, and (ii) there could be other cases that remnants of plasmids on chromosomes positively affect the stability of other plasmids.

Personally, I am pretty surprised to know that E. coli HB101 has a remnant of F-plasmid on its chromosome because HB101 has been known for F-plasmid-free strain for decades!

References:

Funnell B. E., and R. A. Slavcev. 2004. Partition Systems of Bacterial Plasmids in Plasmid Biology, ASM press, Washington D.C.

Graumann P. L. 2007. Cytoskeletal Elements in Bacteria. Annu. Rev. Microbiol. 61:589-618

Lin M.H. and S.T. Liu. 2008. Stabilization of pSW100 from Pantoea stewartii by the F conjugation system. J. Bacteriol. 190: 3681-3689

posted by H.Yano (University of Idaho)

Qui gladio ferit, gladio perit

2009-01-31T13:54:00.000-08:00

Bacterial conjugation-based antimicrobial agents.
Marcin Filutowicz, Richard Burgess, Richard L. Gamelli, Jack A. Heinemann, Brigitta Kurenbach, Sheryl A. Rakowski, Ravi Shankar
Plasmid 60 (2008) 38–44

Horizontal gene transfer is an essential mechanism in the adaptive evolution of bacteria. In genomic era it is more evident that the antibiotic resistance genes are spread across microbial population. It is also known that these genes are often located on mobile genetic elements, of which conjugative plasmids represent a major group and are found in nearly all Prokaryotes. Broad host range plasmids and conjugation are the main ways of spreading antibiotic resistance through bacterial population. But “He who lives by the sword shall die by the sword”, so could we take advantage of conjugation and use it to kill pathogenic bacteria.
In this short review paper authors summarize the work on so called bacterial conjugation-based technologies (BCBT). These technologies exploit plasmid biology for combating the rising tide of antibiotic-resistant bacteria. Specifically, the concept utilizes conjugationally delivered plasmids as antimicrobial agents, and it builds on the accumulated work of many scientists dating back to the discoveries of conjugation and plasmids themselves.
It is easy to imagine how it works. Genetic information carried by plasmid DNA is expressed in the recipient cells upon conjugation. So if plasmids carry instruction for destruction of the host cells it is executed. Authors present 3different ways to kill the host cell by plasmid.

The first approach is very simple. It uses so called runaway plasmid which can replicate without any control in a host cell. Replication of plasmid DNA acts like a trap to capture all of the cell’s available replication machinery to the exclusion of chromosomal replication.

The second is production of plasmid- or chromosome-encoded bacteriocins. In almost all instances, cells producing a bacteriocin also produce a bacteriocin-specific antidote, typically a peptide or RNA. For BCBT purpose, an anti-kill antidote, which can neutralize the expression of a plasmid-encoded antimicrobial agent, can be integrated into the chromosome of the donor bacteria. Susceptible recipients are killed after plasmid transfer from the protected donor cells.

In the third approach, a donor might be rendered insensitive to a killer plasmid by using a tightly regulatable promoter-operator system in which the expression of a lethal bacteriocin gene is prevented by a repressor made only in the donor cell. An engineered example of a plasmid with multiple toxins that are independently regulated has been built and employed in the proof-of-concept experiments which are described in the paper.

The BCBT has been successfully used by ConjuGon Inc. (Madison, WI), and the Loyola University Medical Center’s Burn and Shock Trauma Institute (Maywood, IL), in eradicating Acinetobacter baumannii in vitro and in an in vivo murine burn sepsis model. A. baumannii is a Gram-negative opportunistic human pathogen that is found in soil and water and is easily transmitted in health care settings. Wounds such as burns are routinely treated with topical antibiotics at high enough doses to achieve therapeutic concentration; however, such antibiotic treatment is compromised if the wound is infected with multidrug- resistant bacterial strains. Many clinically-isolated strains of A. baumannii are pan resistant (resistant to all antibiotics) and the incidence of nosocomial infections caused by such strains is increasing in critically injured and immunocompromised patients who are hospitalized for prolonged periods. So BCBT could be extremely useful in such cases.
The other target for BCBT is to kill pathogenic bacteria which are living inside the host cells. These bacteria establish themselves in the intracellular milieu of their host, thereby evading administered antibiotics as well as the host’s immune system. To target those pathogens the intra- or inter-species conjugation can be used. In that case a non pathogenic strain (such as Salmonella) acts a donor of killer-plasmid. In the case of Mycobaterium tuberculosis and M. avium infections bacteriophages were used instead of plasmid to reduce number of pathogens.

There are also some disadvantages of this technology. The efficiency of killing pathogenic bacteria depends first on plasmid transfer efficiency, and second, on plasmid killing properties itself. Thus it is important to increase the ability of plasmid to be transfer to the specific target host. It is also good to find highly efficient killing system. But even with high efficient conjugation and killing system it seems to be very unlikely to eliminate all susceptible bacteria in the environment, because they still have some “defense” systems like restriction systems to protect. On the other hand the commercial antibiotics also do not kill all susceptible bacteria. But the decreased number of pathogenic cells allows immunological system to finish the job.

Another thing is the use of bacterial strains containing modified genetic information and “releasing” them to the “environment”. Authors present an assortment of applications of live bacteria approved by U.S. government agencies for use or further study.

To summarize, in some cases the BCBT could be an alternative method of dealing with bacterial infections and as a new technology can be developed in all possible ways...

dr Jaroslaw E. Krol
UofI

Plasmid gene content analysis

2009-01-23T01:28:00.000-08:00

Plasmid gene content analysis

Shared gene content patterns, also called phylogenetic profiles, have been used to build phylogenetic trees (Snel et al. 1999), to predict protein function (Pellegrini et al. 1999), and to reconstruct gene content of ancestral species (Kunin et al. 2003) for prokaryotic genomes. The phylogenetic reconstruction based on gene content is useful particularly for mobile genetic elements such as phages and plasmids where universally shared homologous sequences, a prerequisite for phylogenetic analyses, are not always available. Recently, the gene content analysis has been applied to phages (Lima-Mendez et al. 2008). Most recently, Brilli et al. (2008) applied this to plasmids from Enterobacteriaceae family of gamma-Proteobacteria including Escherichia, Salmonella and Shigella genera. The authors stated that 'most of plasmids does not form tight clusters coherent with the taxonomic status of their respective host species (E. coli, Salmonella or Shigella). This finding suggest a complex evolutionary history of such plasmid replicons with massive horizontal transfer and gene rearrangements.'

In contrast to other researchers, Brilli et al. (2008) did not discuss the performance of the phylogenetic profiling methods for plasmids. For example, Snel et al. (1999) demonstrated the correlation of prokarytic phylogeny based on gene content with that based on sequence similarity of 16S rRNA. Also, Lima-Mendez et al. (2008) clustered phage genes based on their phylogenetic profiles to define evolutionary cohesive modules, and showed that in temperate phages evolutionary modules correspond better to functional modules, whereas in virulent phages they span several functional categories. This suggests that the phylogenetic profiling does not always work well at predicting protein function in phages.

This has inspired us to validate the performance of the gene content analysis with the set of genes shared as orthologs by all members of an evolutionarily coherent plasmid group (such as IncFI, IncFII, IncI1, IncN, IncP-1, and IncW), and by focusing on functionally linked proteins such as those involved in the replication, maintenance, and conjugative transfer of plasmids.

PRIMARY ARTICLE:
Brilli M, Mengoni A, Fondi M, Bazzicalupo M, Lio P, Fani R. BMC Bioinformatics. (2008) 9(1):551. Analysis of plasmid genes by phylogenetic profiling and visualization of homology relationships using Blast2Network.

ADDITIONAL REFERENCES:
Snel B, Bork P, Huynen MA. Nat Genet. (1999) 21(1):108-10. Genome phylogeny based on gene content.

Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO. Proc Natl Acad Sci U S A. (1999) 96:4285-8. Assigning protein functions by comparative genome analysis: protein phylogenetic profiles.

Kunin V, Ouzounis CA. Bioinformatics. (2003) 19:1412-6. GeneTRACE-reconstruction of gene content of ancestral species.

Lima-Mendez G, Van Helden J, Toussaint A, Leplae R. Mol Biol Evol. (2008) 25:762-77. Reticulate representation of evolutionary and functional relationships between phage genomes.

Dr. Haruo Suzuki
University of Idaho

Changing each other’s lives and transcription

2009-01-15T15:31:00.000-08:00

One of the challenges of biology is that life does not function as a vacuum, but rather constantly influences and is influenced by other organisms and the environment. Therefore, in order to unravel some of the mysteries of the functions and evolution of living things, one must examine not only the organism itself, but also those things that may be influencing it. For bacteria, horizontal gene transfer (HGT) is a key process by which a bacterium is influenced by its environment and the genetic organization of neighboring bacteria. Through HGT a bacterium can acquire plasmids that confer upon the bacterial host new phenotypic traits. In the case of plasmid pCAR1, hosts receive the genetic information necessary to degrade carbazole and therefore use it as a carbon source. However, the gain or loss of a plasmid and its inherent phenotypic properties are not the extent of how plasmids and bacteria influence one another. As the authors of this paper pointed out, the host’s chromosomal transcriptome can be influenced by the presence of a plasmid and that, conversely, transcription of plasmid backbone and accessory genes are affected by the chromosome of the host in which the plasmid finds itself.

Past research by these authors showed that pCAR1 could successfully transfer to and function in Pseudomonas putida KT2440 from its original host, Pseudomonas resinovorans CA10. They also found that the introduction of the pCAR1 affected chromosomal transcription. In this study, they pursued the question of if and how plasmid transcription of pCAR1 is affected by different host chromosomes, again using P. putida KT2440 and P. resinovorans CA10. To do this they used a microarray to map and quantify PCAR1 transcripts in the two hosts when grown on succinate or carbazole as the only available carbon source. They also verified their results from the microarrays through real-time PCR. Through these two methods, the authors verified that growth on carbazole induced the catabolic operons antA and antR to the same extent, regardless of the host. However, other genes, such as the car operons, were expressed at significantly different levels depending on which host the plasmid was acting in.

This article adds to the growing body of evidence that the expression of genomes are not static bodies with occasional changes due only to mistakes made from one generation of cells to another, but is rather a dynamic entity that is, like the organism itself, influenced by its immediate environment at any given time.

Primary article:

Miyakoshi M, H Nishida, M Shintani, H Yamane, H Nojiri. (2009). High-resolution mapping of plasmid transcriptomes in different host bacteria. BMC Genomics, 10:12.

Additional reading:

Frost LS, Leplae R, Summers AO, Toussaint A. (2005). Mobile genetic elements: the agents of open source evolution. Nat Rev Microbiol 3:722-32.

Harr B, Schlötterer C. (2006). Gene expression analysis indicates extensive genotype-specific crosstalk between the conjugative F-plasmid and the E. coli chromosome. BMC Microbiol 6:80.

Miyakoshi M, Sintani M, Terabayashi T, Kai S, Yamane H, Nojiri H. (2007). Transcriptome analysis of Pseudomonas putida KT2440 harboring the completely sequenced IncP-7 plasmid pCAR1. Bacteriol. 189: 6849-60.

Ramos JL, Marqués S, Timmis KN. (1997). Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rec Microbiol. 51: 341-72.

Thomas CM. (2006). Transcription regulatory circuits in bacterial plasmids. Biochem Soc Trans. 34:1072-4.

Julie M. Hughes
University of Idaho

Cupriavidus metallidurans: evolution of a metal-resistant bacterium

2009-01-10T09:24:00.000-08:00

Torsten von Rozycki Æ Dietrich H. Nies

Cupriavidus metallidurans CH34 is a gram-negative bacterium that is frequently found in soils and sediments with a high content of heavy metals and is therefore resistant to multiple heavy metals such as Zn, Cd, Co, Pb, Cu, Hg, Ni and Cr. C. metallidurans CH34 has a large mega-plasmid and two large plasmids (pMOL28 and pMOL30) that have low copy numbers and can be maintained in the cell even without selective pressure. Resistance to these metals is mediated by transmembrane protein complexes, which export cations from the cytoplasm to the exterior of the cell. The main question this work is attempting to answer is: “ How did C. metallidurans CH34 acquire so many metal resistance genes? ”

To answer this question, the authors decided to analyze the genomes of seven bacteria to study the occurrence of orthologous and paralogous proteins coding for metal resistance. All seven bacteria belong to the β-proteobacterial family Burkholderiaceae of the order Burkholderiales. These include the hydrogen oxidizing C. eutrophus strain H16 (Pohlmann et al. 2006) and the xenobiotic degrader C. eutrophus JMP134 along with 2 phytopathogenic bacteria Ralstonia solanacearum strain GMI1000 (Salanoubat et al. 2002) and strain UW551. The last two organisms that were included were Burkholderia xenovorans strain LB400 and Burkholderi cepacia strain AMMD and were taxonomically distinct from Ralstonia and Cupriavidus. For this analysis, a standardized database for transporter proteins TCDB (http://www.tcdb.org/) was used as a reference. The latest releases of protein sequences of all seven strains were obtained from JGI and NCBI. These were then blasted against the TCDB database (Busch and Saier 2002). A total of seven transporter protein classes (channels/pore, electrochemical potential-driven
transporters, primary active transporters, PTS-group translocators, transport electron carriers, accessory factors involved in transport, incompletely characterized transport
systems) were found in all of the seven genomes. These transporter proteins differed from each other based on the method of transport and also on the mechanism of energy utilization (Saier 2000; Saier et al. 2006). It was also seen that the number of transporter proteins per Mb was similar in all of the strains (and most of the plasmids) analyzed. Thus, the authors conclude that metal resistance in C. metallidurans is not due to a higher number of transport proteins.

In the next step the authors analyzed the paralogs in all seven strains. Paralogs arise by gene duplication in an organism. A high percentage of protein coding paralogs were found on the plasmids of CH34 (34%), H16 (31%) and JMP134 (21%). Moreover, half of the transport proteins found on plasmids of CH34 were paralogs. For instance, the plasmid pMOL30 had a higher percentage of paralogous proteins than any of the other plasmids or chromosomes. The authors surmise that evolution of CH34 has been due to the duplication of transport proteins on its plasmids. The same mechanism may have been responsible for the evolution of the strains H16 and JMP134. Orthologs were investigated next. Here also, pMOL30 exhibited an unusually low percentage (17%) of orthologous proteins. This fact along with the high number of paralogs on plasmid pMOL30 may indicate that gene duplication and horizontal gene transfer played important roles in the evolution of this plasmid.

A total of 700 transport proteins were common among the three Cupriavidus strains. The transport proteins of CH34 could be assigned to twenty protein families based on the classification of the TCDB database. The twenty protein families had orthologs in all strains, however; some protein families were present more than once in CH34. Examples include the Mot/Exb complex components that energize active transport across the outer membrane, ABC transport systems, and metal inorganic transport (MIT) systems, RND, MFP and OMF protein families, P-type ATPases, proteins of the major facilitator superfamily (MFS), and components of the type III (TTS) and the type IV (TFS) secretion systems. Since all of the above proteins export cations, this shows that CH34 has twice as many of these proteins as the other six strains.
Next, the number of protein families involved in the transport of transition metals such as CDF, MerTP, MFP, MIT, NiCoT, OMF, OMR, P-type ATPase, CHR, HME/RND, and ZIP protein families was studied in the seven strains. The authors found that CH34 had a much higher number of the above protein families than the other bacterial strains (i.e., 83 compared to between 44 and 69). When genome size was taken into consideration it was shown that CH34 had 12 transition metal transport proteins per Mb while all the other six bacteria had 6–8 such proteins per Mb. Thus CH34 seems to have evolved its metal resistance by horizontal gene transfer and gene duplication.
RND proteins are a superfamily of proteins that are part of multi subunit protein complexes involved in efflux reactions (Tseng et al. 1999). A subgroup of this family called the HME-RND proteins are involved in the efflux of metals. CH34 has twelve HME-RND operons (Nies 2003), while the other six bacteria have fewer than twelve. This means that the number of operons has steadily increased in CH34 probably by horizontal gene transfer. Three of these twelve operons were vigorously expressed in CH34 and code for the following: the chromosomal copper/silver HME4-RND system, cnr for cobalt/nickel resistance on plasmid pMOL28, and czc for cobalt/zinc/cadmium resistance on plasmid pMOL30. The cobalt/zinc/cadmium resistance operon is czcICBA (Nies 2003) which is found not only on pMOL30, but also on CH34 chromosome 2 and has homologs on chromosome 2 of both C. eutrophus strains. Thus the authors conclude that all three strains might have inherited a czcICBA-like operon on chromosome 2 from an ancestral Cupriavidus strain and in CH34 this operon was duplicated onto plasmid pMOL30. Another operon, czcDRSE, (Große et al. 1999, 2004) is located downstream of the czcICBA operon on pMOL30 and encodes the CDF protein CzcD which transports divalent cations. The authors suggest that this operon was probably assembled by the horizontal transfer of czcD and regulatory genes czcRS along with the duplication of the copH gene (from the copper resistance cluster on pMOL30) to form czcE. Since czcE binds copper, it may form a link between the czcDRSE and czcICBA operons. Similarly, nickel/cobalt resistance is encoded on pMOL28 by the cnrYXHCBA operon (Liesegang et al. 1993), which has no homologs on any of the other bacterial strains. This operon, too, may have been acquired by horizontal gene transfer. P-type ATPases form a family of membrane-bound primary transport systems (Fagan and Saier 1994). Strain CH34 contains a high number of 13 predicted P-type ATPases. The other two Cupriavidus strains 7 or 8 orthologs including Ca2+ and Zn2+/Cd2+/Pb2+ exporting enzymes. Cupriavidus metallidurans contains four CHR proteins that export chromate from the cytoplasm (Nies 2003; Nies et al. 1998). This too could have been a result of gene duplication after speciation from the ancestral Cupriavidus strain. During speciation of C. metallidurans CH34 two MerT proteins duplicated into four, yielding three active mercury-detoxification systems. The authors summarize by saying that “ the ancestral Cupriavidus strain might have been a facultatively hydrogen-oxidizing, moderately metal-resistant degrader of aromatic compounds and organic acids rather than a dweller on sugars .” This strain evolved by the acquisition of plasmids such as those that carry hydrogen-oxidizing genes, metal resistance genes such as nickel, cobalt, chromate, and mercury, as well as genes coding for degradation of organic compounds such as 2,4-D. CH34 in particular probably evolved by a combination of horizontal gene transfer and gene duplication events along with rearrangements on Pmol30 which lead to adaptation of this strain to a wide range of metals.
It is now a known fact that horizontal gene transfer plays a crucial role in prokaryotic evolution. Studies such as these are important since they provide evidence of the role of horizontal gene transfer in the evolution of a complex strain such as CH34. Detailed analysis of each operon on the CH34 strain made it possible to trace its origin from the ancestral strain.

References:

Pohlmann A, Fricke WF, Reinecke F et al (2006) Genome sequence of the bioplastic-producing ‘‘Knallgas’’ bacterium Ralstonia eutropha H16. Nat Biotechnol 24:1257–
1262.

Salanoubat M, Genin S, Artiguenave F et al (2002) Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415:497–502.

Busch W, Saier MHJ (2002) The transporter classification (TC) system. Crit Rev Biochem Mol Biol 37:287–337.

Saier MHJ (2000) A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev 64:354–411

Saier MHJ, Tran CV, Barabote RD (2006) TCDB: the transporter classification database for membrane transport protein analyses and information. Nucleic Acids Res
34:D181–D186

Tseng T-T, Gratwick KS, Kollman J, Park D, Nies DH, Goffeau A et al (1999) The RND superfamily: an ancient, ubiquitous and diverse family that includes human disease
and development proteins. J Mol Microbiol Biotechnol 1:107–125.

Nies DH (2003) Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27:313–339.

Große C, Grass G, Anton A, Franke S, Navarrete Santos A, Lawley B et al (1999) Transcriptional organization of the czc heavy metal homoeostasis determinant from Alcaligenes eutrophus. J Bacteriol 181:2385–2393

Große C, Anton A, Hoffmann T, Franke S, Schleuder G, Nies DH (2004) Identification of a regulatory pathway that controls the heavy metal resistance system Czc via promoter
czcNp in Ralstonia metallidurans. Arch Microbiol 182:109–118

Liesegang H, Lemke K, Siddiqui RA, Schlegel H-G (1993) Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes
eutrophus CH34. J Bacteriol 175:767–778

Fagan MJ, Saier MH Jr (1994) P-type ATPases of eukaryotes and bacteria: sequence comparisons and construction of phylogenetic trees. J Mol Evol 38:57–99.

Nies DH, Brown N (1998) Two-component systems in the regulation of heavy metal resistance. In: Silver S, Walden W (eds) Metal ions in gene regulation. Chapman Hall,
London, pp 77–103

Diya Sen
University of Idaho

Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

2008-12-28T10:51:00.000-08:00

De Bast M. S., Mine N. and Van Melderen L. (2008) J. Bacteriol. 190:4603-4609

Plasmids are known as carriers of pathogenic determinants and antibiotic resistance genes that help bacteria survive in specific circumstances. However, if the circumstances changed, the bacteria would no longer need such genes, so that the plasmid would become just a burden for bacteria. As a result, plasmid-free bacterial cells would increase their number more rapidly than plasmid-carrying cells. Most of naturally occurring plasmids have special systems to prevent such an event from happening. One of the systems is the Toxin-Antitoxin (TA) system, in which one gene encodes a stable toxin protein and the other gene encodes an unstable antitoxin protein that counteracts the toxin activity. If the plasmid carrying the TA system was lost from a cell, the cell would be immediately killed or damaged by the more stable toxins which persist in the cell; this phenomenon is called postsegregational killing [PSK]. Gene pairs comprising TA systems are called addiction modules. Addiction modules were originally discovered on a plasmid (Hiraga et al., 1986), but recently they have also been discovered on chromosomes (reviewed by Kobayashi I., 2004). Here, we have a question: What is the biological function of chromosomally-located addiction modules?

The first hypothesis proposed is the so-called programmed cell death (PCD) hypothesis: the addiction module induces cell death under stress conditions and the dead cells release nutrients for other cells to remain viable (Aizenman et al., 1996). Recently, this hypothesis has been shown to be unlikely by several research groups (Tsilibaris et al., 2007; Szekeres et al., 2007; Dudde et al., 2007; Pedersen et. al., 2002). The second and more reasonable hypothesis is that addiction modules contribute to stabilize a genome: the toxin reduces the number of bacteria that have lost the chromosomal DNA segment containing the addiction modules, which ensures that the DNA in the region of the addiction module is maintained in the bacterial population (Szekeres et al., 2007).

In this paper, the authors propose a third theory: the "anti-addiction module" hypothesis. In this hypothesis, addiction modules on a chromosome protect bacteria against PSK induced by orthologous addiction modules on a plasmid, which confers selective advantage on a host bacterium under PSK conditions.

To test the anti-addiction module hypothesis, the authors used the CcdB(F)/CcdA(F) TA system of F-plasmid (Hiraga et al., 1986) and its homologous system [CcdB(Ech)/CcdA(Ech) TA system] found in the Escherichia chrysanthemi chromosome; CcdB(F) toxin is 61% identical to CcdB(Ech) while CcdA(F) antitoxin is 65% identical to CcdA(Ech). In this article, the authors first showed that the ccdB(Ech) and ccdA(Ech) genes indeed act as toxin and antitoxin genes in E. coli MG1655 where the ccdB(F)/ccdA(F) homologous genes are absent. However, unlike F-plasmid's ccdB(F)-ccdA(F) gene pair, the ccdB(Ech)-ccdA(Ech) gene pair could not mediate PSK when it was cloned on a plasmid. This suggests that the two homologous TA systems have evolved for different purposes. The authors also showed that CcdA(Ech) can antagonize CcdB(F) toxic activity as efficiently as CcdA(F) can. Furthermore, they showed that the E. coli MG1655 derivative that carries the ccdB(Ech)-ccdA(Ech) gene pair on its chromosome (designated MG1655ccdEch) are more viable than the original MG1655 after the induction of PSK mediated by F-plasmid's ccdB(F)-ccdA(F) gene pair on a plasmid. The following competition assays between MG1655 and MG1655ccdEch in PSK conditions indicated that MG1655ccdEch has a 25% fitness advantage over MG1655. Therefore, all experiments performed in this article support anti-addiction module hypothesis. A related idea was also proposed by Takahashi et al. (2002), using a restriction-modification TA system, in which restriction enzymes act as toxins and modification methyltransferases act as antitoxins. They showed that dcm methyltransferase gene located on the E. coli chromosome protected cells against PSK mediated by a restriction enzyme and DNA modification gene pair on a plasmid.

If the anti-addiction module hypothesis is valid, there would be few cases in nature that counteracting addiction modules are found on both plasmid and chromosome in the same cell, because PSK does not happen in such a situation and consequently there would be no advantage for plasmids to carry the addiction module. However, in the genome of E. coli O157:H7, two homologous ccdB-ccdA gene pairs exist. One ccdB-ccdA gene pair is located on plasmid pO157 and the other is located on the chromosome. The ccd genes of pO157 are identical to F-plasmid's counterparts. Chromosomal ccdB and ccdA gene products, CcdB(O157) and CcdA(O157), are 35% and 30% identical to CcdB(F) and CcdA(F), respectively. As we can expect, the chromosomal ccdA-ccdB gene pair of O157:H7 does not counteract CcdB(F) toxicity and O157:H7 is susceptible to PSK mediated by the CcdB(F)/CcdA(F) TA system (Wibaux et al., 2007).

The integration of addiction modules into the chromosome can protect bacteria from plasmids that may have a high cost under some conditions. However, as the authors state in this article, that in turn drives the evolution of plasmid TA systems so as not to be counteracted by chromosomal TA systems. It thus appears to me that the primal role of chromosomal TA systems is maintaining the integrity of chromosomes in bacterial populations and the secondary role may be protecting bacteria against PSK mediated by invader DNA elements such as phages and plasmids. Do you have another hypothesis? If so, please let me know.

References:
Aizenman E, Engelberg-Kulka H, Glaser G. (1996) An Escherichia coli chromosomal "addiction module" regulated by guanosine 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc. Natl. Acad. Sci. 93:6059-6063.

Budde PP, Davis BM, Yuan J, Waldor MK. (2007) Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae. J Bacteriol. 189:491-500

Hiraga S, Jaffé A, Ogura T, Mori H, Takahashi H. (1986) F plasmid ccd mechanism in Escherichia coli. J. Bacteriol. 166:100-104.

Kobayashi I. (2004) Genetic Addition: a principle of Gene symbiosis in a Genome, in Plasmid Biology (Funnell B. E. and Phillips G. J. eds), pp.105-144 ASM press, Washigton D.C.

Pederson K, Christensen SK, and Gerdes K (2002) Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Mol. Microbiol. 45:501-510.

Szekeres S, Dauti M, Wilde C, Mazel D, Rowe-Magnus DA. (2007) Chromosomal toxin-antitoxin loci can diminish large-scale genome reductions in the absence of selection. Mol. Microbiol. 63:1588-1605.

Tsilibaris V, Maenhaut-Michel G, Mine N, Van Melderen L (2007) What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome? J. Bacteriol. 189:6101-6108.

Takahashi N, Naito Y, Handa N, Kobayashi I. (2002) A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex. J. Bacteriol. 184:6100-6108.

posted by Hirokazu Yano, University of Idaho

Who will win this war…….?

2008-12-16T21:04:00.000-08:00

High in vitro antimicrobial activity of synthetic antimicrobial
peptidomimetics against staphylococcal biofilms.

Kristina Flemming, Claus Klingenberg, Jorun Pauline Cavanagh, Merethe Sletteng, Wenche Stensen, John Sigurd Svendsen and Trond Flægstad

Journal of Antimicrobial Chemotherapy (2009) 63, 136–145

It is more than 80 years when the first antibiotic was discovered by Sir Alexander Fleming (September 28, 1928). Since the mid 40’s antibiotics have been widely used to prevent bacterial outbreaks in humans, but they also play a role as growth promoting agents in agriculture. The years of positive selection pressure have caused the global spread of antibiotic resistance in the microbial population. Special threats are bacteria that form a biofilm. Biofilm is defined as microbial-derived sessile communities attached to a surface and embedded in a self-produced polymeric matrix. Bacteria in biofilms are usually less susceptible to antimicrobial agents than rapidly growing planktonic cells. There are several hypotheses to explain the strong antimicrobial tolerance of biofilm cells such as the limitation of agent penetration, the existence of dormant cells, phenotypic variations, a quorum sensing system, and multidrug efflux pumps. So there is always need to develop new effective antimicrobial agents that can kill bacteria.

Cationic antimicrobial peptides (CAPs) are widespread in nature and play an important role as part of innate immunity. In general, CAPs are fairly large molecules that carry a net positive charge and contain ~50% hydrophobic residues. Their mode of action involves binding to negatively charged structural molecules on the microbial membrane. Once bound, CAPs form pores that increase the cell membrane permeability and ultimately lead to cell lysis. There is also evidence for other antimicrobial mechanisms such as interaction with intracellular targets or activation of autolytic enzymes in the bacteria, or induction of the immune response in the host. CAPs have a broad spectrum of antimicrobial activity and development of resistance is rare. Unfortunately, CAPs are difficult and expensive to produce in large quantities and are usually sensitive to protease digestion. Modifications of CAPs have resulted in the development of extremely short synthetic antimicrobial peptidomimetics, also called SAMPs. SAMPs mimic the effect of CAPs, but have improved pharmacokinetic properties and are thus a promising new group of antimicrobial substances.

In this study the authors investigated the antimicrobial activity of clinically relevant antibiotics like linezolid, tetracycline, rifampicin and vancomycinand, and newly designed SAMPs against biofilms of three different staphylococcal species (six strains). They also evaluate a simple screening method to quantify the metabolic activity of biofilms before and after the biofilm had been subjected to treatment with antimicrobial agents.

Two methods were used for quantify the biofilm metabolic activity. The first method used Alamar Blue (AB) to measure metabolic activity. AB is a redox indicator that both fluoresces and changes color in response to chemical reduction that can be measured by monitoring absorbances at 570 and 600nm. The AB method showed excellent applicability and it is simple, fast, non-toxic and suitable for high-throughput quantification, at least for biofilms grown in microtitre plates. It shows also great reproducibility and good sensitivity which is very important in antibiotic effect studies. To confirm the killing properties of the antibiotics used, the second method based on LIVE/DEAD biofilm staining was used. This use two stains: Syto9 (green fluorescence) and propionium iodide (PI – red fluorescence). Syto9 stains DNA in living cells while PI reduces green fluorescence only in dead cells. Fluorescence is observed with a confocal laser scanning microscope (CLSM).

Using those two methods authors showed that all SAMPs were clearly more effective in reducing metabolic activity in staphylococcal biofilms at low concentrations compared with antibiotics, even though they generally had higher MICs under planktonic growth conditions. In general, antibiotics were rarely able to cause a complete suppression of metabolic activity. In contrast, SAMPs were frequently able to suppress metabolic activity completely, indicating effective killing. It seems that SAMPs caused damage of the bacterial cell membranes even in slow growing or dormant bacteria embedded in a biofilm. In contrast, the antimicrobial agents used in this study predominantly affected growing bacteria by inhibiting their cell wall development (vancomycin) or by inhibition of their protein synthesis (linezolid, rifampicin and tetracycline).

In conclusion the SAMPs are potential new therapeutic agents in biofilm-associated infections. They could be especially attractive for topical treatment of chronic wound infections. The possible clinical applicability of SAMPs to prevent medical device-associated staphylococcal infections warrants future in vivo studies.

So maybe we can win this war…….

dr Jaroslaw E. Krol

UoI, Moscow

Functions of horizontally transferred genes

2008-12-08T21:18:00.000-08:00

Horizontal gene transfer (HGT) is thought to play an important role in the evolution of species and innovation of genomes. Different researchers examined functional propensity in HGT of protein families.

First, Jain et al. (1999) proposed under the complexity hypothesis that HGT may have occurred preferentially among operational genes (those that maintain cell growth such as metabolism-related genes) than among informational genes (those involved in DNA replication, transcription, and translation) which are part of more complex protein-interaction networks.

Second, Nakamura et al. (2004) observed that only parts of genes in functional categories such as mobile element, cell surface, DNA binding, and pathogenicity-related, were preferred.

Third, Beiko et al. (2005) found extensive evidence for the preferential transfer of metabolic genes, while informational genes (e.g. ribosomal proteins, and proteins involved in DNA replication and repair, cell wall synthesis, and cell division) are susceptible or resistant to HGT.

Recently, Choi et al. (2007) suggested that there is no strong preference of HGT for protein families of particular cellular or molecular functions. They reconfirmed previous findings that HGT was biased toward cell surface and DNA binding functions (Nakamura et al., 2004), but the biases are marginal. They suggest that HGT is nearly neutral to all genes and that a random HGT process is followed by selection due to environment or other factors.

These discrepancies may be due to differences in the methods (e.g. phylogenetic versus compositional methods) and databases used, the genome samples tested, and possibly other reasons. For example, functions were assigned to protein families by using different databases: that is, (i) The Institute for Genomic Research role categories database (Peterson et al., 2001), (ii) The NCBI clusters of orthologous groups (COG) database (http://www.ncbi.nlm.nih.gov/COG/), and (iii) Gene Ontology (GO) terms (Camon et al., 2003).

This has inspired us to examine functional correlates of the vertically versus horizontally transferred genes using uniform approaches (methods and databases).

REFERENCES:
Jain R, Rivera MC, Lake JA. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3801-6. Horizontal gene transfer among genomes: the complexity hypothesis.

Nakamura Y, Itoh T, Matsuda H, Gojobori T. Nat Genet. 2004 Jul;36(7):760-6. Biased biological functions of horizontally transferred genes in prokaryotic genomes.

Beiko RG, Harlow TJ, Ragan MA. Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14332-7. Highways of gene sharing in prokaryotes.

Choi IG, Kim SH. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4489-94. Global extent of horizontal gene transfer.

Dr. Haruo Suzuki
University of Idaho

Host roles in plasmid partitioning

2008-12-01T09:08:00.000-08:00

In order to gain a clear idea of how plasmids or bacteria function on their own, it is necessary to have some understanding of how the two interact. There are, for example, obvious ways in which one influences the other, such as the conference of useful phenotypic traits (e.g. antibiotic resistance) to the host and the molecular maintenance (e.g. DNA replication) of plasmids by the host. There are also much more subtle interactions between the two, which are just now being elucidated for the first time. One such interaction is described by Kolatka et. al. in their recent publication.

This article describes the interactions of the broad-host-range IncP-1 plasmid RK2 and the partitioning systems of Pseudomonas putida and Escherichia coli. The authors found that the subcellular location of a given type of plasmid in a given strain of host will have a particular location within the cell. Also, and even more interestingly, this position depends on the protein products of both the host and plasmid.

Comparing the subcellular location of an RK2 mini-derivative in E. coli and P. putida showed this interaction between host and plasmid. Because the plasmid lacked an active partitioning system, the partitioning machinery of its host determined its position. In the case of E. coli, the plasmid was found to cluster at the cell poles, whereas in P. putida it was located either at a mid-cell or one quarter of the way into the cell on either side. The location within P. putida was explained by the interactions between the centromere-like sequences on the plasmid and the ParB protein encoded by the host’s chromosome. Indeed, when the parB gene was inserted into the E. coli chromosome the resultant partitioning mirrored that of P. putida. Conversely, when the par genes in P. putida where made nonfunctional then the plasmids were found at the poles. The position of RK2 itself showed similar locational disruptions when its position was determined in the P. putida par mutants. In all cases, plasmid location was determined by fluorescence in situ hybridization (FISH) and fluorescence microscopy. Protein interactions between the plasmids and bacteria were determined by formaldehyde cross-linking and chromatin immunoprecipitation.

It is shown here that, as always, the actions and activity of an individual organism (or even a mobile genetic element) are by no means completely independent, but rather that they are constantly influenced by the organisms and environments that they come into contact with. Indeed, the basic tenets of evolution involve, not isolated individuals, but the interaction between individuals. Therefore, in the case of bacteria and plasmids, it is necessary to not only study one and then the other, but also the influence that they have on one another.

Primary Article:

Kolatka, K., M. Witosinska, M. Pierechod, and I. Konieczny. 2008. Bacterial partitioning proteins affect the subcellular location of broad-host-range plasmid RK2. Microbiology. 154:2847-56.

Additional References:

Ebersbach, G. & Gerdes, K. (2005). Plasmid segregation mechanisms. Annu Rev Genet 39, 453–479.

Funnell, B. E. (2005). Partition-mediated plasmid pairing. Plasmid 53, 119–125.

Gordon, S., Rech, J., Lane, D. & Wright, A. (2004). Kinetics of plasmid segregation in Escherichia coli. Mol Microbiol 51, 461–469.

Julie Hughes
Graduate Student
Department of Biological Sciences
University of Idaho

2008-11-26T13:16:00.000-08:00

Piggery manure used for soil fertilization is a reservoir for
transferable antibiotic resistance plasmids

Chu Thi Thanh Binh, Holger Heuer, Martin Kaupenjohann & Kornelia Smalla
FEMS MICROBIOL. ECOL. 66:25-37

Overuse of antibiotics has been responsible for the spread of antibiotic resistance among bacteria all over the world. Continual use of antibiotics has maintained a strong selective pressure for the persistence of antibiotic resistance genes, while horizontal gene transfer has resulted in the spread of these genes across phylogenetically diverse bacteria (Witte, 1998; Rhodes et al., 2000; Schmidt et al., 2001; Tennstedt et al., 2003). Studies on plasmid content from manures have shown the presence of transferable plasmids carrying antibiotic resistance genes (Gotz et al., 1996; Smalla et al., 2000; Heuer et al., 2002; van Overbeek et al., 2002). This study looks at manures from 15 pig farms, where each farm represents a different size of herd or different quantity of meat production.

16 manure samples were taken from 15 different farms across Germany. Exogenous biparental matings were carried out in the laboratory by using E. coli CV601 as the recipient and manure as donor. Mixtures of recipient and donor were incubated overnight and then plated on agar supplemented with either amoxicillin, sulfadiazine or tetracycline.
A total of 228 transconjugants were picked. Eight antibiotics were tested on all transconjugants using the disc diffusion method (Barry et al.,). Based on different combinations of antibiotic resistance phenotypes, 37 unique patterns were found. 204 transconjugants showed sulfadiazine resistance. The frequent use of sulfadizine in animal husbandry may be the reason for this observation. 40 transconjugants showed resistance to six antibiotics and 4 were resistant to all 8 antibiotics used. This is a frightening scenario, since only 8 antibiotics were tested and many more resistance genes may be present on these plasmids. A previous study (Normark & Normark, 2002) had shown that selection for one antibiotic might co-select other antibiotics. The authors hypothesize that this may be the reason for the appearance of multiple antibiotic resistances on these plasmids. In order to make their study simpler, they decided to use a subset of the 228 transconjugants. Hence, one transconjugant was chosen per manure for each antibiotic resistance pattern. This gave them 81 plasmids which they decided to analyse further.
Plasmids extracted from transconjugants were dot-blotted and hybridized with probes specific for replicon sequences of the broad-host-range (BHR) plasmid classes IncN, IncW, IncP-1 and IncQ. 28 plasmids were found to be IncN, 1 was IncW, 13 were IncP-1, 19 were similar to the recently discovered pHHV216-like plasmids (Heuer et al., 2008) and 20 plasmids could not be assigned to any of the known Inc groups. Next the authors wanted to see which genes were conferring resistances to amoxicillin and sulfadiazine in these plasmids. Dot-blotted plasmid DNA was hybridized with labeled probes for bla-TEM, sul1, sul2 and sul3 genes. While bla-TEM genes are most often associated with resistance to amoxicillin, a combination of sul1, sul2 and sul3 genes may be responsible for sulfadiazine resistance. From this experiment they saw that all transconjugants with the amoxicillin resistance phenotype carried the bla-TEM gene, confirming the findings of Binh et al., who showed the frequent occurrence of bla-TEM genes in manure and amoxicillin resistance soils. An interesting observation was the repeated occurrence of these genes on similar plasmids, for example the occurrence of bla-TEM genes on all 28 IncN plasmids. The authors conclude that IncN plasmids that were captured from 10 different manures could be responsible for the dissemination of bla-TEM genes. Similarly, the sul2 gene was found on all 19 pHHV216-type plasmids captured from 6 manures and the sul1 gene was found on 12 of 13 IncP-1 plasmids. The authors state that their work shows that antibiotic resistance genes are associated preferably with BHR plasmids. Next the authors tested the transferability of the 81 plasmids by carrying out matings where they used their transconjugants as donors and E. coli J53 as the recipient. They found that 73 could be transferred to the recipient and only 8 could not. 6 of these 8 were the pHHV216-like plasmids.

In order to compare the method of direct PCR-based detection of plasmids in total DNA of manure to the method of plasmid capture, they used primers specific to repA for IncN, trfA2 for IncP-1, or oriV for IncQ and IncW for PCR of total DNA of manure.
No correlation was observed between the frequency of plasmid capture and plasmid abundance as noted from total DNA of manure. For example, although one third of the plasmids captured from 15 manures were characterized as IncN, this class of plasmid was detected in only 5 manures by PCR and Southern blot hybridization. The authors attribute this to the low abundance of IncN plasmids in manure, which could have resulted in making PCR based detection difficult. Using the exogenous plasmid isolation method, they were able to capture IncN plasmids from these soils. Thus, they suggest that the exogenous isolation method captures plasmids even when they are not abundant and PCR-based detection of plasmid types may not be as efficient.

This study is important because it shows how prevalent broad host range plasmids are. Moreover, association of antibiotic resistance genes with such plasmids ensures their rapid spread in an environment with antibiotics that maintain a strong selection. We get some idea of the prevalence of resistance to antibiotics in bacteria. All transconjugants were found to confer resistance to one or more antibiotics. Co-selection of antibiotics is also a phenomenon that we should be looking at closely.

References:

Witte W. (1998): Medical consequences of antibiotic use in agriculture. Science 279: 996–997.

Rho des G., Huys G., Swings J., McGann P., Hiney M., Smith P. & Pickup R.W.
(2000): Distribution of oxytetracycline resistance plasmids between Aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol 66: 3883–3890.

Schmidt A.S., Bruun M.S., Dalsgaard I. & Larsen J.L. (2001): Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl Environ Microbiol 67: 5675–5682.

Tennstedt T., Szczepanowski R., Braun S., Puhler A. & Schlüter A. (2003): Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant. FEMS Microbiol Ecol 45:239–252.

Gotz A., Pukall R., Smit E., Tietze E., Prager R., Tschape H., van Elsas J.D. & Smalla K. (1996) Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl Environ Microbiol 62: 2621–2628.

Smalla K., Heuer H., Gotz A., Niemeyer D., Krogerrecklenfort E. & Tietze E., 2000: Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids. Appl Environ Microbiol 66: 4854–4862.

Heuer H., Krogerrecklenfort E., Egan S. et al. (2002): Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42: 28-302.

Van Overbeek L.S., Wellington E.M.H., Egan S., Smalla K., Heuer H., Collard J.M., Guillaume G., Karagouni A.D., Nikolakopoulou T.L. & van Elsas J.D. (2002): Prevalence of streptomycin-resistance genes in bacterial populations in European habitats. FEMS Microbiol Ecol 42: 277–288.

Normark B.H. & Normark S (2002): Evolution and spread of antibiotic resistance. J Intern Med 252: 91–106.

Heuer H., Kopmann C., Binh C.T. T., Top E.M., Smalla K.(2008): Spreading antibiotic resistance through spread manure: characteristics 1 of a novel 2 plasmid type with low %G+C content. In press.

Barry A. L., Garcia F., and Thrupp L.D. (1970): An improved single-disk method for testing the antibiotic susceptibility of rapidly-growing pathogens. Am. J. Clin. Pathol. 53:149-158.

Diya Sen
Graduate Student,
Department of Biological Sciences,
University of Idaho

Lessons from the first comprehensive survey of prokaryote genomics

2008-11-22T22:26:00.000-08:00

Genomics of bacteria and archaeal: the emerging dynamic view of the prokaryotic world. Eugene V. Koonin & Yuri I. Wolf, Nucleic Acids Research (2008)

The first bacterial genome (Haemophilus influenzae) was published in 1995, ushering in the so-called age of genomics. Since then, exponentially increasing numbers of whole-genome sequencing projects have generated a huge amount of raw data. While this holds great promise for developing our understanding of how prokaryotic genomes are formed and function, extracting meaningful observations from that mountain of data is a big challenge.

Drs Eugene Koonin and Yuri Wolf recently tackled this daunting task and embarked on a comprehensive survey of the genomic data produced to date by microbial sequencing projects. Their latest paper, titled “Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world”, presents their findings in a dense but rich monograph that offers deep insight into processes of genome evolution and defines general principles of prokaryotic genome organization.

Although there can be no substitute for reading the original paper, a point-by-point summary of the paper that may interest readers is provided here.

Dr. Geraldine A. Van der Auwera
Harvard Medical School

Bacteriophages SPP1 contains two tail tube proteins produced by programmed translational frameshift

2008-10-15T17:49:00.000-07:00

Origin and function of the two major tail proteins of bacteriophage SPP1. Auzat et al., Molecular Microbiology (2008) 70, 557-569

Bacteriophages are viruses that infect bacteria and are known to play roles in horizontal gene transfer. The majority of known bacteriophages have a head and long non-contractile tail that serves as a pipeline for phage genome delivery into bacterial cell. Here, the authors report that the tail tube of Bacillus subtilus bacteriaphage SPP1 is comprised of two proteins, gp17.1 and gp17.1* , that are produced by a translational frameshift. This mosaic construction of tail tube was found to be important for assembly of the functional tail tube, but its significance is not fully uncovered yet.

Previously, it was shown that the key event of phage DNA injection in bacterial cell is a rearrangement of the inner wall of the tail tube (EMBO J, (2007) 26, 3720-3728). In this article, the authors separated SSP1 tail proteins by SDS-PAGE and found a protein band which was not expected from phage DNA sequence. Protein sequencing analysis indicated that the unexpected protein designated gp17.1* has the same amino-terminal sequence as that of tail protein gp17.1. gp17.1* is 10 kDa larger than gp17.1. The tail tube is made up of these two proteins at the ratio of 1:3. Based on the molecular size of gp17.1* and the sequences of gene 17.1, authors postulated that 17.1* is produced by a translational frameshift. Using site-directed mutagenesis of coding sequence 17.1 with protein profile analysis of the mutant phages, authors found out that 5'-CCCUAA-3' sequence located at the end of coding sequence 17.1 was the frameshift position.

To get insight into the function of gp17.1*, the authors constructed mutant phages that have tail tubes comprised exclusively of gp17.1 or gp17.1* and analyzed their structures by electron microscopy. When phages are assembled under the condition which either gp17.1 or gp17.1* are exclusively expressed, significant numbers of tailless heads (capsids) are made. This suggests the 3:1 ratio of gp17.1 and gp17.1* is important for correct phage assembly. Interestingly, both mutant phages had infection activity. The length and flexibility of mutant tails composed of either gp17.1 or gp17.1* were identical to SSP1 wild-type tails. gp17.1*-specific tail has protrusions on surface while gp17.1-specific tail has smooth surface. Authors postulate that carboxyl-terminus of gp17.1* causes protrusions on tail surface which facilitate initial contact of phages and attachment to the bacterial surface, while gp17.1 is ensures correct assembly of tail tube.

The potential translational frameshift site producing a carboxyl-terminus extension in a protein are also found in other phage surface protein genes (Mol Microbiol. (2003) 50, 303-317). Why do phages need two types of surface proteins which are identical except the carboxyl end extension of one of the proteins? What is the significance of strict ratio of the two proteins? Why is this the best strategy for phages? What is the target of the protrusion on tail tube surface? These are still interesting mysteries. It might be interesting to compare the host ranges of mutant phages that have either one of the two surface proteins.

References
Origin and Function of the Two Major Tail Proteins of Bacteriophage SPP1. Auzat I, Dröge A, Weise F, Lurz R, Tavares P. Molecular Microbiology (2008) 70: 557-569

Structure of bacteriophage SPP1 tail reveals trigger for DNA ejection. Plisson C, White HE, Auzat I, Zafarani A, São-José C, Lhuillier S, Tavares P, Orlova EV,
EMBO J (2007) 26:3720-8.

Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis. Zimmer M, Sattelberger E, Inman RB, Calendar R, Loessner MJ., Molecular Microbiology. (2003) 50:303-317.

Hirokazu Yano (university of idaho)

Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains

2008-10-07T00:04:00.000-07:00

Mette Burmølle, Martin Iain Bahl, Lars Bogø Jensen, Søren J. Sørensen and Lars Hestbjerg Hansen
Microbiology (2008), 154, 187–195

In this paper researchers from University of Copenhagen and The National Food Institute in Denmark bring our attention to a conjugative plasmid pOLA52 which features and genetic content occurred to be disturbing as they regard human and animal health.
pOLA52 plasmid was first isolated from swine manure and was shown to encode multidrug efflux pump which provides resistance to many antimicrobial agents such as olaquindox (which was or still is commonly used as a growth factor in pig farming), chloramphenicol, ethidium bromide, other antibiotics, detergents and disinfectants. It also carries bla gene conferring resisitance to ^β-lactam antibiotics, such as ampicilin. Apart from multidrug resistance, pOLA52 plasmid carries 5.6 kb operon consisting of five genes, homologous to mrkABCDF genes contained in the mrk operon of Klebsiella pneumoniae, encoding type 3 fimbriae, which are known to be involved in attachment of bacteria to different kinds of biotic and abiotic surfaces, and thus increased biofilm formation.
Authors of the paper have previously observed that E. coli CSH26 strain harbouring pOLA52 plasmid formed higher amounts of biofilm and thus wanted to investigate if the operon conferring type 3 fimbriae could be responsible for observed feature.
In this study authors randomly introduced entranceposon pENTRANCEPOSON (Kan^R) into pOLA52 plasmid, electroporated it into E. coli Genehogs, selected tranformants on Kan and checked their ability to form biofilms on urinary catheters. Some clones occured to be biofilm negative and sequencing revealed that inserts were located inside type 3 fimbriae operon. Not surprisingly, biofilm positive clones had the inserts outside the operon.
Researchers have checked expression of mrk genes with RT-PCR and showed with immunoblotting that biofilm positive clones expressed type 3 fimbriae, whereas biofilm negative did not.
Later, they have conducted conjugation of pOLA52 plasmid into potentially pathogenic Enterobacteriaceae strains (such as Klebsiella pneumoniae, Salmonella typhimurium, Kluyvera sp., Enterobacter aerogenes) and tested the ability of transconjugants to form biofilms. It occured that transconjugants harbouring plasmid with transposon inside mrk operon showed significantly lower rate of plasmid transfer comparing to strains carrying wild type plasmid. Futhermore, transconjugants harbouring wild type pOLA52 plasmid formed biofilms, whereas strains with operon mrk mutated plasmid showed much lower biofilm formation.
This study proves how important and potentially dangerous pOLA52 plasmid is, as it can be transferred via conjugation to other bacteria, including pathogenic strains, providing them with new antibiotic resistances and type 3 fimbriae increasing their ability of spread plasmids and to form biofilms. Those newly accuaired features can lead to higher antibiotic persistence and increased spreading of pathogens on biological surfaces, such as tissues, as well as abiotic ones, for example catheters or artificial heart valves.
As pOLA52 plasmid is the first of probably many more plasmids with similar genetic content, there is a risk that one day they could be used as another dangerous weapon in hands of pathogenic bacteria. Let's hope we will be well prepared if this day would come...

Sylwia Deneka
Visiting Scholar
University of Idaho

What a man can make …….

2008-10-01T09:19:00.000-07:00

RESEARCH ARTICLES

Genome Transplantation in Bacteria: Changing One Species to Another
Carole Lartigue, John I. Glass, Nina Alperovich, Rembert Pieper, Prashanth P. Parmar, Clyde A. Hutchison III, Hamilton O. Smith, and J. Craig Venter 2007. Science 317, 632.

Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasmagenitalium Genome.
Daniel G. Gibson, Gwynedd A. Benders, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Holly Baden-Tillson, JayshreeZaveri, Timothy B. Stockwell, AnushkaBrownley, David W. Thomas, Mikkel A. Algire, Chuck Merryman, Lei Young, Vladimir N. Noskov, John I. Glass, J. Craig Venter, Clyde A. Hutchison III, Hamilton O. Smith 2008Published Online January 24, 2008 Science DOI: 10.1126/science.1151721

In these two papers, researchers from J. Craig Venter Institute in Rockville Maryland showed us the possibility of changing the entire genetic information of a living microorganism to create a new artificial organism.
In first paper they used two closely related Mycoplasmas: Mycoplasma mycoides large colony (LC) and M. capricolum. They used mycoplasma because of its specificity. Mycoplasmas do not have a cell wall, which makes them resistant to some therapeutic antibiotics like B-lactams. This also makes them more susceptible for uptake of different substances from the environment. One also cannot underestimate the biological importance of Mycoplasmas as pathogenic microorganisms, causing some nasty diseases in human and animals. Another interesting fact is that Mycoplasmas, compared to other bacteria, have relatively small genome, ranging from 0.6 to 1.4Mb. Up to now, 12 Mycolasma genomes have been completely sequenced and 11 more are in progress.
Small genome size and lack of the cell wall makes them ideal candidates for the genome “switching” experiment.
So, Carole Lartigue with coworkers took the large colony of M. mycoides and prepared intact genomic DNA in the way usually used for PFGE (pulsed field gel electrophoresis). All proteins were removed by proteinase treatment. This purified DNA was used for transformation of M. capricolum strain. After a few days some transformants grew on selective media. These transformants had genetic markers specific for donor strain but no sign of host specific markers was detected. That means that it is possible to replace a whole genome, at least using closely relative bacteria.
Cloning large genomic fragments is not a technical problem. Some bacterial artificial chromosome (BAC) clones already contain inserts of about 300kb in size. Such clones can be easily introduced into E. coli cells by electroporation. Using some specific recombination systems it could be possible to join two or three large clones inside E. coli cells to generate single DNA molecule, then isolate such a genome and introduce it into a new bacterial host.
But there is another way to build an entire genome. In second of the presented papers, the authors described this alternative way. They split the whole Mycoplasma genitalium genome into 101 cassettes, each of about 5000-7000bp. Those cassettes were chemically synthesized by three companies. All cassettes have specific overlapping sequences so they can be stuck together. The in vitro recombination event yielded intermediate assembliesof approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4genome"), which were all cloned as bacterial artificial chromosomes(BACs) in The complete synthetic genomewas assembled by transformation-associated recombination (TAR)cloning in the yeast Saccharomycescerevisiae, then isolatedand sequenced. This method allowed construction of an entire “artificial” bacterial genome.
Mycoplasmas have small genomes, but we already know some bacteria with even smaller genomes. The amphid endosymbiont Buchnera aphidicola genome consists of ~422kb, but a psyllidendosymbiont, Carlsonella ruddii, has even smaller genome (~160kb). Such small genomes are specific for pathogenic and especially endosymbiotic style of life. These bacteria reduce their genomes by deleting genes that are not necessary, like some anabolic pathways. On the other hand, they keep genes that are useful for themselves and for their hosts. These genes encode basic functions like replication, transcription and translation, as well as some of the biosynthetic pathways encoding for amino acids, cofactors and other essential compounds that their host cannot obtain from their diet. Surprisingly, also lots of genes encoding transport (uptake) systems have been eliminated.
Studying such small genomes we can learn which genes are really core genes and cannot be removed from the genome and which genes are not necessary. Furthermore, we can induce such big reductions in larger bacterial genomes. Analysis of these data as well as data obtained from analysis of the eukaryotic organelle (mitochondria and plastids) genomes could be used to construct an artificial endosymbiont.
Perhaps, it could be possible to make a new endosymbiont,which could be specific for some body tissue like liver or pancreas. They could live inside the cell,producingspecific proteins like insulin, clotting factors or other factors deficient in genetic diseases. This is fantasy but who knows the future…….

Additional articles:
S. G. E. Andersson 2006. TheBacterial World Gets Smaller. Science 13, pp.: 259 – 260.

A. I. Nilsson,S. Koskiniemi,S. Eriksson,E. Kugelberg,J. C. D. Hinton,D. I. Andersson 2005, Bacterial genome size reduction by experimental evolution. Proc. Natl. Acad.Sci. 102, pp.: 12112-12116.

Quanzhou Tao and Hong-Bin1998 Zhang 1998 Cloning and stable maintenance of DNA fragmentsover 300 kb in Escherichia coli with conventional plasmid-based vectors. NAR 26, 21, pp.: 4901-4909.

dr Jaroslaw Krol
UofI

Contribution of horizontal gene transfer to microbial evolution

2008-09-22T16:43:00.001-07:00

“Modular networks and cumulative impact of lateral transfer in prokaryote genome evolution” by Dagan et al. (2008)

A horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the process by which genetic material is transferred between distinct evolutionary lineages, through (plasmid-mediated) conjugation, (virus-mediated) transduction, and transformation (extracellular DNA uptake). Although HGT may occur at the cellular level frequently, transferred genes cannot be always inherited to the subsequent generations.

Generally, a gene is thought to be acquired by HGT if gene tree conflicts or unusual nucleotide composition is observed. The major caveat of these approaches is that the observations can also be explained by other reasons, such as inaccurate phylogenetic reconstruction methods, gene loss in multiple lineages, novel sequences arising from the divergence of gene duplications, and varying mutation rates for different proteins (Kechris et al., 2006).

HGT is an important source of genetic diversity among microorganisms, but the degree of its contribution on microbial genome evolution is still debated. Dagan et al. (2008) conducted a network analysis of shared gene content across prokaryotic genomes to estimate the contribution of HGT to microbial evolution. Their result suggests that on average, 81 ± 15% of the genes in each genome were involved in HGT at some point in their history. Once acquired, genes can be vertically inherited within a group, and their result suggests that this has occurred for the vast majority of genes.

The Dagan's work have inspired us to estimate relative contributions of different mechanisms (conjugation, transduction, and transformation) on horizontal gene transfer among prokaryotes.

PRIMARY ARTICLE:
Dagan T, Artzy-Randrup Y, Martin W. Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10039-44. Modular networks and cumulative impact of lateral transfer in prokaryote genome evolution.

ADDITIONAL REFERENCES:
Choi IG, Kim SH. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4489-94. Global extent of horizontal gene transfer.

Dagan T, Martin W. Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):870-5. Ancestral genome sizes specify the minimum rate of lateral gene transfer during prokaryote evolution.

Kechris KJ, Lin JC, Bickel PJ, Glazer AN. Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9584-9. Quantitative exploration of the occurrence of lateral gene transfer by using nitrogen fixation genes as a case study.

Beiko RG, Harlow TJ, Ragan MA. Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14332-7. Highways of gene sharing in prokaryotes.

Kunin V, Goldovsky L, Darzentas N, Ouzounis CA. Genome Res. 2005 Jul;15(7):954-9. The net of life: reconstructing the microbial phylogenetic network.

Dr. Haruo Suzuki
University of Idaho

Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5

2008-09-16T16:53:00.000-07:00

Bahl MI, Hansen HL & Sørensen SJ (2007. FEMS Microbiol Lett 266:250-6.

“With great power comes great responsibility”. As strange as it may seem, Churchill’s famous quote can be applicable to the bacterial as well as the human world. Many bacteria contain plasmids that confer to them the “power” to do many impressive things: resist antibiotics or heavy metals, break down toxins in the environment, become virulent, and some even give bacteria the power to conjugate (mate) with other bacteria. All this power comes at a cost, however. Having a plasmid that gifts a bacterium with novel traits also means that the bacterium has to invest in maintaining the plasmid. A variable amount of a bacterium’s resources will have to be diverted to keeping the plasmid and its various functions up and running. Thus, plasmids often affect bacterial growth rates, causing the plasmid bearing bacteria to grow and reproduce more slowly than their plasmid free neighbors. Therefore, it is commonly thought that a plasmid is only truly beneficial if there’s an immediate selective advantage to having it (for instance, having a plasmid that keeps you alive in the presence of antibiotics is great when being actively doused in antibiotics, but the costs of the plasmid might not be worth it when the flow of antibiotics stops). In times where such selective pressures are removed the percentage of plasmid harboring cells will often decrease due to factors such as the slower growth rate and the occasional loss of plasmids in one of the daughter cells during segregation (Bergstrom et. al., 2000).

All this said, the authors of a recent paper suggest that plasmid loss in the absence of selective pressures may not be such a sure thing after all. They propose that when bacteria have a plasmid and frequent access to each other (as is the case in bacterial biofilms or microcolonies) conjugation can more than compensate for plasmid loss in a population. By constructing fluorescing bacteria the authors of this paper were able to see and quantify plasmid stability in bacterial populations that could and could not conjugate. They therefore were able to study the impact of conjugal transfer on the stability of an IncP-1 plasmid in bacterial populations, as the name of their article implies.

The authors carried out this study using Escherichia coli MC4100 and Kluyveria sp. MB101. A gene cassette coding for kanamycin (Km) and streptomycin (Sm) resistance, as well as a green fluorescing protein (GFP) was inserted into the chromosome of each of the above-mentioned bacteria. In liquid broth the constitutively expressed gfp can be detected by flow cytometry using an argon ion laser, whereas an epi-flouresence microscope was used for visual detection of fluorescing colonies on solid media. The production of GFP is regulated by a lac operon, and so is repressed in the presence of a functional lacI gene (which is not present in either of the constructed strains of bacteria). In order to test the importance of conjugation on plasmid stability, the authors inserted an entranceposon containing a lacIq1 gene into plasmid pKJK5. The entire genome of pKJK5 had been previously sequenced, and so the authors were able to use PCR to screen for neutral insertions (e.g. pMIB4) and insertions that disrupted conjugation (e.g. pMIB8) (Haase et al., 1997). The authors introduced these lacI-containing plasmids into E. coli MC4100 and Kluyveria sp. MB101. Therefore, any bacterium containing pKJK5 or one of its derivatives would not fluoresce, due to the lacI suppression of GFP production. Thus, this method allowed the authors to quantify the percentage of plasmid harboring and plasmid free cells.

Using this system, the authors were also able to compare the stability of plasmid pKJK5 in the presence and absence of conjugation. In a culture initially containing 100% pMIB4, three days (and many generations) later more than 99.99% of the cells still contained the lacI plasmid even without selection for it. On the other hand, in bacteria that couldn’t conjugate (those containing pMIB8) only around 99.43% or 99.13% of the E. coli and Kluyvera sp., respectively, still were plasmid harboring in bacterial mats. As with similar experiments involving stability of conjugation-deficient bacteria conducted by Sia et al (1995), this suggests that conjugation plays a significant role in sustaining an IncP-1 plasmid in bacterial mats. But that’s not all. Not only can conjugation promote plasmid persistence in a population, but according to the authors it can also account for the infectious spread of plasmids throughout a mat population within three days, even when starting from only an initial 25% of the population containing the plasmid. Again, this is only true if conjugation is possible. With the pMIB8 plasmid the total plasmid-containing population actually decreased, likely due to segregational loss.

Whereas conjugation may compensate for segregational loss in high-density bacterial mats, the same cannot be said of lower density, well mixed liquid broth cultures. It appears that the percentage of plasmid containing cells decreased in populations harboring either pMIB4 or pMIB8, although the decline was less dramatic in those populations that could conjugate. So what does it matter if plasmid stability in bacterial mats differs from that in liquid media is different? Well, for one, other than the thermos of chicken soup that’s been rolling around in the back of your car for a week, bacterial populations in nature may not be accurately modeled by the perfectly mixed broth cultures common to most labs. This means that in general, we may be underestimating the role of conjugation in plasmid stability due to unrepresentative experimental systems.

There is a vast range of applications of studies in horizontal gene transfer in general. In some cases we may want to limit plasmid stability in populations such as in the fight against antibiotic resistant strains. In other cases, as with bioremediation, we may want to encourage plasmid stability so that plasmids that we introduce into bacteria allow the bacteria to do our clean-up work for us. In either case, we need a solid understanding of how, when, and under what conditions plasmids are more or less stable. That’s not to say that conjugation is the only important factor in stability. As mentioned above, and in the author’s paper, segregational loss, relative growth rates, and transfer frequency all contribute to overall plasmid stability. This article doesn’t discount the importance of these other factors, but rather emphasizes the need to respect conjugation as a major player that can, given the right conditions, act parasitically in its spread through a population, even when it doesn’t benefit the host bacterium. So maybe the bacteria aren’t as “responsible” for the process as we originally thought. Maybe the plasmids themselves are the ones with the real power after all.

This study also opens up a question for the philosophers of science out there (although it’s a question much too broad for one blog, so an answer won’t be attempted here). That question is one raised by Richard Dawkins, and pertains to the idea of the selfish gene. If plasmids behave parasitically, does that support the selfish gene idea? Could the results of this article be applied to an argument that the population isn’t always the level that we should think about when considering evolution, if it’s the plasmids and not their host bacteria that run the show? Maybe, maybe not, but it’s a fun debate either way, and something to think about.

References:

Bahl MI, Sørensen SJ &Hansen HL (2004) Impact of conjugal transfer on the stability of IncP-1plasmid pKJK5 in bacterial populations. FEMS Microbiol Lett 232:45-49.

Bergstrom CT, Lipsitch M & Levin BR (2000) Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics 155: 1505–1519.

Haase J & Lanka E (1997) A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus synthesis. J Bacteriol 179.

Sia EA, Roberts RC, Easter C, Helinski DR & Figurski DH (1995) Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2. J Bacteriol 177: 2789–2797. 5728–5735.

Julie Hughes, Ph.D. student
Department of Biological Sciences, University of Idaho

Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine

2008-09-04T19:44:00.000-07:00

Schjørring, S., Struve, C., & Krogfelt, K.A. (2008). (e-publ. Aug. 13, 2008) Journal of Antimicrobial Chemotherapy doi: 10.1093/jac/dkn323.

Nosocomial infections, commonplace in health care systems including intensive care units (Çaatay et al., 2007), are becoming more of a pressing issue as bacteria continue to exhibit multiple antimicrobial resistances. Such cases have been reported in hospitals as well as extended care facilities such as nursing homes (Wiener et al., 1999). While the development of antibiotics are important in our fight against pathogens, it is equally important to focus on the mechanisms involved in developing increased resistance. In this paper by Schjørring, et al., one of the most common nosocomial pathogens was studied: Klebsiella pneumoniae, gram-negative bacteria shown to exhibit multiple antimicrobial resistances. The authors studied the effects of introducing antimicrobial genes and monitoring the colonization of K. pneumonia in mice intestines. The findings reveal several pieces of information about K. pneumonia, including the nature of the pathogen as well as its’ ability to transfer resistance genes to other bacteria (Schjørring, et al., 2008).

The authors of this article, created an intestinal colonizational model in order to observe this transfer more readily, so the plasmid transfer procedure was observed both in vitro and in vivo (Schjørring, et al., 2008). K. pneumoniae strain MGH75875 was used to follow the transfer process including colonization, and horizontal gene transfer (Schjørring, et al., 2008). This strain was originally isolated from an intensive care unit (ICU) patient with pneumonia. K. pneumoniae MGH75875 is currently known to be resistant to ampicillin, streptomycin, tetracycline, nalidixic acid, ticarcillin, trimethoprim/ sulfamethoxazole, cefotaxime and gentamicin, and is susceptible to imipenem (Schjørring, et al., 2008).

The plasmids monitored in this experiment presented interesting results on the basis of in vitro and in vivo examination. Several of the plasmids monitored (only named by their relative size) showed that environmental conditions do influence the nature of transfer. For example, the in vitro experiments showed transfer of the 108 or 157 kb plasmid, while in vivo only showed transfer of the 89 kb plasmid (Schjørring, et al., 2008).

The mice used in this study were individually caged and had unlimited access to resources, including food and water; antibiotics were administered through the water, at dosages described in the protocol. To begin, mice were first inoculated with the strain K. pneumoniae. This was done by growing up overnight cultures and resuspending the cultures in a 20% sucrose solution. Each mouse was given 100μL of this solution orally and subsequently their fecal matter was measured for bacteria; up to 109 cfu/g feces was found (Schjørring, et al., 2008). To determine the effects of antimicrobial treatment on the intestinal flora, three mice were treated with only K. pneumoniae and later treated with ampicillin added to their drinking water to represent treatment of infection. To determine the colonization of the intestine, two mice per experiment were treated with 0.5g/L ampicillin prior to exposure to the strain. Finally, to monitor gene transfer in the intestine three mice per experiment were treated with 0.5 g/L streptomycin sulphate in their drinking water prior to inoculation with the recipient strain as well as during the experiment (Schjørring, et al., 2008). A verification of transconjugants was done, via biochemical marker assays and by DNA isolation to provide a plasmid profile to detect the E. coli transconjugants. Also, a PCR was used to detect the presence of the plasmid-encoded extended spectrum β-lactamases (ESBL) genes, or the genes that code for antimicrobial resistance. In mice without any antimicrobial pretreatment, the inoculated strain quickly dropped below the detection limit due to the competitive nature of the other strains of bacteria present in the intestine. With the introduction of an antimicrobial treatment, there was an immediate increase in the population of the MGH75875 strain up to 109 cfu/g feces (Schjørring, et al., 2008). This experiment thus shows a direct relationship between selection factors and the immediate colonization of the gastrointestinal tract (GI) by the resistant pathogen (Schjørring, et al., 2008). There was also an observable higher transfer frequency of different plasmids into E. coli from K. pneumoniae during colonization of the mouse intestine. K. pneumoniae is thus an excellent colonizer in the GI tract of antibiotic-pretreated mice, and highly promiscuous with respect to numerous plasmids. The observed increase in the number of resistant bacteria, which can inherently lead to an increased risk of spreading resistance genes (Schjørring, et al., 2008).

After reading this paper I became very interested in the concept of evolution in our everyday lives. Most of us imagine evolution as a long and gradual process; however, in microbiology a normal 24-hour period can consist of several generations of bacteria. In this way, evolution can be easily observed and measured especially in the presence of selection factors. As a future physician, I recognize the importance of studying the relative effects of antibiotic use, including those associated with overuse, underuse and more recently the effects associated with resistant strains of bacteria in medicine. While studying antibiotic use is important, equally important is gaining a better understanding of what mechanisms are associated with resistance. Through this research and others, we all may come to appreciate what role evolution plays in our everyday lives, including our health care.

Related Articles of Interest:

Çaatay, A.A., Özcan PE, Gulec L, et al. Risk Factors for Mortality of Nosocomial Bacteraemia in Intensive Care Units. Med Princ Pract 2007;16:187-192.

Wiener, J., Quinn, J.P., Bradford, P.A., Goering, R.V., Nathan, C., Bush, K., Weinstein, R.A. JAMA 1999; 281: 517-523.

Nick Hardin, Undergraduate Researcher
University of Idaho

Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in Pseudomonads

2008-08-25T09:38:00.000-07:00

Slater, Frances R.; Bruce, Kenneth D.; Ellis, Richard J.; Lilley, Andrew K.; Turner, Sarah L.
2008
Applied & Environmental Microbiology-74 (10). 3189-3197. doi:10.1128/AEM.02383-07

Bacteria live in natural environments that have a spatial structure where their movement is restricted by the surrounding soil or water. Such a spatial structure may create physicochemical gradients leading to heterogeneous patches. This heterogeneity is very important to the biodiversity of microorganisms as studied by Rainey and Travisano (1998). The present study by Slater et al aims to compare the effects of uniform and heterogeneous mercuric chloride HgCl(2) on a model community of plasmid-carrying and plasmid-free pseudomonads. The authors describe a novel experimental system for quantification of the different spatially variable selection pressures that are present in natural environments.

Plasmids may carry a variety of genes for antibiotic and heavy metal resistance that benefit their hosts. These genes are beneficial only in the presence of selection for those particular substances. In their absence the plasmid-carrying bacteria has a reduced growth rate due to the metabolic cost of maintaining the plasmid. Thus a tradeoff exists between benefit and burden resulting in persistence or loss of the plasmid as described by Lilley and Bailey (1997).

To study this phenomenon of plasmid persistence in heterogeneous environments, the authors investigated the effects of spatially heterogeneous Hg pollution on selection for P. fluorescens SBW25 carrying the Hg(r) plasmid pQBR103 relative to the plasmid-free strain.

1) The authors labeled the plasmid-free SBW25 chromosome with an RFP cassette and the plasmid pQBR103 with a GFP cassette.
2) They calculated maximum specific growth rates in liquid cultures of SBW25::rif and SBW25 (pQBR103::gfp).
3) Liquid monocultures of SBW25::rif and SBW25 (pQBR103::gfp) were prepared. Cells were harvested and resuspended in PBS (O.D=0.5). Mixtures having 1:1 ratio of Hg(r)-to-Hg(s) were diluted in PBS (to approximately 2 x 104 CFU /ml) and used to inoculate membranes.
4) Black Isopore polycarbonate membrane filters were pre-washed with sterile distilled water (SDW). The filters were incubated with inoculum on 0.7 R2A for 3 days at 21-23°C.
5) For heterogeneous treatment, the authors soaked 5 gm of carboxymethyl cellulose fibers with either 30 ml SDW or 1.15 or 7.66 mM HgCl(2) to have a final concentration of 0, 1,875 or 12,500 μg HgCl(2) g / cellulose. The cellulose mixtures were dried and crushed to separate the fibers. For random distribution of Hg foci, the cellulose fibers were sprayed down a sealed pressurized container onto the pre-grown bacterial culture on the membrane filter.
6) For uniform treatment, the authors placed the membranes on R2A supplemented with HgCl(2) to give a final concentration of 0, 2.5, 5 or 7.5 μM.
7) Visualization of the colonies was done by using an Eclipse E600 microscope. Images of at least 10 fields of view were captured.
8) Calculation of a(Hg)(r) gives the area of each field of view (FOV) occupied by Hg(r) relative to Hg(s) ( where Hg(r) is strain resistant to Hg and Hg(s) is strain sensitive to Hg) . W(Hg)(r) which is the relative fitness of Hg(r) populations was also calculated.

The results showed that when starting with 1:1 ratio of SBW25::rif and SBW25 (pQBR103::gfp), in the absence of Hg, a(Hg)(r) was 0.33 and W(Hg)(r) was 0.78 where mean area of FOVS occupied by Hg(s) was 74.42%. In the presence of uniform and heterogeneous Hg, a(Hg)(r) was found to increase with Hg concentration. However the value of W(Hg)(r) which is calculated over the entire population, increased only for the uniform Hg treatment and not for the heterogeneous treatment. They explain this by localized selection for Hg(r) in the heterogeneous condition, which did not increase the value of W(Hg)(r) considerably. To test this, they repeated the experiment with either a heterogeneous treatment of 9,375 μg HgCl(2) g/cellulose or a no-Hg control treatment and a variety of starting ratios of Hg(r) to Hg(s) bacteria. Changes in a(Hg)(r) between no-Hg control and heterogeneous treatments were proportional to the starting inoculum of Hg(r) bacteria. Starting with a smaller inoculum of Hg(r) resulted in a greater increase in selection. This lead them to conclude that negative frequency-dependent selection for the plasmid carrying bacteria was taking place in heterogeneously distributed spatial environments. Thus when the number of Hg(r) cells is low there is a higher selection for Hg(r) bacteria than when the number of Hg(r) cells is high.
Their conclusion supports previous work by Ellis, R. J., et al (2007) that demonstrated negative frequency-dependent selection in structured and unstructured environments under uniform Hg conditions. This study goes a step ahead and proves that negative frequency-dependent selection is taking place when Hg is distributed heterogeneously in a spatial environment. In order to determine the time periods over which coexistence of plasmid-bearing and plasmid-free cells takes place and other variables, the authors indicate that further work is to be carried out.
This paper is important because the authors carry out experimental work to demonstrate plasmid persistence in heterogeneous environments, which would be more typical of the “real-world” growth conditions for bacteria, rather than the usually more homogeneous laboratory conditions. A novel method was developed for creating a spatially heterogeneous environment using cellulose fibers imbued with HgCl(2) sprayed onto preinoculated membranes.

Primary Article
Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in Pseudomonads

Additional references:

Rainey, P.B., and M. Travisano. 1998. Adaptive radiation in a heterogeneous environment. Nature 394:69-72.

Ellis, R. J., A. K. Lilley, S.J. Lacey, D. Murrell, and H.C.J. Godfray.2007.
Frequency-dependent advantages of plasmid carriage by Pseudomonas sp in homogeneous and spatially structured environments. ISME J. 1:92-95

Lilley, A.K., and M.J. Bailey.1997. Impact of plasmid pQBR103 acquisition and carriage on the phytosphere fitness of Pseudomonas fluorescens SBW25: burden and benefit. Appl. Environ. Microbiol.63:1584-1587.

Diya Sen, Graduate Student
University of Idaho

2008-08-08T17:53:00.000-07:00

Acclimation of Subsurface Microbial Communities to Mercury
De Lipthay, J.R., Rasmussen, L.D., Oregaard, G., Simonsen, K., Bahl, M.I., Kroer, N., & Sørensen, S.J. (2008). FEMS Microbiology Ecology, 65, 145-155.

As contamination of our oceans, freshwater sources, and soils continues to be an increasing concern, the need for research addressing the potential of remediating contaminated environments also becomes increasingly evident. Mercury (Hg) contamination of soil and water is a particular concern, since mercury is toxic even at very low concentrations. In this article by Lipthay, et al., the authors studied the adaptation of microbial communities to mercury in both mercury-contaminated and non-contaminated subsurface soils. The findings in this study point to the importance of the role of plasmids in the acclimation to mercury of the soil bacteria communities and, on the other hand, their importance in bioremediation efforts.
The authors’ analysis of contaminated and non-contaminated soils included diverse microbiological techniques:
(1) the appearance of colonies on R2A agar plates with or without Hg(II) amendment (to test the abundance of culturable bacteria),
(2) substrate utilization patterns in Biolog ECOplates with or without Hg(II) amendment (to measure the ability of soil bacteria to utilize single carbon sources),
(3) substrate utilization patterns in Biolog mt-2 plates amended with increasing concentrations of Hg(II) (to estimate the minimal inhibitory concentration of Hg(II) in the soil communities),
(4) merA PCR (to test for the presence of merA genes in whole community soil DNA extracts),
(5) IncP trfA1 PCR (to test for the presence of trfA1 genes of the IncP-1 incompatibility group of plasmids in whole community soil DNA extracts), and
(6) 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) analysis (to analyze the genetic diversity of the various bacterial soil communities).
As expected, the abundance of culturable, mercury-resistant bacteria was higher in the mercury-contaminated soils than in the non-contaminated soils and the soil communities from the contaminated site were better adapted to mercury. Results also suggested a higher diversity of mercury-resistant bacteria in the surface soil communities than in the subsurface soil communities. Treatment of the soils with mercury though, resulted in an equally diverse mercury-resistant population in both subsurface and surface soil communities. This increase in diversity following mercury “stimulation” is similar to results found in another study (Muller et al., 2001a), and the authors point out that it is probably the result of either 1) growth of previously low abundance resistant bacteria or 2) transfer of the mer operon by horizontal gene exchange, leading to elevated mercury resistance in the bacterial soil community.
The mercury tolerance of the soil bacteria communities decreased with depth in both the contaminated and non-contaminated soils, with tolerance levels being greater overall in microbial communities from the contaminated soils. Mercury tolerance increased significantly after mercury amendment, indicating a shift in community composition. This increase in tolerance is also most likely due to horizontal gene transfer of the mer operon. Before mercury was added to soils, PCR targeting merA genes resulted only in PCR products from the contaminated soils (cultivation-based methods were used to show that mercury-resistant bacteria were present in non-contaminated soils). After mercury-amendment merA genes were found in all soils, thus demonstrating the adaptive potential of the soil bacteria communities.
IncP-1 trfA1 genes were detected only in DNA extracted from the contaminated soils, not from the non-contaminated soils, and were also more abundant in the surface soil than in subsurface soils.
Another interesting finding of this study to note here is that the microbial diversity did not generally decrease with soil depth, thus contradicting one of the authors’ original hypotheses.
Through biparental exogenous plasmid isolation, the authors found four different mercury-resistance plasmids (pCPH-001, -002, -003, and -004). Further analysis of these plasmids confirmed that all four had identical partial trfA sequences (and belonged to the IncP-1B group), yet had different merA genes. All four plasmid types were isolated from the contaminated surface soil; however, only plasmid pCPH-001 was isolated from the two subsurface soils. This finding of a higher diversity of mercury-resistance plasmids in the surface soil compared with the subsurface soils corresponds well with the findings of a higher diversity of mercury-resistant bacteria in the contaminated surface soil. In contrast, no mercury-resistance plasmids were retrieved from the non-contaminated soils. The observations in this study, that IncP-1 plasmids are present in contaminated but not in non-contaminated soils, are similar to those in other studies (Smalla et al., 2000; Campbell et al., 1995) and are evidence of the importance of IncP-1 plasmids in the dissemination of adaptive functional traits in microbial communities.
The authors suggest that the findings of this study are important to bear in mind when considering bioremediation of mercury-polluted environments.

Additional References:
Campbell, J.I.A., Jacobsen, C.S., & Sørensen, J. (1995). Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils. FEMS Microbiology Ecology, 18, 51–62.

Muller, A.K., Rasmussen, L.D., & Sørensen, S.J. (2001a). Adaptation of the bacterial community to mercury contamination. FEMS Microbiology Letters, 204, 49–53.

Smalla, K., Krogerrecklenfort, E., Heuer, H., et al. (2000) PCR-based detection of mobile genetic elements in total community DNA. Microbiology, 146, 1256–1257.

Matt Bauer, B.S.
University of Idaho

The Role of Plasmid-encoded H-NS-like Protein

2008-07-25T13:27:00.000-07:00

"An H-NS-like stealth protein aids horizontal DNA transmission in bacteria" Doyle M et al., Science 315: 251-252 (2007)

H-NS is one of the abundant DNA-binding proteins that are found in gram-negative bacterial cells. H-NS is known to bind to A+T-rich sequences and regulate expression of a large number of chromosomal genes (Fang FC and Rimsky S, 2008) . Recently, it has become clear that some narrow-host-range plasmids derived from gram-negative bacteria encode H-NS-like proteins. Yet, their roles were still obscure.

So far, it has been shown that Sfh, an H-NS paralog encoded by IncHI1 group plasmid pSf-R27, interacts with host H-NS, and both proteins are functionally exchangeable (Deighan P et al., 2003; Beloin C et al., 2003). People thus might think that plasmid-encoded H-NS-like proteins influence global gene expression of host bacteria. Interestingly, the results shown in this article look to be in the contrary: the absence of Shf disturbed global gene expression of transconjugants. In this article, authors proposed that plasmid-encoded H-NS is a stealth protein that allows host bacteria to carry A+T-rich plasmids with minimal effect on global gene expression and "fitness", by preventing plasmids from titrating cellular pool of H-NS.

Authors introduced pSf-R27, with or without the sfh gene, from original host Shigella flexeneri into Salmonella Typhimurium, and analyzed the transcriptome as well as several phenotypes of transconjugants. Interestingly, the transfer of wild-type pSf-R27 resulted in a few change in the recipient, but transfer of pSf-27Δsfh resulted in the drastic changes in expression of a wide range of genes. Noteworthy phenotypes of the recipient carrying sfh mutant were increased resistance to UV, increased virulence (persistence in macrophage) and reduced motility. These phenotypes are reminiscent of the chromosomal hns mutant (Navarre WW et al., 2006). Authors then showed that the sfh mutation significantly reduced the fitness of recipient (this phenotype was completely complemented by supplying Sfh in trans from another plasmid). To figure out if the reduction of fitness resulted from the titration of "host" H-NS by A+T-rich sequence on the plasmid, authors constructed a pUC18 derivative that carried chromosome-derived A+T-rich DNA fragment and introduced it into the recipient cells, instead of pSf-R27Δsfh. The recipient that carries the pUC18 derivative caused reduction in the fitness, and this reduction was complemented in the presence of Sfh, as in the case of pSf-R27.

Based on these observations authors proposed that sfh is a "stealth" gene that allows the A+T-rich pSf-R27 to invade a new bacterial host with a minimal impact on global gene expression patterns and fitness. They added that the positive effects of sfh on the fitness can be applied to biotechnology to construct more stable cloning vectors.

PRIMARY ARTICLE
Doyle M, Fookes M, Ivens A, Mangan MW, Wain J, Dorman CJ.
An H-NS-like stealth protein aids horizontal DNA transmission in bacteria.
Science 2007, 315:251-2.

ADDITIONAL REFERENCES
Fang FC, Rimsky S.
New insights into transcriptional regulation by H-NS.
Curr. Opin. Microbiol. 2008, 11:113-20.

Deighan P, Beloin C, Dorman CJ.
Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T.
Mol. Microbiol. 2003, 48:1401-16.

Beloin C, Deighan P, Doyle M, Dorman CJ.
Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein.
Mol. Genet. Genomics 2003, 270:66-77.

Navarre WW, Porwollik S, Wang Y, McClelland M, Rosen H, Libby SJ, Fang FC. Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella.
Science. 2006 Jul 14;313(5784):236-8.

Hirokazu Yano (Ph. D.)
University of Idaho

Direct Visualization of Plasmid Transfer

2008-07-19T09:05:00.000-07:00

Direct evidence of extant in situ plasmid transfer in natural environments has typically been obtained by identifying plasmid-encoded phenotypes following the introduction of donor strains. Many plasmids do not encode any known functions (cryptic plasmids); in many unknown plasmids, functions are not determined or there is no easy method to select plasmid-containing cells. So there is the necessity to use known reporter markers, like antibiotic resistance, lacZ, gusA, luxAB or fluorescent proteins genes to label plasmids prior to study. The main advantage of using luciferase and especially fluorescent proteins is that it is possible to detect the presence of plasmid DNA without plating (non-culturable bacteria) and adding additional substrates to the environment. Using of fluorescent proteins enables direct visualization of plasmid transfer but has also some limitations. First is using epifluorescence microscopy or flow-cytometry-based method to detect fluorescent cells, both of which are technically demanding and require expensive equipment. The second limitation arises from the properties of fluorescent proteins. They need some time - from a few to several hours, to produce the strong, detectable signal. So this make impossible to detect precisely the time of plasmid DNA entering the recipient cell.

In the paper “Direct visualization of horizontal gene transfer” by Ana Babic et al. (2008) authors developed an experimental system that enables them to distinguish the transferred donor DNA from both donor and recipient DNA, and to visualize DNA transfer and recombination by means of fluorescence microscopy in real time, at the level of individual living cells. This tool also allowed them to quantify the ongoing transfer of DNA during conjugation and to acquire time-lapse movies that follow the fate of the newly acquired DNA in individual cells through any number of cell divisions.

This method uses fusion of YFP gene with seqA gene. This translational fusion driven from the native seqA promoter was introduced into E.coli chromosome replacing the wild type seqA allel. SeqA protein has strong affinity for DNA which is hemimethylated by Dam methylase at GATC sequences. Such a hemimethylated DNA usually occurs in the replication forks during replication and SeqA-YFP fusion protein had been found bound to chromosomal DNA previously. When the host strain lacks Dam methylase, the chromosomal DNA is not methylated and SeqA-YFP protein is dispersed in the cytoplasm giving dim background fluorescence.
During conjugation single stranded DNA is transferred from a donor strain to the recipient cells and the second DNA strand is synthesized. When plasmid DNA is methylated by Dam methylase and transferred to a Dam deficient strain, stable hemimethylated duplex is formed. Such a duplex is recognized by SeqA-YFP fusion protein and gives strong fluorescence foci.
Using this technique authors were able to detect presence of transferred plasmid DNA in transconjugants as quickly as 5 minutes after mixing together parental strains. After 30-40 minutes almost all recipient cells in the vicinity of donors showed fluorescent foci. In comparison, the RFP protein expressed from the transferred plasmid from tetracycline promoter showed visible signal 2 hours after transfer. They also showed that in the case of F plasmid direct cell wall contact is not necessary for transfer; this means that single stranded plasmid DNA is transferred from cell to cell thru the sex pili.
To summarize this method allows them to visualize and quantify the DNA of any sequence as it is being transferred from one individual cell to another, and to watch its stable genomic acquisition via genetic recombination (horizontal gene transfer) in real time. This experimental system can be applied to monitor horizontal gene transfer by indefinitely following the fate of DNA acquired in intra- and interspecies crosses.

PRIMARY ARTICLE:
Ana Babic, Ariel B. Lindner, Marin Vuli, Eric J. Stewart and Miroslav Radman 2008, Direct Visualization of Horizontal Gene Transfer. Science 319, pp. 1533 - 1536.

On Line Supplements And Movies:

ADDITIONAL REFERENCES:
Søren J. Sørensen, Mark Bailey, Lars H. Hansen, Niels Kroer and Stefan Wuertz 2005, Studing Plasmid Horizontal Transfer In Situ: A Critical Review. Nature Rev. 3, pp.700 - 710.

Sota Hiraga, Chiyome Ichinose, Hironori Niki and Mitsuyoshi Yamazoe 1998, Cell Cycle–Dependent Duplication and Bidirectional Migration of SeqA-Associated DNA–Protein Complexes in E. coli. Mol. Cell 1, pp.381 - 397.

Dr Jaroslaw E. Krol
University of Idaho

F-A

Reticulate classification of mobile genetic elements

2008-07-13T11:16:00.000-07:00

“Reticulate representation of evolutionary and functional relationships between phage genomes.” by Lima-Mendez et al. (2008)

In this paper, the authors note that mobile genetic elements (MGE) in prokaryotes (such as phages, plasmids, conjugative transposons, and genomic islands) show mosaic structures, indicating the importance of horizontal gene exchange in their evolution. These elements represent unique combinations of modules, each of them with a different phylogenetic history. The traditional classification schemes cannot be applied to these genetic elements in part due to the intrinsic inability of tree-based methods to efficiently deal with mosaicism.

To solve the problem, Lima-Mendez et al. (2008) proposed a framework for a reticulate classification of phages based on gene content; i.e., presence (1) or absence (0) of protein family. First, the authors built a graph, where nodes represent phages and lines represent similarities in gene content between phages. Then, the authors applied a two-step clustering [Markov clustering (MCL) and fuzzy clustering] to this graph to generate a reticulate classification of phages: each phage is represented by a membership vector, which quantitatively characterizes its membership in the set of clusters. Phages within the same MCL cluster are likely descendant from a unique module combination, and one phage could belong to several clusters (Lawrence et al. 2002); for example, phage lambda belongs almost equally to two different clusters. Lima-Mendez et al. (2008) stated that “The weight of the intracluster connections represents 79% of the total weight of the connections of the network. This number can be taken as a rough estimate of the contribution of vertical evolution in this network. However, phages from different MCL clusters may be also be related through vertical evolution, but they might have diverged so much that sequence similarities are no longer recognizable or only some [evolutionary cohesive] modules may have been vertically inherited, whereas others have been replaced through horizontal gene transfer.” Thus, it is still difficult to estimate the contribution of different evolutionary events (i.e., vertical and horizontal gene transfer). Kunin and Ouzounis (2003) suggested a framework for the inference of presence or absence of individual protein families at any node on a phylogenetic tree, and assumed that: (1) A protein family shared by most of the clade members would be vertically transmitted; (2) If a protein family is present in most of the descendants of a particular ancestor, but is not found in some subclade, the observed gene absence would normally result from gene loss; and (3) A protein family interspersed across distantly related clades would be horizontally transferred. This assumption cannot detect horizontal gene transfer (HGT) among closely related species, as is true for most methods used to identify HGT (those based on phylogenetic information and compositional features). However, it is well recognized that phage and plasmid transfers (and consequently HGT) should be more likely among closely related species than among distantly related species.

Phylogenetic profiles have been widely applied to bacterial genomes to predict functional links between proteins on the assumption that proteins interacting in metabolic pathways or physical structure would be required to co-occur in genomes (Pellegrini et al. 1999). Lima-Mendez et al. (2008) clustered genes based on their “phylogenetic profiles” to define “evolutionary cohesive modules.” In virulent phages, evolutionary modules span several functional categories, whereas in temperate phages they correspond better to functional modules, suggesting that the phylogenetic profile method does not work well at predicting protein function in virulent phages. The Lima-Mendez analysis reminds us that we must be careful to consider the total context of the MGE, and not only the genome content.

The Lima-Mendez analysis was implemented using Network Analysis Tools (NeAT) (Brohée et al. 2008), available at http://rsat.ulb.ac.be/rsat/index_neat.html.

PRIMARY ARTICLE:
Lima-Mendez G, Van Helden J, Toussaint A, Leplae R. Mol Biol Evol. (2008) 25:762-77. Reticulate representation of evolutionary and functional relationships between phage genomes.

ADDITIONAL REFERENCES:
Lawrence JG, Hatfull GF, Hendrix RW. J Bacteriol. (2002) 184:4891-905. Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches.

Kunin V, Ouzounis CA. Bioinformatics. (2003) 19:1412-6. GeneTRACE-reconstruction of gene content of ancestral species.

Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO. Proc Natl Acad Sci U S A. (1999) 96:4285-8. Assigning protein functions by comparative genome analysis: protein phylogenetic profiles.

Brohée S, Faust K, Lima-Mendez G, Sand O, Janky R, Vanderstocken G, Deville Y, van Helden J. Nucleic Acids Res. (2008) 36(Web Server issue):W444-51. NeAT: a toolbox for the analysis of biological networks, clusters, classes and pathways.

Dr. Haruo Suzuki
University of Idaho

Introduction to this blog

2008-07-11T22:55:00.000-07:00

There is no doubt that bacteria evolve and adapt to local environments in part by horizontal (or lateral) gene transfer between closely and very distantly related organisms. The detection of very similar genes in distinct bacteria showed that large fractions of bacterial genomes have arisen through horizontal gene transfer. Consistent with this, studies done in the laboratory and in the field have shown that the horizontal transfer of genes between bacterial populations readily occurs in various habitats. Thus bacteria have access to a common pool of genes, called the ‘virtual genome’ (VG) or horizontal gene pool (‘HGP’). This sharing of genes from the VG allows ‘wholesale’ acquisition of useful traits such as drug resistance, heavy metal resistance, pollutant degradation, virulence factors, and many more.
Of the various MGEs that play a role in genetic exchange among bacteria, plasmids are of particular interest because many are self-transferable and able to replicate in a wide range of hosts. They often carry antibiotic resistance, pollutant degradation or other provide a fitness advantage to their host. Because plasmids can transfer among different bacterial species, they play an important role in the ability of bacteria to degrade environmental contaminants and to become resistant to drugs used to treat infectious diseases of plants, animals and humans. However, little is known about the genetic structure of these mobile elements and the full range of functions that they encode. To gain insight into the issues outlined above, our plasmid genome sequencing project is analyzing the genome sequences of 100 plasmids that have a broad host-range (also called BHR plasmids). The sequenced plasmids were obtained from soil, water, and sewage sludge samples from around the globe. The project includes finishing the sequencing and annotation of the plasmids, and then interpreting the sequence information to better understand the evolutionary history of plasmids, and their role in bacterial chromosome evolution and adaptation to new environments. The information collected during this project will add significant new data to the paltry plasmid sequence database that now exists, which is also skewed towards plasmids relevant to human infectious diseases.

With this blog we hope to spark your interest in horizontal gene transfer among bacteria, and the diversity, ecology and evolution of bacterial plasmids, and generate discussion about the latest findings in the field.

Dr. Eva Top
University of Idaho