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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
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