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