APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2007, p. 7703–7710 Vol. 73, No. 23
0099-2240/07/$08.00
0 doi:10.1128/AEM.01577-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Sequence Characterization and Comparative Analysis of Three
Plasmids Isolated from Environmental Vibrio spp.
†
Tracy H. Hazen,1 Dongying Wu,2,3 Jonathan A. Eisen,2,3 and Patricia A. Sobecky1*
School of Biology, Georgia Institute of Technology, Atlanta, Georgia 303321; The Institute for Genomic Research, Rockville,
Maryland 208502; and UC Davis Genome Center, University of California, Davis, Davis, California 956163
Received 11 July 2007/Accepted 26 September 2007
The horizontal transfer of genes by mobile genetic elements such as plasmids and phages can accelerate
genome diversification of Vibrio spp., affecting their physiology, pathogenicity, and ecological character. In this
study, sequence analysis of three plasmids from Vibrio spp. previously isolated from salt marsh sediment
revealed the remarkable diversity of these elements. Plasmids p0908 (81.4 kb), p23023 (52.5 kb), and p09022
(31.0 kb) had a predicted 99, 64, and 32 protein-coding sequences and G
C contents of 49.2%, 44.7%, and
42.4%, respectively. A phylogenetic tree based on concatenation of the host 16S rRNA and rpoA nucleotide
sequences indicated p23023 and p09022 were isolated from strains most closely related to V. mediterranei and
V. campbellii, respectively, while the host of p0908 forms a clade with V. fluvialis and V. furnissii. Many predicted
proteins had amino acid identities to proteins of previously characterized phages and plasmids (24 to 94%).
Predicted proteins with similarity to chromosomally encoded proteins included RecA, a nucleoid-associated
protein (NdpA), a type IV helicase (UvrD), and multiple hypothetical proteins. Plasmid p0908 had striking
similarity to enterobacteria phage P1, sharing genetic organization and amino acid identity for 23 predicted
proteins. This study provides evidence of genetic exchange between Vibrio plasmids, phages, and chromosomes
among diverse Vibrio spp.
The Vibrionaceae are gram-negative Gammaproteobacteria
that occur in temperate to tropical, coastal, and estuarine ma-
rine systems (62). Vibrio spp. occupy a diverse range of eco-
logical niches, including sediments, the water column, and in
association with organisms either as symbionts (48) or patho-
gens (26, 37). Phages contribute to Vibrio evolution and ecol-
ogy by regulating host abundance (29) and transferring viru-
lence genes, such as the cholera toxin encoded by ctxAB of the
CTX phage of V. cholerae (64). Plasmids such as pJM1 of V.
anguillarum (20) have also been shown to play a role in Vibrio
pathogenicity. In recent years, sequencing has revealed the vast
diversity of phage genomes (10) and their globally significant
contributions to horizontal gene transfer within marine envi-
ronments (35). In contrast to the demonstrated genetic diver-
sity of vibriophages (16, 66), much less is known of Vibrio
plasmid diversity and the role of plasmids in gene transfer. A
few studies have reported the occurrence of plasmids among
Vibrio populations (19–21, 44, 63), and several have reported
complete sequences of Vibrio plasmids associated with patho-
genic vibrios; however, the distribution and sequence diversity
of Vibrio plasmids has not been studied as extensively as vibrio-
phages.
As of September 2007, there are 16 plasmid and 20 phage
sequences in GenBank that were isolated from vibrios (12–14,
20, 21, 23, 25, 27, 28, 31, 38, 41, 43, 45–48, 51, 67). These
sequences are biased toward small elements (i.e., nine plas-
mids of 8 kb and 10 phages of 9 kb) and are primarily
associated with well-characterized human and fish pathogens.
Among these are plasmids isolated from V. anguillarum (20,
67), V. cholerae (46, 47), V. vulnificus (14), V. parahaemolyticus
(41), and V. salmonicida. The lack of plasmid sequence data,
particularly of plasmids from Vibrio hosts isolated from coastal
water and sediment, limits our understanding of Vibrio plasmid
evolution and diversity.
In the present study we provide a comparative assessment of
plasmids with diverse sizes and gene contents isolated from
vibrios. Similarities of replication initiation and hypothetical
proteins revealed relatedness of plasmids from vibrios occupy-
ing diverse niches. In addition, these elements contained nu-
merous phage-like proteins, including proteins with consider-
able similarity and conserved gene order to enterobacteria
phage P1. To our knowledge, this is the first report of P1-like
phage sequences isolated from a marine bacterium. A previous
study identified two P1-like genes as part of a marine viral
metagenome (10); however, no additional P1 genes or nearly
complete P1 genomes have been characterized from the ma-
rine environment.
MATERIALS AND METHODS
Bacterial strains, media, and plasmid isolation. Vibrio sp. strains 0908, 23023,
and 09022 were isolated from salt marsh sediment of Charleston, SC, in Decem-
ber 1998 (17). DNA for sequencing was obtained by purification of supercoiled
plasmid DNA by cesium chloride density gradient centrifugation as previously
described (52).
Plasmid sequencing and sequence analysis. Plasmids were sequenced using
whole-genome shotgun sequencing and finishing methods (26). Initial open read-
ing frame designations and annotation of select open reading frames was done
using an automated annotation system (26). Protein-coding sequences (CDSs)
were confirmed by independent analysis using GeneMark software (7). Putative
similarity to known proteins was determined by amino acid sequence comparison
and identification of common motif and domain structure using a combination of
PSI-BLAST (3) from the National Center for Biotechnology Information,
SMART (50), COG (57), and Pfam (6) Web-based software. PSI-BLAST anal-
* Corresponding author. Mailing address: 311 Ferst Drive, Atlanta,
GA 30332-0230. Phone: (404) 894-5819. Fax: (404) 385-4440. E-mail:
patricia.sobecky@biology.gatech.edu.
† Supplemental material for this article may be found at http://aem
.asm.org/.

Published ahead of print on 5 October 2007.
7703

ysis was performed with the default threshold E-value of 0.005 and a maximum
threshold of 1.0 over one to two iterations. ClustalW was used to generate all
alignments (61).
Phylogenetic analyses and sequence alignments. Host strains were identified
by a concatenated phylogenetic analysis of 16S rRNA and rpoA nucleotide
sequences as previously described (18). The neighbor-joining tree was generated
using MEGA with the Jukes-Cantor (30) distance estimation model with 1,000
replications for the nucleotide concatenation or the Poisson correction for the
amino acid RecA tree (42). Percent identities of the nucleotide sequences to the
most related organism were determined using BLASTN (3) and BLAST2 (58)
sequences. Sequencing was performed by the University of Nevada, Reno,
Genomics Center and the Core Genomics Facility at the Georgia Institute of
Technology.
Identification of phage-like proteins. Prophage Finder (9) was used with
BLAST analysis (3, 49) of a phage sequence database to identify prophages and
proteins with similarities to phage-associated proteins for all sequenced Vibrio
plasmids available in GenBank as of July 2007. An E value of 0.001 with 10
hits/prophage and a hit spacing of 3,500 were used as parameters for all plasmids
examined.
Nucleotide sequence accession numbers. The plasmid sequences have been
submitted to the GenBank database under accession numbers CP000755 to
CP000757. All additional sequences have been submitted to the GenBank data-
base under accession numbers EU022567 to EU022572.
RESULTS AND DISCUSSION
Host phylogeny and plasmid features. In this study we ex-
amined the sequence diversity of plasmids previously isolated
from three Vibrio hosts (17). A concatenation of 16S rRNA
sequences and rpoA nucleotide sequences was used for greater
resolution of related Vibrio spp. (60). The 16S rRNA and rpoA
nucleotide sequences of Vibrio sp. strains 0908, 23023, and
09022 were 98 and 97%, 98 and 99%, and 99 and 98% identical
to those of V. fluvialis, V. mediterranei, and V. campbellii, re-
spectively. Phylogenetic analysis of concatenated 16S rRNA
and rpoA nucleotide sequences of Vibrio sp. strains 23023 and
09022 indicated they were most related to V. mediterranei and
V. campbellii, respectively (Fig. 1). Vibrio sp. strain 0908 forms
a clade with the closely related V. furnissii and V. fluvialis group
(11). To date, the only report of mobile genetic elements
(MGEs) associated with any of these Vibrio species is an SXT-
like element of V. fluvialis with similarity to the multiple anti-
biotic resistance element SXT previously characterized from V.
cholerae (2). This previous study indicated there may be trans-
fer of MGEs among well-characterized pathogens such as V.
cholerae and emerging marine pathogens such as V. fluvialis
(11, 34, 55).
The nucleotide sequences of the Vibrio plasmids p0908,
p23023, and p09022 were 81,413 bp, 52,527 bp, and 31,036 bp
in length with overall G
C contents of 49.2%, 44.7%, and
42.4%, respectively (see Table S1 in the supplemental mate-
rial). With the exception of p0908, the G
C contents of the
plasmids were within the range of percentages reported for
Vibrio genomes (38 to 47%) (14, 26, 37, 48). The plasmids
p0908, p23023, and p09022 encoded 99, 64, and 32 predicted
CDSs, respectively (see Table S1 in the supplemental mate-
rial). The predicted proteins were assigned primarily to the
following functional categories: replication, stable mainte-
nance, partitioning, and recombination. Additional predicted
proteins identified on one or more of the plasmids may be
involved in mobilization, restriction modification, or transcrip-
tional regulation (see Tables S1 to S3 in the supplemental
material). The only genes common to at least two of the three
plasmids were the putative replication initiation and partition-
ing proteins. The predicted replication initiation protein of
p09022 encoded by CDS19 was 94% identical to the replication
initiation protein of plasmid pKA1 from V. cholerae and 39%
identical to the replication initiation protein of p0908. The
predicted protein of p23023 most closely resembling a replica-
tion initiation protein was that encoded by CDS11, although it
had little similarity to predicted replication proteins from char-
acterized Vibrio or other marine plasmids.
Plasmids encoding putative proteins for self-mobilization,
FIG. 1. A concatenation of 16S rRNA and rpoA nucleotide sequences of the plasmid hosts, Vibrio sp. strains 0908, 23023, and 09022, was used
to determine relatedness of the hosts to other Vibrio spp. as examined in a previous study (60). The neighbor-joining method with the Jukes-Cantor
model of distance estimation (30) was used to generate the tree with a concatenation of 16S rRNA (1,452 nucleotides) and rpoA (772 nucleotides)
sequences. Bootstrap values represent 1,000 replications, and only those with values of
50 are shown.
7704 HAZEN ET AL. APPL. ENVIRON. MICROBIOL.

such as p23023, may be frequently transferred between Vibrio
hosts. In contrast, plasmids such as p0908 and p09022, without
identifiable proteins aiding transfer, may rely on transmission
by phages or other mechanisms. V. cholerae was recently shown
to naturally transform 22-kb segments of genomic DNA, sug-
gesting mechanisms of DNA uptake may facilitate incorpora-
tion of large DNA molecules (39). Additional studies would be
required to determine mechanisms promoting transmission of
the plasmids described in this study.
The significant amino acid identity (
83%) (see Table S3 in
the supplemental material) and conserved gene order of six
predicted proteins encoded by p09022 compared to those of V.
cholerae plasmid pKA1 suggest Vibrio plasmids from diverse
hosts may undergo frequent gene exchange. Alternately, this
may indicate a common rep family exists among diverse Vibrio
hosts, as the conserved genes included three proteins likely to
be involved in replication initiation and partitioning. The re-
maining three predicted proteins were hypothetical (see Table
S3 in the supplemental material). An additional protein en-
coded on p09022 had 94% amino acid identity to a hypothet-
ical protein of plasmid p0471 from an uncharacterized marine
bacterial host (1).
Identification of P1-like proteins on Vibrio plasmid p0908.
The phage P1 of enterobacteria has been isolated from enteric
bacteria (40) and has been shown to infect diverse bacteria under
certain laboratory conditions (40). P1-like proteins and evidence
of intact P1 phage have been identified in freshwater (5); how-
ever, to date none has been identified in marine systems. We
identified 23 CDSs on p0908 with similarity to P1-encoded pro-
teins and 20 additional proteins with similarity to other phage-
encoded proteins. The remaining 16 predicted proteins were sim-
ilar to chromosomally or plasmid-encoded proteins, and 41 had
no similarity to previously characterized proteins. The 23 CDSs of
p0908 encoding P1-like proteins also occur in the same genomic
arrangement as reported for the P1 genome, with a few differ-
ences, possibly due to rearrangements (Fig. 2) (36). The majority
of these P1-like proteins (20 of 23) exhibited the same direction of
transcription (Fig. 2). The G
C contents of CDSs encoding the
P1-like proteins (47 to 54%) were more similar to the overall
G
C contents of p0908 (49.2%), P1 (49%) (36), and the Esche-
richia coli host (50%) (8) than Vibrio chromosomes (39 to 47%)
(14, 26, 37, 48).
Of the proteins encoded on p0908 with similarity to P1
proteins, there were 16 structural proteins, 6 antirestriction
and head-processing proteins, and 1 involved in DNA packag-
ing (see Table S1 in the supplemental material). The structural
proteins included those described as base plate and tail tube
(gp16, BplA, PmgB, Tub, PmgC, BplB, PmgG, gp5, gp6, gp24,
gp25, and gp26), sheath (gp21 and gp22), and head (PmgS and
gp23) components. The P1-like proteins involved in antirestric-
tion and head processing include DarA and DarB (36). Anti-
restriction proteins such as DarA and DarB prevent damage of
phage DNA by host restriction enzymes (36). Identification of
a protein encoded on p0908 with similarity to DarA strongly
suggests p0908 may have acquired the P1-like genes from a P1
phage, since DarA was shown to be unique to P1 (36). An
additional indication of gene exchange between P1 and p0908
is the presence of a CDS encoding a protein similar to DarB.
The predicted protein of CDS26 (2,350 amino acids) is com-
parable to DarB, which is the largest P1-encoded protein
(2,255 amino acids) (36) (see Table S1 in the supplemental
material). Most of the P1-like genes of p0908 encode proteins
for phage structures, such as tub, encoding a tail protein, and
pro, involved in cleavage of head proteins during phage for-
mation (36). P1 genes involved in prophage addiction, phd and
doc (26), were noticeably absent from p0908. Although there
were many phage structural proteins encoded on p0908, it is
unlikely this is a functional phage, as critical proteins for pack-
aging and dispersal were absent. These included lydA and lydB,
encoding a holin and antiholin for host cell lysis (36). Of the
proteins known to be required for functional packaging, PacB
was identified; however, PacA was absent. The gene encoding
PacA includes the pac cleavage site, which is cleaved by the
pacase enzyme, which is composed of PacA and PacB proteins
(36). A few proteins of KVP40, a T4-like phage, were similar to
proteins of P1; however, this similarity was attributed to the
relatedness of P1 and T4, both of which are in the viral family
Myoviridae (38). Two of these shared proteins were identified
on p0908, BplA and Tub (38); however, numerous additional
proteins similar to those encoded in the P1 genome were
identified on p0908 that were not present on KVP40. Also
encoded on p0908 are integrase-like proteins, indicating the
potential for integration of this element into a host chromo-
some (see Table S1 in the supplemental material). Although
Vibrio phages have been characterized with similarity to T4
(38), T7 (25), and P2 (43) phages, none have been character-
FIG. 2. Genetic organization and amino acid conservation of predicted CDSs of p0908 compared to CDSs of enterobacteria phage P1. Shading
indicates regions with amino acid similarity, while the protein lengths (number of amino acids) are designated under each arrow and approximated
by the arrow size. The orientation of each arrow indicates the direction of transcription. Vertical lines indicate the presence of additional genes
that are not shown.
VOL. 73, 2007 ENVIRONMENTAL VIBRIO PLASMIDS 7705

ized with similarity to P1 (36). The P1 integrase (cre) was
identified in bacterial lysogens from a freshwater pond, indi-
cating the presence of P1 in a freshwater environment (5). A
viral metagenomic study produced two sequences with similar-
ity to P1 PacA and PacB in estuarine waters of southern Cal-
ifornia (10). To our knowledge, no additional P1-like se-
quences or nearly complete P1 genomes have been identified
from marine environments. The complete P1 sequence was
finished after the viral metagenome was performed and, there-
fore, some of the P1 genes may have not been identified in the
viral metagenome; however, a recent comparison of the P1
nucleotide sequence to the viral metagenome database primar-
ily yielded hits to prophage from fish ponds (F. Rohwer, per-
sonal communication). This indicates that additional P1 genes
were not present in the marine viral metagenome. The patho-
genic nature of some Vibrio spp. and possible residence in the
gut may have facilitated a Vibrio MGE to exchange genes with
P1 of an enteric bacterium, resulting in an element such as
p0908.
Identification of additional phage-like proteins on Vibrio
plasmids. The prevalence of P1-like proteins on p0908 led us
to examine the occurrence of additional proteins typical of
phage on all available Vibrio plasmids. Several non-P1 phage-
like proteins were identified on the plasmids and in some cases
had a conserved gene order as well as amino acid identity.
BLAST searches (3, 49) of the plasmid genomes to a phage-
only sequence database using Prophage Finder (9) identified
plasmid CDSs encoding proteins similar to phage proteins.
There were 43, 5, and 6 predicted proteins with similarity to
phage proteins encoded by CDSs of p0908, p23023, and
p09022, respectively (see Tables S1 to S3 in the supplemental
material). Functions assigned to these proteins included rep-
lication, partitioning, transcriptional regulation, methylation,
and recombination.
Of the 16 complete Vibrio plasmid sequences currently avail-
able (pES100, pYJ106, pJM1, pEIB1, pPS41, pSA19, pSIO1,
pTC68, pVS43/pVS54, pES213, pTLC, pC4602-1, pC4602-2,
pMP-1, and pR99), we detected the highest frequency of phage
proteins on plasmids described in this study. These proteins
encoded by CDSs of p0908, p23023, and p09022 represented
43, 8, and 20% of the total predicted CDSs, respectively. In
contrast, the other large plasmid sequences available, pES100
(45.8 kb) (48), pJM1 (65 kb) (20), pEIB1 (66.1 kb) (67), and
pC4602-1 (56.6 kb), pC4602-2 (66.9 kb), pR99 (68.4 kb), and
pYJ106 (48.5 kb) (14), isolated from V. fischeri, V. anguillarum,
and V. vulnificus, respectively, encoded proteins with similarity
to phage proteins that comprised between 4 and 14% of the
predicted CDSs. Of the remaining nine plasmids, all less than
8 kb in size, pMP-1 (7.6 kb) had three proteins and pTLC (4.7
kb) (47), pVS43 (4.3 kb), and pVS54 (5.4 kb), isolated from a
single strain of V. salmonicida, encoded a protein with similar-
ity to a protein associated with a phage.
The additional non-P1 phage proteins identified by BLAST
analysis included recombinases, transcriptional regulators, trans-
posases, and hypothetical proteins. Specifically, CDSs 53 to 56 of
p0908 encoded proteins with 49 to 72% amino acid identity to
CDSs 35 to 38 of phage VHML of V. harveyi (see Table S1 of the
supplemental material) (43). The comparable predicted amino
acid sizes, identical gene order, and high amino acid identities of
these proteins suggest recombination between Vibrio phage and
plasmid elements. Additional proteins identified on phages and
other plasmids include hypothetical proteins with a helix-turn-
helix (H-T-H) motif. The H-T-H motif is typical of transcriptional
regulators and other proteins with DNA-binding activity (6, 56).
The amino acid sequence of CDS27 of p0908 is one example with
30% amino acid identity to a hypothetical protein of Photorhab-
dus luminescens and 25% identity to the luxR of V. parahaemo-
lyticus. CDSs 10 and 21 of p23023 were shown to have similar
H-T-H motifs. Hypothetical proteins with H-T-H motifs were
also reported for predicted proteins of vibriophages VP16C and
VP16T (51). To our knowledge, the function and role of these
putative transcriptional regulators for plasmid or phage stability
have not been characterized.
Identification of conserved Vibrio chromosomal genes on
Vibrio plasmids. The three plasmids examined in this study
encoded numerous CDSs with significant amino acid identity
(33 to 81%) to chromosomally encoded genes of vibrios (see
Tables S1 to S3 in the supplemental material). To our knowl-
edge, these chromosomally encoded genes have exclusively
been identified on chromosomes and not on MGEs. Among
those with significant amino acid identity were RecA (81%), a
nucleoid-associated protein, NdpA (65%), a type IV helicase,
UvrD (65%), and a number of hypothetical proteins (50 to
80%). A RecA protein was previously reported on plasmid
pNP40 (65 kb) from Lactococcus lactis (24); however, none has
been identified to date on Vibrio plasmids. Also, the plasmid-
encoded RecA described in this study has greater protein iden-
tity (81%) to Vibrio RecAs than the lactococcal plasmid RecA
had to other characterized lactococcal RecAs (45% amino acid
identity) (24). The RecA (CDS24) encoded on p23023 (see
Table S2 in the supplemental material) was more similar to
RecA of other vibrios (81% amino acid identity) than to that of
related Gammaproteobacteria, such as Photobacterium spp.
(Fig. 3). This indicates the plasmid-encoded recA was likely
from a Vibrio host. Sequence alignment of the predicted
amino acid sequence of CDS24 to RecA sequences of V.
mediterranei, V. splendidus, V. parahaemolyticus, and E. coli
shows the extent of conservation of CDS24 to Vibrio RecAs
(Fig. 4). The RecA signature motif characteristic of RecA
proteins is present in all the aligned sequences (Fig. 4) (6).
Also, the P-loop motif for ATP binding, which is character-
istic of ATPase-like proteins, is present in CDS24 (4, 65)
(Fig. 4). The DNA-binding loops L1 and L2, which are
involved in double-stranded and single-stranded DNA bind-
ing, respectively (24, 54), are also present in CDS24. The
DNA-binding loop L1 of CDS24 is identical to the same
motif found in other RecAs (24, 54). In contrast, loop L2
contains a gap and two other amino acid changes that may
alter the single-stranded DNA-binding activity of the pro-
tein encoded by CDS24. Based on sequence analysis of
CDS24, the predicted protein likely has the recombinase
(15) and proteolytic cleavage activities that have been char-
acterized to date for other RecAs (22, 32). Future experi-
mental studies are required to confirm these predicted func-
tions of CDS24.
RecA protein sequences have been shown for some bacterial
species to provide greater resolution than phylogenetic analy-
ses of an equal number of 16S rRNA sequences (22). Phylo-
genetic assessments of vibrios have previously demonstrated
that recA nucleotide sequences can be used as an alternate
7706 HAZEN ET AL. APPL. ENVIRON. MICROBIOL.

phylogenetic marker to 16S rRNA (53, 59, 60). Several studies
revealed considerable sequence variation (0 to 6%) of recA for
certain Vibrio spp. (60), with as low as 94% recA nucleotide
identity within a species. In contrast, Photobacterium spp., also
within the Vibrionaceae, had less than 94% recA identity to the
closest-related Vibrio recA. Overall, CDS24 is highly conserved
compared to other RecA sequences; however, the N- and C-
terminal regions have significantly diverged (Fig. 4). Sequence
analyses of RecAs from diverse bacteria revealed the majority
of the protein to be highly conserved while the N and C termini
were significantly variable (22). The observed sequence diver-
gence of the termini of the predicted protein sequence of
CDS24 may have occurred by recombination with alleles hav-
ing greater sequence divergence after the sequence was ac-
quired by the plasmid. Alternately, selection pressure for a
specialized role of the plasmid-encoded RecA for plasmid sta-
bility or uncharacterized functions may have led to the se-
quence divergence in the terminal regions.
This study is the first report of a recA encoded on a plasmid
isolated from a Vibrio host. The potential for horizontal trans-
mission of recA by Vibrio plasmids raises questions of whether
recA provides reliable resolution for discriminating between
related Vibrio spp. (59) or determining the extent of O-antigen
gene exchange (53). Additional plasmid-encoded proteins with
similarity to conserved chromosomal genes include the nucle-
oid-associated protein NdpA and a UvrD-like helicase. The
plasmid-encoded NdpA reported here, CDS97 of p0908, had
65% amino acid identity (79% similarity) to NdpA of V. vulni-
ficus (see Table S2 in the supplemental material). Also, CDS48
of p23023 had 65% amino acid identity (79% similarity) to a
UvrD-like helicase of Vibrio splendidus (see Table S2 in the
supplemental material). Recombination of host and plasmid-
encoded uvrD-like genes may increase diversity of uvrD, po-
tentially disrupting function of the host protein. To our knowl-
edge this is the first description of an NdpA-like protein and a
UvrD-like helicase encoded on a plasmid. These results indi-
cate Vibrio plasmids may be involved in horizontal dissemina-
tion of conserved genes, such as recA and uvrD, both involved
in host adaptive responses. Further investigation of the diver-
sity encoded by Vibrio plasmids would be necessary to deter-
mine the extent that these elements transfer conserved
genomic regions among diverse Vibrio spp.
In addition to high amino acid identity, several proteins had
an identical gene order in the plasmid as that found in the
Vibrio chromosomes. Specifically, CDSs 51 to 54 and 59 to 60
of p23023 encoded predicted proteins with amino acid identi-
ties and a conserved gene order to those reported for hypo-
thetical proteins of V. fischeri (see Table S2 in the supplemen-
tal material).
FIG. 3. Phylogenetic comparison of the predicted amino acid se-
quence encoded by CDS24 of p23023 with RecA amino acid sequences
representing most Vibrio spp. and additional members of the Vibrion-
aceae available in GenBank as of March 2007. Distantly related pro-
teobacteria were included as outgroups. A neighbor-joining tree was
constructed in MEGA (33) using the Poisson correction (42). Boot-
strap values were generated over 1,000 replications and are indicated
where the value was
50.
VOL. 73, 2007 ENVIRONMENTAL VIBRIO PLASMIDS 7707

Conclusion. This study provides evidence for a role of Vibrio
plasmids in gene exchange among diverse Vibrio spp., as evi-
denced by the gene content and unique genomic signatures of
Vibrio plasmids relative to Vibrio chromosomes. Identification
of P1-like proteins and other phage-like proteins on Vibrio
plasmids supports the mosaicism of Vibrio MGEs and the po-
tential for recombination between Vibrio plasmids and phages.
The considerable diversity of recA among strains of certain
Vibrio spp. may be facilitated by recombination of plasmid-
encoded genes, such as the p23023 recA. Further studies into
the genetic diversity of Vibrio plasmids as well as their potential
host range are needed to better understand the evolution of
MGEs and their role in diversification of Vibrio spp. This will
serve as the basis for future molecular investigations into the
role of plasmids for unique phenotypes promoting adaptation
to fluctuating environmental conditions and the potential
emergence of pathogens.
ACKNOWLEDGMENTS
We thank Mary Barnstead, Susan van Aken, Grace Pai, and M.
Brook Craven for coordinating the library construction and sequencing
of the plasmids. Also, we thank Ryan Mills and Heath Mills for the
initial annotation work performed on the plasmids and Forest Rohwer
for valuable suggestions for the sequence analysis and manuscript
preparation.
This work was supported by Office of Naval Research grant N00014-
02-1-0228 to P.A.S. and Office of Naval Research grant N00014-99-1-
0860 to J.A.E. T. H. Hazen was supported by an NSF IGERT graduate
fellowship.
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FIG. 4. Sequence alignment of RecA amino acid sequences of CDS24 from p23023 with those sequences encoded on the genomes of V.
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binding of double-stranded DNA (24, 54), and the loop L2 motif for binding single-stranded DNA (24, 54) are all indicated.
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