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Stalking the Wild Plasmid Jeremy Koenig and Remy Chait Microbial Diversity, 2006. MBL, Woods Hole MA, US. Abstract Describing microbial evolutionary relationships in phylogenetic trees can be (and is in many instances) a great misrepresentation of organismal relatedness. The reason for this is that generally speaking microbial genomes can partake in exceptionally high rates of Lateral Gene Transfer thereby blurring evolutionary relationships to the extent that the branches of phylogenetic trees become highly unresolved bushes. Evolutionary networks are quickly becoming a more excepted approach of documenting microbial evolution. Therefore the question that deserves consideration is: what genes travel along these networks? In this study we present an approach to studying genes that are likely to exhibit high transfer rates, specifically, those genes found on plasmids. Furthermore, we attempt to consider plasmids in an environmental context i.e. using metagenomics in order to assess plasmid mediated microbial interactions in the wild. Our results indicate that it is possible to build wild plasmid libraries. Introduction Phylogenetic trees have provided a great deal of insight into the evolutionary paths and relatedness of life. However, these trees with mathematically derived branching patterns pay little attention to the potential genetic interactions that may occur between branches. While a tree may be a reasonable depiction for short time scales between vertebrates, it has been suggested to be a gross misrepresentation of the evolutionary histories of microbes more specifically, Phylogenetic trees do not accurately consider the quantity of genetic exchange that has occurred between microbial “species” (1). Many microbes employ complex mechanisms of taking up, integrating, and dispensing DNA into the environment, and these microbes are typically found living in a veritable soup of extracellular DNA and viral bodies providing them with essentially limitless genetic capacity to facilitate environmentally derived phenotypic plasticity and adaptation. The era of genomic sequencing has revealed many instances of Lateral Gene Transfer (LGT) between microbes and it is now a widely accepted fundamental process that contributes to microbial evolution. One of the great modern advances of genomics is the development of metagenomics, the “omics” that allows us to gain access to the sum of environmental DNA and as a result to assess the unculturable organisms and to observe the workings of the environment –at least in the context of genomics. However, metagenomics has never been used to study the role of plasmids in the environment. It is thought that plasmids likely represent a significant proportion of inter-species gene transfer. They are typically small compared to genomes, they are often present in greater numbers relative to whole genomes (stoichiometrically speaking), they encode an exciting class of genes that are likely indicative of niche pressures, and these genetic elements likely have a profoundly interesting ecology. Studying plasmids in a metagenomic context hinges on extractability from the environment and stabilization in a host. These are not trivial processes and perhaps this is why wild plasmids have not been studied. In this study we apply some general molecular biology tools in a unique combination specifically, plasmid isolation and in addition, insertion of an origin of replication that is compatible in Escherichia coli as well as a selection marker as developed in (2). We hope that this strategy will be useful in identifying the mobility of different genes and as well as aid in identifying between species transfer of plasmids, i.e. plasmid networks. This approach could give us a great deal of insight into an otherwise unresolved area of microbial evolution and could supplement our understanding of prevalent pandemic issues such as the spread of resistance of antimicrobials. Methods Sample collection and cultivation: Three spatially isolated soil samples from School Street Marsh Woods Hole MA were pooled and homogenized. These samples were used for DNA isolation directly from the environment and this sample was also used to grow microbes on R2A agar plates both with and without antibiotic. A broad spectrum of antibiotics were used in order to screen the of drug-resistance capability of Bacteria cultivated from soil as well as to increase chances of isolating plasmids that encoded drug resistance determinants. 20ug/ml of the following antibiotics were used: kanamycin, vancomycin, chloramphenicol, trimethoprim cephalothin and tetracycline. Cells that had grown O/N on the R2A agar plates with and without antibiotic were scraped off the agar plates and suspended in 500 uL dH2O and also subjected to DNA extraction. Putative Streptomyces isolated from a previous study were also grown on R2A agar media with and without the above listed antibiotics. The Streptomyces grown on the R2A agar plates were scraped off and suspended in 500ul dH2O and subjected to DNA extraction. Individual Streptomyces isolates that illustrated multi-drug resistance were picked and then grown in 5mL R2A liquid media O/N with aeration. These Streptomyces isolates were also subjected to the same DNA extraction. DNA extractions: Homogenized soil samples and cultivated cells were subjected to three DNA isolation protocols: bead beating (Ultra Clean Soil DNA Kit Cat. No. 12800-100), whole cell extraction (Wizard genomic DNA purification Kit Cat. No. 206043), and miniprep (QIAprep Spin Miniprep Kit Cat. No. 27104) that was preceded by a 30 min lysozyme incubation as well as a freeze-thaw step in order to weaken more ridged microbial cell wall structures. Enrichment of plasmid DNA: Chromosomal DNA of one aliquot of each of the extracted DNA samples was digested with Epicentre’s Plasmid safe ATP-dependent DNase (Cat. No. E3101K) and an additional aliquot was miniprepped (QIAprep Spin Miniprep Kit Cat. No. 27104) in order to compare the plasmid recoverability of the two methods. Transposition of Plasmid DNA: The purified plasmid DNA from both the plasmid friendly DNase and miniprep treatments were transposed with Epicenter EZ-Tn5 <R6Kγori / KAN-2> insertion kit Cat. No. EZI011RK. This transposon contains an E. coli compatible origin of replication as well as a kanamycin resistance marker. Transformation: Transposed plasmids where electroporated into Epicentre’s Transformax EC100D pir+ electrocompetent E. coli as well as in TOPO chemically competent E. coli. Transformants were plated on LB agar plates with 50ug/mL kanamycin as well as on 50ug/mL kanamycin plus 20ug/mL of one of each of the following antibiotics: ampicillin, vancomycin, chloramphenicol, trimethoprim cephalothin or tetracycline. Experimental Design We considered two approaches when attempting to identify plasmids in the wild microbial community. In order that the results of our antibiotic screen saw the effects of plasmids in their natural hosts, we carried out a selection directly on community strains. This was done on two groups (Fig 1). The first, a consortium of fast-growing strains, was picked from overnight growth of soil extracts on plain R2A plates, as well as some R2A plates containing antibiotics. The strains from the antibiotic plates were designated ‘resistant’, and selected isolates were assayed for plasmid content. A duplicate screen was carried out on a set of Streptomyces isolates from soil near the Loeb Laboratory (MBL) by Erika Quintana, these Streptomyces were selected because they more closely parallel the strains used in the extensive assessment of drug resistance described in D’Costa et al., 2006 (2). Selected strains, once characterized for resistance, were also assayed for plasmids. By isolating and characterizing strains prior to plasmid extraction, this method permits correlation of plasmids to strains that can be identified by 16S rDNA sequence. In addition, one can be more certain that antibiotic resistance by exact combinations of host and plasmid mirrors that seen in the environment. The major disadvantage of this method is the nature of its low throughput when compared to whole community isolations of wild DNA plasmids and therefore this approach only considers those plasmid-cell systems in which the cells can be brought into culture, perhaps <1% of the total community. We attempted to address these issues and complement results from the above protocol via isolation of plasmid DNA in bulk from the environment without culturing the hosts (Fig 2). Using Epicenter EZ-Tn5 <R6Kγori/KAN-2> we attempted to make plasmids obtained directly from the environment capable of replication in Escherichia coli. We then assayed transformants for resistance on kanamycin and we performed a plasmid friendly DNase treatment as well as mini-preps in order to isolate plasmids for sequencing. The tradeoff made when considering this method is that functional genomic assays are difficult to perform as plasmid expression is assayed in strains that may be unable to express their products or may be toxic to their E. coli host. Therefore, functional metagenomic studies may not necessarily be a completely unbiased representation of plasmid-encoded genes, though this is a limitation of every metagenomic functional assay. However, despite this limitation sequence driven analysis is possible because the Epicenter EZ-Tn5 <R6Kγori / KAN-2> encodes internal primers for sequencing. Thus it is feasible to construct the first ever plasmid metagenomic library. Results and Discussion Plasmid DNA from Bacterial Isolates Colonies that grew O/N from described treatments are indicated in (Fig.3). These were re-streaked for isolation. 20 Previously isolated strains of Streptomyces were also assayed for resistance and these profiles are summarized in Table 1. 5ul aliquots of DNA extractions from isolates were run on a gel (Fig 4a). Although faint bands indicating the presence of plasmids are present, there was strong background signal of chromosomal DNA. We improved plasmid purity by attacking linear DNA with an exonuclease, ineffective against circular and nicked circular DNA. A second gel (Fig 4b) was run following this treatment and plasmid bands are apparent in a much-reduced chromosomal DNA background. At this point the plasmids, still identifiable with specific strains (Fig 4c) have been isolated, and can be cloned by insertion of an E. coli-compatible origin of replication and selection marker, and sequenced. Plasmid DNA and Whole Wild Community and Cultivated Heterogeneous Colony Plasmid DNA The results of DNA extractions form soil and community DNA from heterogeneous colonies grown on R2A plates are indicated in (Fig 5). This DNA was ethanolprecipitated, concentrated and a transposition reaction was carried out to insert the R6KAlpha origin of replication and a Kanamycin resistance gene into plasmid DNA. This plasmid DNA with the EZ-Tn5 <R6Kγori / KAN-2> transposon was then transformed and clones where selected for on LB plates with 50ug/ml kanamycin alone and with kanamycin plus one of 5 other drugs (tetracycline, cephalothin, vancomycin, chloramphenicol and trimethoprim) at 20ug/ml. Transformation was performed twice using cells capable of permitting R6K replication (pir+), and once in pir- cells where only plasmids having an inherent capacity to replicate in E. coli could be selected for. Although the control worked well albeit at lower efficiency, there were no transformants on the pir- strain, indicating that at least E. coli-compatible plasmids did not make up a significant portion of the environmental plasmid population. For the pir+ strain, many colonies were observed on all but the tetracycline and chloramphenicol plates, though it was later also determined that the titers of vancomycin and trimethoprim were too low to inhibit our untransformed host strain (vancomycin likely because E. coli has a higher MIC than Streptomyces and our stock of trimethoprim was expired and likely significantly degraded). Further studies should select at a range of multiples of the MIC for each drug rather than at a single concentration. Therefore the only transformants under additional selection for properties of plasmids were those plated on cephalothin. Approximately 60 transformants were picked, grown overnight in LB + kanamycin and mini-prepped (Fig 6). Approximately 16 of these were end-sequenced out from the transposon in both directions, and the transposon removed from sequence computationally. Many of the plasmid preps yielded a band around 2000 base pairs which, when sequenced, aligned perfectly with the transposon, indicating its circularization (Fig 7a). Unfortunately, this component of the reactions was highly representative in out plasmid library. The fact that the cephalothin selection worked was fortunate in this case, as it selected against this component, and thus hits from this set of colonies appeared much more likely to be real environmental plasmids. Several plasmids ran longer on a gel than the circularized transposon, and we analyzed the assembled sequence of these around the insertion site. These were checked for homology to known sequences, using BLASTn and BLASTx and ORFs were defined using NCBI’s ORFfinder (Fig 7b). These results are summarized in figure 7c. Plasmids isolated from the kanamycin and cephalothin-resistant transformants were typically larger and believed likely to carry some resistance factor(s) to cephalosporin. Two of these sequences unfortunately were likely vector impurities that were present in the Epicenter transposase kit component. However, in the case of plasmid #11, high-scoring blast hits were found for two factors implicated in drug resistance: an uroporphyrin, and an ABC transporter. Although a more robust sequencing effort of this representative is required we are fairly confident that we have isolated a wild plasmid. Future work We find the initial results of these techniques encouraging however, there is substantial work to be considered. Specifically, the transposition reaction needs to be optimized to increase the concentrations of desired transposed plasmid product. We believe that if the quantities of DNA in the transposition reaction were better tuned, the circularized transposon component in the product could be significantly reduced. If there were any remaining in the future, we would likely make our selection criterion of plasmids to be sequenced based on size as bona fide plasmids will typically be substantially larger. In addition methods that can define host versatility of plasmids should be considered in order to determine the best representation of each plasmid’s genetic capabilities. Furthermore, controls will be needed to optimize selection and reduce bias for environmental plasmids. Once these techniques are made to work reliably, a myriad of questions become accessible. For example, we would like to know what genes are transferred between species and what species/environments are responsible for moving them around. In addition, labeling plasmid libraries with different fluorescence proteins and releasing them back into the wild may allow one to quantitatively measure interactions between wild plasmids and to follow them in populations and ask about intra- and inter-cellular plasmid ecologies, and symbioses. Immediately practical questions are also apparent for example, if clinical pathogenic drug-resistance is facilitated via a plasmid intermediate, is it possible to block transfer of resistance genes by targeting specific species or transfer pathways? Little is understood about plasmid populations in the wild, but theoretically speaking their importance is difficult to dispute, we hope that the method can be used in order to examine the importance ecological importance of plasmids within wild microbial communities. Acknowledgements We would like to thank Tom Schmidt and Bill Metcalf, and the entire staff of the 2006 Microbial Diversity course at MBL, Woods Hole MA for vastly useful advice and inspiration, and especially David Walsh for help with the molecular biology and not killing us when we made him work past his bedtime, and Dion for immeasurable calmness and making the impossible less so. In addition, we were blessed to be part of a truly incredible group of students and would like to thank them for a most amazing, enlightening, and fun summer. In particular, pertaining to this project, a vast amount of help with the Streptomyces was given to us by Erika Quintana, and the same by Nicky Caiazza for anything pertaining to nucleic acids. Many thanks to the ‘handful of people who know a handful about a handful of things’, and shared it with all of us. We’ll miss you. References Doolittle WF, Phylogenetic Classification and the Universal Tree Science 25 June 1999:Vol. 284. no. 5423, pp. 2124 - 2128 Agron P, Sobecky P, Andersen G, Establishment of Uncharacterized Plasmids in Escherichia Coli by In Vitro Transposition FEMS Microbiol Lett. 2002. Vol 217 (249254) D'Costa V, McGrann K, Hughes D, Wright G, Sampling the Antibiotic Resistome Science 20 January 2006 311: 374-377 Tables and Figures