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

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.

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

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.

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.

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

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

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

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

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

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

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

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

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.

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

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