Comparative ICE genomics: Insights into the evolution of the SXT/R391 family of ICEs

Abstract

Integrating and conjugative elements (ICEs) are one of the three principal types of self-transmissible mobile genetic elements in bacteria. ICEs, like plasmids, transfer via conjugation; but unlike plasmids and similar to many phages, these elements integrate into and replicate along with the host chromosome. Members of the SXT/R391 family of ICEs have been isolated from several species of gram-negative bacteria, including Vibrio cholerae, the cause of cholera, where they have been important vectors for disseminating genes conferring resistance to antibiotics. Here we developed a plasmid-based system to capture and isolate SXT/R391 ICEs for sequencing. Comparative analyses of the genomes of 13 SXT/R391 ICEs derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes that serve as a scaffold capable of mobilizing an array of variable DNA. Furthermore, selection pressure to maintain ICE mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions. The variable genes confer diverse element-specific phenotypes, such as resistance to antibiotics. Functional analysis of a set of deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of most of the dispensable core genes are unknown. Several lines of evidence suggest that there has been extensive recombination between SXT/R391 ICEs, resulting in re-assortment of their respective variable gene content. Furthermore, our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of mobile genetic elements.

Full text

Comparative ICE Genomics: Insights into the Evolution of
the SXT/R391 Family of ICEs
Rachel A. F. Wozniak
1,2,3
, Derrick E. Fouts
3
, Matteo Spagnoletti
4
, Mauro M. Colombo
4
, Daniela
Ceccarelli
5
, Genevie
`ve Garriss
5
, Christine De
´ry
5
, Vincent Burrus
5
*, Matthew K. Waldor
1,2,6
*
1Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 2Department of Genetics, Tufts
Medical School, Boston, Massachusetts, United States of America, 3J. Craig Venter Institute, Rockville, Maryland, United States of America, 4Dipartimento di Biologia
Cellulare e dello Sviluppo, Universita
´di Roma La Sapienza, Rome, Italy, 5Centre d’E
´tude et de Valorisation de la Diversite
´Microbienne, De
´partement de Biologie,
Universite
´de Sherbrooke, Sherbrooke, Que
´bec, Canada, 6Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America
Abstract
Integrating and conjugative elements (ICEs) are one of the three principal types of self-transmissible mobile genetic
elements in bacteria. ICEs, like plasmids, transfer via conjugation; but unlike plasmids and similar to many phages, these
elements integrate into and replicate along with the host chromosome. Members of the SXT/R391 family of ICEs have been
isolated from several species of gram-negative bacteria, including Vibrio cholerae, the cause of cholera, where they have
been important vectors for disseminating genes conferring resistance to antibiotics. Here we developed a plasmid-based
system to capture and isolate SXT/R391 ICEs for sequencing. Comparative analyses of the genomes of 13 SXT/R391 ICEs
derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes
that serve as a scaffold capable of mobilizing an array of variable DNA. Furthermore, selection pressure to maintain ICE
mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions. The
variable genes confer diverse element-specific phenotypes, such as resistance to antibiotics. Functional analysis of a set of
deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of
most of the dispensable core genes are unknown. Several lines of evidence suggest that there has been extensive
recombination between SXT/R391 ICEs, resulting in re-assortment of their respective variable gene content. Furthermore,
our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of
mobile genetic elements.
Citation: Wozniak RAF, Fouts DE, Spagnoletti M, Colombo MM, Ceccarelli D, et al. (2009) Comparative ICE Genomics: Insights into the Evolution of the SXT/R391
Family of ICEs. PLoS Genet 5(12): e1000786. doi:10.1371/journal.pgen.1000786
Editor: Ivan Matic, Universite
´Paris Descartes, INSERM U571, France
Received October 7, 2009; Accepted November 24, 2009; Published December 24, 2009
Copyright: ß2009 Wozniak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: MKW acknowledges support from NIH (AI R37-42347) and HHMI. GG is the recipient of a PhD research scholarship from FQRNT. VB holds a Canada
Research Chair in molecular biology, impact and evolution of bacterial mobile elements and is grateful for support from the Natural Sciences and Engineering
Research Council of Canada (Discovery Grant Program). MS was supported by a grant from PRIN 2007 - Italy, and DC was supported by a fellowship from Cenci
Bolognetti-Institut Pasteur Foundation, Italy. Sequencing at the JCVI was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Department of Health and Human Services under contract number N01-AI-30071. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Vincent.Burrus@USherbrooke.ca (VB); mwaldor@rics.bwh.harvard.edu (MKW)
Introduction
There are three types of self-transmissible mobile genetic
elements: plasmids, bacteriophages and integrative conjugative
elements (ICEs). All three classes of elements enable horizontal
transmission of genetic information and all have had major
impacts on bacterial evolution [1–4]. ICEs, (aka conjugation
transposons), like plasmids, are transmitted via conjugation;
however, unlike plasmids, ICEs integrate into and replicate along
with the chromosome. Following integration, ICEs can excise
from the chromosome and form circular molecules that are
intermediates in ICE transfer. Plasmids and phages have been the
subject of more extensive study than ICEs and while there is
growing understanding of the molecular aspects of several ICEs
[5–10], to date there have been few reports of comparative ICE
genomics [11,12] and consequently understanding of ICE
evolution is only beginning to be unraveled.
Diverse ICEs have been identified in a variety of gram-positive
and gram–negative organisms [13]. These elements utilize a
variety of genes to mediate the core ICE functions of chromosome
integration, excision and conjugation. In addition to a core gene
set, ICEs routinely contain genes that confer specific phenotypes
upon their hosts, such as resistance to antibiotics and heavy metals
[14–18], aromatic compound degradation [19] or nitrogen
fixation [20].
SXT is an ,100 Kb ICE that was originally discovered in Vibrio
cholerae O139 [16], the first non-O1 serogroup to cause epidemic
cholera [21]. SXT encodes resistances to several antibiotics,
including sulfamethoxazole and trimethoprim (which together are
often abbreviated as SXT) that had previously been useful in the
treatment of cholera. Since the emergence of V. cholerae O139 on
the Indian subcontinent in 1992, SXT or a similar ICE has been
found in most clinical isolates of V. cholerae, including V. cholerae
serogroup O1, from both Asia and Africa. Other vibrio species
besides V. cholerae have also been found to harbor SXT-related
ICEs [22]. Furthermore, SXT-like ICEs are not restricted to
vibrio species, as such ICEs have been detected in Photobacterium
damselae,Shewanella putrefaciens and Providencia alcalifaciens [23–25].
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Moreover, Hochhut et al [26] found that SXT is genetically and
functionally related to the so-called ‘Inc J’ element R391, which
was derived from a South African Providencia rettgeri strain isolated
in 1967 [27]. It is now clear that Inc J elements are SXT-related
ICEs that were originally misclassified as plasmids. In the
laboratory, SXT has a fairly broad host range and can be
transmitted between a variety of gram-negative organisms [16].
The SXT/R391 family of ICEs is now known to include more
than 30 elements that have been detected in clinical and
environmental isolates of several species of c- proteobacteria from
disparate locations around the globe [28]. SXT/R391 ICEs are
grouped together as an ICE family because they all encode a
nearly identical integrase, Int. Int, a tyrosine recombinase, is
considered a defining feature of these elements because it enables
their site-specific integration into the 59end of prfC, a conserved
chromosomal gene that encodes peptide chain release factor 3
[29]. Int mediates recombination between nearly identical element
and chromosome sequences, attP and attB respectively [29]. When
an SXT/R391 ICE excises from the chromosome, Int, aided by
Xis, a recombination directionality factor, mediates the reverse
reaction - recombination between the extreme right and left ends
(attR and attL) of the integrated element - thereby reconstituting
attP and attB [6,29]. The excised circular SXT form is thought to
be the principal substrate for its conjugative transfer. The genes
that encode activities required for SXT transfer (tra genes) were
originally found to be distantly related to certain plasmid tra genes
[30–32]. The tra genes encode proteins important for processing
DNA for transfer, mating pair formation and generating the
conjugation machinery. Regulation of SXT excision and transfer
is at least in part governed by a pathway that resembles the
pathway governing the lytic development of the phage lambda.
Agents that damage DNA and induce the bacterial SOS response
are thought to stimulate the cleavage and inactivation of SetR, an
SXT encoded lcI-related repressor, which represses expression of
setD and setC, transcription activators that promote expression of
int and tra genes [5].
The complete nucleotide sequences of SXT (99.5kb) and R391
(89kb) were the first SXT/R391 ICE family genomes to be
reported [14,32]. Comparative [33] and functional genomic
analyses [5,32] revealed that these 2 ICEs share a set of conserved
core genes that mediate their integration/excision (int and xis),
conjugative transfer (various tra genes), and regulation (setR,setCD).
In addition to the conserved genes, these 2 ICEs contain element
specific genes that confer element specific properties such as
resistance to antibiotics or heavy metals. Interestingly, many of
these genes were found in identical locations in SXT and R391,
leading Beaber et al [33] to propose that there are ‘hotspots’ where
SXT/R391 ICEs can acquire new DNA. The genomes of two
additional SXT/R391 ICEs, ICEPdaSpa1, isolated from Photo-
bacterium damselae [23], and ICESpuPO1, derived from an
environmental isolate of Shewanella putrefaciens [24] are now also
known. These two genomes also share most of the conserved set of
core genes present in SXT and R391 and contain element specific
DNA.
Determination of the sequences of SXT/R391 family ICE
genomes was a fairly arduous task due to their size and
predominantly chromosomal localization. Here, we developed a
method to capture and then sequence complete SXT/R391 ICE
genomes. In addition, we identified 3 as yet unannotated SXT/
R391 ICE genomes in the database of completed bacterial
genomes. Comparative analyses of the 13 SXT/R391 genomes
now available allowed us to greatly refine our understanding of the
organization and conservation of the core genes that are present in
all members of this ICE family. Comparative and functional
analyses also facilitated our proposal of the minimal functional
SXT/R391 ICE genome. Furthermore, this work provides new
knowledge of the considerable diversity of genes and potential
accessory functions encoded by the variable DNA found in these
mobile elements. Finally, this comparative genomics approach has
allowed us to garner clues regarding the evolution of this class of
mobile elements.
Results/Discussion
An ICE capture system
To date, ICE sequencing has been cumbersome because it has
typically required construction of chromosome-derived cosmid
libraries and screening for sequences that hybridize to ICE probes
[23,32]. We constructed a vector (pIceCap) that enables capture of
complete SXT/R391 ICE genomes on a low-copy plasmid to
simplify the protocol for ICE sequencing. This plasmid is a
derivative of the single-copy modified F plasmid pXX704 [34,35],
which contains a minimal set of genes for F replication and
segregation but lacks genes enabling conjugation. We modified
pXX704 to include an ,400bp fragment that encompasses the
SXT/R391 attachment site (attB) and thereby enabled Int-catalyzed
site-specific recombination between attB on pIceCap and attP on an
excised and transferred ICE to drive ICE capture (Figure 1).
Conjugations between an SXT/R391 ICE-bearing donor strain
and an E. coli recipient deleted for prfC (and thus chromosomal attB)
and harboring pIceCap yielded exconjugants containing the
transferred ICE integrated into pIceCap (Figure 1). We used the
DprfC recipient to bias integration of the transferred ICE into
pIceCap rather than the chromosome. In these experiments, we
selected for exconjugants containing the transferred ICE integrated
into pIceCap, using an antibiotic marker present on the ICE as well
as a marker present in pIceCap. The low copy IceCap::ICE plasmid
was then isolated and used as a substrate for shotgun sequencing.
We also found that the IceCap::ICE plasmids were transmissible.
Thus, in principle this technique should facilitate capture of ICEs
that do not harbor genes conferring resistance to antibiotics, by
mating out the IceCap::ICE plasmid into a new recipient and
selecting for the marker on pIceCap.
SXT/R391 ICEs included in this analysis
A list of the 13 SXT/R391 ICEs whose genomes were analyzed
and compared in this study is shown in Table 1. All of the ICEs
Author Summary
Integrative and conjugative elements (ICEs) are a class of
mobile genetic elements that are key mediators of
horizontal gene flow in bacteria. These elements integrate
into the host chromosome, yet are able to excise and
transfer via conjugation. Our understanding of ICE
evolution is rudimentary. Here, we developed a method
to capture ICEs on plasmids, thus facilitating their
sequencing. Comparative analyses of the DNA sequences
of ICEs from the same family revealed that they have an
identical genetic structure consisting of syntenous, highly
conserved core genes that are interrupted by clusters of
diverse variable genes. Unexpectedly, many genes in the
core backbone proved non-essential for ICE transfer.
Comparisons of the variable gene content in the ICEs
analyzed revealed that these elements are mosaics whose
genomes have been shaped by inter–ICE recombination.
Finally, our work suggests that ICEs contribute to a larger
gene pool that connects all types of mobile elements.
Comparative ICE Genomics
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included in our analyses contain an int gene that was amplifiable
using PCR primers for int
sxt
[29]. They were isolated on 4
continents and from the Pacific Ocean during a span of more
than 4 decades. They are derived from 7 different genera of
c-proteobacteria and the ICEs derived from V. cholerae strains are
from both clinical and environmental isolates of 3 different
V. cholerae serogroups.
Five of these ICE genome sequences were determined at the J.
Craig Venter Institute (JCVI) using the ICE capture system
described above (Table 1, rows 1–5). In addition, we sequenced
ICEVflInd1, also at the JCVI, by isolating cosmids that
encompassed this V. fluvialis derived ICE prior to developing the
ICE capture technique (Table 1, row 6). Table 1 (rows 7–10) also
includes 4 previously unannotated ICE genomes that we found in
BLAST searches of the NCBI database of completed but as yet
unannotated genomes; 3 of these ICEs are clearly members of
SXT/R391 ICE family since they are integrated into their
respective host’s prfC locus and contain int genes that are predicted
to encode Int proteins that are 99% identical to Int
sxt
. The fourth
element, ICEVchBan8 does not encode an Int
sxt
orthologue;
however, this element contains nearly identical homologues of
most of the known conserved core SXT/R391 ICE family genes.
ICEVchBan8 will be discussed in more detail below but since it
does not contain an Int
sxt
orthologue it is not considered a member
of the SXT/R391 family of ICEs and thus not included in our
comparative study. Finally, Table 1 also includes the 4 SXT/R391
ICEs that were previously sequenced (Table 1, rows 11–14).
Despite the diversity of our sources for SXT/R391 ICEs, the
genomes of two pairs of ICEs that we analyzed proved to be very
similar. SXT
MO10
and ICEVchInd4 only differed by 13 SNPs in 7
genes and by the absence from ICEVchInd4 of dfr18, a gene
conferring trimethoprim resistance. These ICEs were derived from
V. cholerae O139 strains isolated in India from different cities at
different times: SXT
MO10
from Chennai in 1992 and ICEVchInd4
from Kolkata in 1997. The high degree of similarity of these two
ICE genomes suggests that ICEs can be fairly stable over time.
ICEVchBan9 and ICEVchMoz10 were also extremely similar
although ICEVchMoz10 lacks dfrA1, another allele for trimethoprim
resistance. These two ICEs were derived from V. cholerae O1 strains
from Bangladesh (1994) and Mozambique (2004) respectively. The
great similarity of these ICEs suggests that there has been spread of
SXT-related ICEs between Asia and Africa in recent times. Studies
of CTX prophage genomes have also suggested the spread of V.
cholerae strains between these continents [36].
General structure and sizes of SXT/R391 genomes
The ICEs listed in Table 1 were initially compared using
MAUVE [37] and LAGAN [38], programs that enable visuali-
zation of conserved and variable regions on a global scale. All of
the SXT/R391 ICEs we analyzed share a common structure and
have sizes ranging from 79,733 bp to 108,623 bp (Table 1 and
Figure 2). They contain syntenous sets of 52 conserved core genes
(Figure 2A) that total approximately 47kb and encode proteins
with an average of 97% identity to those encoded by SXT. All of
the individual ICEs also contain DNA that is relatively specific for
individual elements (Figure 2B); the differences in the sizes of the
variable regions accounts for the range in ICE sizes.
Five sites within the conserved SXT/R391 ICE structure have
variable DNA present in all of the ICEs in Figure 2. Four of these
sites were previously termed ‘hotspots’ for ICE acquisition of new
DNA [33]. Due to similarities between SXT and R391, the fifth
hotspot only became apparent through our comparison of the 13
ICEs examined here. Each of these hotspots (HS1 to HS5 in
Figure 2B) is found in an intergenic region (see below), suggesting
that the acquisition of these variable DNA regions has not
interrupted core ICE gene functions. In addition, some of the
ICEs have variable DNA inserted in additional intergenic
locations or in rumB (labeled I–IV in Figure 2B). Previous analyses
[32] indicated that the insertion in rumB, did not impair SXT
transmissibility. Overall, comparison of these 13 SXT/R391 ICE
genomes suggests that: 1) these elements consist of the same
perfectly syntenous and nearly identical 52 core genes that serve as
a scaffold (see below) capable of mobilizing a large range of
variable DNA; and 2) selection pressure to maintain ICE mobility
has restricted insertions of variable DNA into sites that do not
interrupt core functions.
Figure 1. Schematic of the ICE capture system. Conjugation between a donor strain bearing a chromosomal ICE and a DprfC recipient strain
harboring pIceCap, which contains attB, yields exconjugants that contain the transferred ICE integrated into pIceCap. Exconjugants were selected for
using a marker on pIceCap and on the ICE. attR and attL represent the right and left ICE-chromosome junctions.
doi:10.1371/journal.pgen.1000786.g001
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Table 1. SXT/R391 ICE family members analyzed in this study.
ICE Host strain Site and year of isolation
Size
(bp)
% Identity to
Int
SXT
Resistance profile Notable Variable Genes
Genbank Accession
Number
Strain or ICE
References
ICEVchMex1 Vibrio cholerae non
O1-O139
San Luis Potosi, Mexico 2001 82839 99% (410/413) -Fic family protein, diguanylate cyclase,
restriction modification system
GQ463143 [66]
ICEVchInd4 Vibrio cholerae O139 Kolkata, India 1997 95491 100% (413/413) floR, strBA, sul2 Toxin-antitoxin system GQ463141 [54]
ICEVchInd5 Vibrio cholerae O1 Sevagram, India 1994 97847 99% (409/413) floR, strBA, sul2, dfrA1 AraC family transcription regulator,
glyoxoylase abx resistance
GQ463142 This study
ICEVchBan5 Vibrio cholerae O1 Bangladesh, 1998 102131 99% (409/413) floR, strBA, sul2, dfrA1 AraC family transcription regulator,
glyoxoylase abx resistance
GQ463140 [29]
ICEPalBan1 Providencia alcalifaciens Bangladesh, 1999 96586 99% (409/413) floR, strBA, sul2, dfrA1 Toxin-antitoxin system, phenazine
biosynthesis protein, lysine exporter
GQ463139 [25]
ICEVflInd1 Vibrio fluvialis Kolkata, India 2002 91369
(a)
99% (409/413) dfr18, floR, strBA, sul2 Toxin-antitoxin system GQ463144 [22]
ICEVchMoz10 Vibrio cholerae O1 Beira, Mozambique 2004 104495 99% (409/413) floR, strBA, sul2, tetA’ AraC family transcription regulator,
glyoxoylase abx resistance,
ATP-dependent Lon protease
ACHZ00000000 [67]
ICEPmiUsa1 Proteus mirabilis Maryland, United States 1986 79733 99% (409/413) -ATP-dependent helicase AM942759 [68]
ICEVchBan9 Vibrio cholerae O1 Matlab, Bangladesh 1994 106124 99% (409/413) floR, strBA, sul2, dfrA1,
tetA’
AraC family transcription regulator,
glyoxoylase abx resistance,
ATP-dependent Lon protease
CP001485 [69]
ICEVchBan8 Vibrio cholerae non O1-O139 Bangladesh, 2001 105790
(a)
25% (76/301) -Toxin-antitoxin system NZ_AAUU00000000 This study
SXT
MO10
Vibrio cholerae O139 Chennai, India 2002 99452 100% dfr18, floR, strBA, sul2 Toxin-antitoxin system AY055428 [16]
R391 Providencia rettgeri Pretoria, South Africa 1967 88532 99% (410/413) kanR, merRTPCA Sulfate transporter, universal
stress protein
AY090559 [27]
ICEPdaSpa1 Photobacterium damselae Galicia, Spain 2003 102985 99% (412/413) tetAR ATP-dependent Lon protease,
heat-shock protein
AJ870986 [23]
ICESpuPO1 Shewanella putrefaciens 630m, Pacific Ocean 2000 108623 99% (409/413) - Zn/Co/Cd efflux system, restriction
modification system
CP000503 [24]
(a)
The sequence is not complete and therefore the true size is not known.
doi:10.1371/journal.pgen.1000786.t001
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The SXT/R391 ICE core genes
The 52 core genes present in all the SXT/R391 ICEs analyzed
include sets of genes that are known to be required for the key ICE
functions of integration/excision, conjugative transfer and regu-
lation [32] as well as many genes of unknown function. Most genes
of known or putative (based on homology) function (coded by gray
shading or hatch marks in Figure 2A) are clustered with genes that
have related functions. For example, int and xis, genes required for
integration and excision, are adjacent and setR, and setC/D, the key
SXT regulators are near each other at the extreme 39end of the
elements, although separated by 4 conserved genes of unknown
function. Each ICE also has four gene clusters implicated in
conjugative DNA processing and transfer (shown in light gray in
Figure 2A). Finally, each of the ICEs has a nearly identical origin
of transfer (oriT), a cis-acting DNA site that is thought to be nicked
to initiate DNA processing events during conjugative transfer [39],
in the same relative location.
The conserved core genes include approximately as many genes
of unknown function as genes of known function. Some of the
genes of unknown function are found either interspersed amongst
gene clusters that likely comprise functional modules (e.g s091
between traD and s043) while others are grouped together (e.g.
most genes between traN and traF). In several cases, the
interspersed genes appear to be part of operons with genes of
known function (e.g. s086-s082 maybe in an operon with setDC).
Variable ICE DNA
In addition to sharing 52 core genes, all of the ICE genomes
analyzed contain variable DNA regions, ranging in size from 676
to 29,210 bp. Most of the variable DNA sequences are found in 5
intergenic hotspots (Figure 2B). However, some ICEs contain
additional variable DNA inserts outside the 5 hotspots. For
example, SXT and five other ICEs in Figure 2 have variable DNA
segments, corresponding to related ISCR2 elements, disrupting
rumB (Figure 2B, site III). ISCR2 elements are IS91-like
transposable elements that tend to accumulate antibiotic resistance
genes [40]. Interestingly, it is unusual for the contents of the
hotspots and other variable regions to be found in only one ICE.
Figure 2. Structure of the genomes of 13 SXT/R391 ICEs. (A) The upper line represents the set of core genes (thick arrows) and sequences
common to all 13 SXT/R391 genomes analyzed. Hatched ORFs indicate genes involved in site-specific excision and integration (xis and int), error-
prone DNA repair (rumAB), DNA recombination (bet and exo) or entry exclusion (eex). Dark gray ORFs correspond to genes involved in regulation
(setCDR). Light gray ORFs represent genes encoding the conjugative transfer machinery, and white ORFs represent genes of unknown function. (B)
Variable ICE regions are shown with colors according to the elements in which they were originally described SXT (blue), R391 (red), ICEPdaSpa1
(green), ICESpuPO1 (purple), ICEVchMex1 (yellow), ICEPalBan1 (orange), ICEVchInd5 (turquoise), ICEPmiUSA1 (olive), ICEVchBan9 (pink), ICEVflInd1
(light green). Thin arrows indicate the sites of insertion for each variable region and HS1–HS5 represent hotspots 1–5. Roman numerals indicate
variable regions not considered true hotspots. Cm, chloramphenicol; Hg, mercury; Kn, kanamycin; Sm, streptomycin; Su, sulfamethoxazole; Tc,
tetracycline; Tm, trimethoprim. * indicates that s073 is absent from ICEPdaSpa1.
a
ICEVchMoz10, which lacks dfrA1 in the integron structure, does not
confer resistance to Tm.
b
The purple gene content of ICEVflInd1 was deduced from partial sequencing, PCR analysis and comparison with ICESpuPO1.
doi:10.1371/journal.pgen.1000786.g002
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Instead, the variable gene content of most of the ICEs shown in
Figure 2B is found in more than one ICE. For example,
ICESpuPO1, ICEPalBan1, and ICEVflInd1, all have identical
contents in hotspot 5 (lavender genes in hotspot 5 in Figure 2B);
however, the contents of the other hotspots in these 3 elements are
almost entirely different. Thus, the variable gene content of the
SXT/R391 ICEs reveals that these elements are mosaics. The
overlapping distribution of variable DNA segments seen in the
ICEs in Figure 2B suggests that recombination among this family
of mobile elements may be extensive. In addition, in some
instances, the variable regions appear subject to additional genetic
modifications. For example, ICEPdaSpa1 and ICEVchBan9
contain ICE-specific DNA nested within the shared sequences
inserted at hotspot 5 DNA (the green and pink genes in hotspot 5
in these elements, Figure 2B).
The variable genes encode a large array of functions and only a
few will be discussed here. A complete list of the diverse genes
found in the hotspots is found in Table S1. Although we cannot
predict functions for many genes found in the hotspots, since they
lack homology to genes of known function, at least a subset of the
known genes seem likely to confer an adaptive advantage upon
their hosts. Most of the ICE antibiotic resistance genes are found
within transposon-like structures (e.g., the ISCR2 elements noted
above) but four ICEs contain a dfrA1 cassette, which confers
resistance to trimethoprim [25], in a class IV integron located in
hotspot 3. A disproportionate number of variable genes are likely
involved in DNA modification, recombination or repair, as they
are predicted to encode diverse putative restriction-modification
systems, helicases and endonucleases. Such genes may provide the
host with barriers to invasion by foreign DNA including phage
infection and/or promote the integrity of the ICE genome during
its transfer between hosts. Three ICEs contain genes that encode
diguanylate cyclases [41] in hotspot 3. These enzymes catalyze the
formation of cyclic-diguanosine monophosphate (c-di-GMP), a
second messenger molecule that regulates biofilm formation,
motility and virulence in several organisms including V. cholerae
[42,43]. Most SXT/R391 ICEs contain mosA and mosT in hotspot
2. These two genes encode a novel toxin-antitoxin pair that
promotes SXT maintenance by killing or severely inhibiting the
growth of cells that have lost this element [44]. Not all ICEs in the
SXT/R391 family contain mosAT; however, those lacking these
genes may encode similar systems to prevent ICE loss. For
instance, R391 and ICEVchMex1 contain two genes (orf2 and orf3)
encoding a predicted HipA-like toxin and a predicted transcrip-
tional repressor distantly related to the antitoxin HipB.
Locations of the ICE variable genes
The variable regions found in the 5 hotspots are found
exclusively in intergenic regions, punctuating the conserved ICE
backbone (Figure 2). The boundaries between the conserved and
variable sequences were mapped on the nucleotide level and
compared (Figure 3A–3E). Each hotspot had a distinct boundary.
Remarkably, even though the contents of the variable regions
markedly differ, with few exceptions the left and right boundaries
between conserved and variable DNA for each hotspot was
identical among all the ICEs (Figure 3). For example, the left
junctions of the inserts in hotspot 2 immediately follow the stop
codon of traA and the right junctions are exactly 79 bp upstream of
the start of s054 (Figure 3B), despite the fact that the DNA
contents within these borders greatly differ. In hotspot 2, the right
junction appears to begin with a 15 bp sequence that has two
variants (Figure 3B, brown & light brown sequence). These
sequences may reflect the presence of earlier insertions that have
since been partially replaced. A similar pattern was found adjacent
to the left boundary of hotspot 4 in several ICEs (Figure 3D, lines
3–6). Once an insertion is acquired, the number of permissive sites
for the addition of new variable DNA likely increases.
There are two exceptions to the precise boundaries between
variable and conserved DNA. Hotspot 1 and hotspot 3 in
ICEVchMex1 and ICEPdaSpa1, respectively, contain variable
DNA that extends beyond the boundary exhibited by all the other
ICEs in these locations (Figure 3A, line 3, and Figure 3C, line 7).
The only boundary that could not be identified was the left border
of hotspot 5, the region containing genes between s026 and traI.As
discussed below, s026 is the least conserved core gene and its
variability obscured any consensus sequence abutting the variable
DNA. Perhaps this border has eroded because s026 is not required
for ICE mobility [32].
The relative precision of most boundaries between conserved
and variable DNA sequences in all the ICEs analyzed suggests that
a particular recombination mechanism, such as bet/exo-mediated
recombination, may explain the acquisition of the variable regions.
However, at this point, we cannot exclude the possibility that the
precise locations for variable DNA insertions simply reflects
selection for optimal ICE fitness; i.e., ICEs can optimally
accommodate variable DNA in these locations while preserving
their essential functions.
Similarity of SXT/R391 ICE and IncA/C plasmid core genes
Unexpectedly, BLAST analyses revealed that most of the
conserved core SXT/R391 genes are also present in IncA/C
conjugative plasmids. These multidrug resistance plasmids are
widely distributed among Salmonella and other enterobacterial
isolates from agricultural sources [45,46]. Recently, members of
this family of plasmids have also been identified in Yersinia pestis,
including from a patient with bubonic plague [47], and in aquatic
c-proteobacteria [48], including Vibrio cholerae [49,50]. To date, the
closest known relatives of the SXT/R391 transfer proteins are
found in the IncA/C plasmids. Every predicted SXT transfer
protein is encoded by the IncA/C plasmid pIP1202 isolated from
Y. pestis [50] and the identities of these predicted protein sequences
vary from 34 to 78% (Figure 4A). Furthermore, there is perfect
synteny between the four gene clusters encoding the respective
conjugative machineries of these two mobile elements (yellow and
orange genes in Figure 4A). Despite the extensive similarity of the
SXT and IncA/C conjugative transfer systems, these plasmids lack
homologues of setR and setD/C as well as int/xis, suggesting that
regulation of conjugative transfer differs between these elements.
The similarity of IncA/C plasmids and SXT/R391 ICEs is not
limited to genes important for conjugal DNA transfer. Ten genes
of unknown function (shown in black in Figure 4A), some of which
are interspersed within likely tra gene operons and some of which
are clustered together between traN and traF, are similar in the two
elements. Furthermore, most of these ten genes are in identical
locations in the two elements. Both elements also contain
homologs of bet and exo (shown in green in Figure 4A); these are
the only known homologs of the lRed recombination genes found
outside of bacteriophages. Together, the similarity of DNA
sequences and organization of SXT/R391 ICEs and IncA/C
plasmids suggests that these elements have a common ancestor.
The fact that the contents of the hotspots in the two classes of
elements are entirely distinct suggests that their evolutionary paths
diverged prior to acquisition of these variable DNA segments.
The minimal functional SXT/R391 ICE gene set
The conservation of the 52 core genes in all 13 SXT/R391
ICEs analyzed suggested that many or even all of these genes
would be required for key ICE functions of excision/integration,
Comparative ICE Genomics
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conjugative transfer and regulation. The presence of ten ICE core
genes of unknown function in IncA/C plasmids (black genes in
Figure 4A) is also consistent with the hypothesis that these genes
might be required for ICE transfer. However, our previous work
demonstrated that not all genes recognized here as part of the
conserved core gene set are required for SXT transfer. Beaber et al
showed that deletion of rumB – s026 (which includes 5 cores genes)
from SXT had no detectable influence on SXT excision or
transfer [32]. Therefore, we systematically deleted all of the core
ICE genes whose contributions had not previously been assessed,
in order to explore the hypothesis that these genes (especially those
also present in IncA/C plasmids) would be essential for ICE
transfer and to define the minimum functional SXT/R391 gene
set.
Surprisingly, deletion of most of the ICE core genes of unknown
function, including genes with homologues in IncA/C plasmids,
did not alter SXT transfer efficiency. Deletion of s002 or s003,
which are located downstream of int in all SXT/R391 ICEs, did
not alter the frequency of SXT transfer; similarly, deletion of
s082,s083, and s084, core genes of unknown function that are
found near the opposite end of SXT/R391 ICEs but not in
IncA/C plasmids, also did not influence SXT transfer frequency
(Figure 4B). Furthermore, deletion of s091, which is found
between traD and s043 in ICEs and IncA/C plasmids, did not
reduce SXT transfer (Figure 4B). In contrast, deletion of s043,
which has weak homology to traJ in the F plasmid (a gene
important in DNA processing) and is located in a transfer cluster
containing traI and traD, abolished transfer (Figure 4B, Dd),
suggesting that s043, here re-named traJ is required for SXT
transfer. It is unlikely that the transfer defect of SXTDtraJ can be
explained by polar effects of the deletion on downstream genes,
since traJ appears to be the last gene of an operon found
immediately upstream of hotspot 1. Similarly, deletion of s054,
which is found immediately 59of traC and is homologous to a
disulfide-bond isomerase dsbC, also abolished transfer (Figure 4B,
De). Interestingly, disulfide bond-isomerases are present in several
other conjugative systems [51]. However, it is not clear at this
point if the deletion of s054 from SXT accounts for the transfer
defect of SXTDs054, since we could not restore transfer by
complementation.
Additionally, Beaber et al found that deletion of s060 through
s073 in SXT, which includes 7 genes that are also found in IncA/
C plasmids reduced SXT transfer more than 100-fold [32]. We
constructed several smaller deletions in this region and found that
Figure 3. The boundaries of the 5 hotspots. The boundaries between conserved and hotspot variable regions are shown. Black typeface
indicates conserved sequence, while color indicates variable sequence. Numbers in parentheses indicate the number of intervening nucleotides. The
thin dotted lines indicate continuations of variable DNA. Bold letters indicate a non-conserved base. (A) Hotspot 1, which is present between traJ and
traL. Line 1: SXT, ICEVchInd4, ICEPalBan1; Line 2: R391, ICEPdaSpa1, ICEVchBan5, ICEVchInd5, ICEPmiUSA, ICESpuPO1, ICEVflInd, ICEVchMoz10,
ICEVchBan9; Line 3: ICEVchMex1. (B) Hotspot 2, which is present between traA and s054. Line 1: SXT, ICEVchInd4, ICEPmiUSA, ICEVflInd, ICEVchInd5,
ICEPalBan1, ICEVchBan5; Line 2: ICEPdaSpa1, ICEVchMoz10, ICEVchBan9; Line 3: R391; Line 4: ICEVchMex1; Line 5: ICESpuPO1. (C) Hotspot 3, which is
present between s073 and traF. Line 1: SXT, ICEVchInd4; Line 2: ICEVchMex1, ICEVflInd; Line 3: ICEVchMoz10, ICEVchBan9, ICEVchInd5, ICEPalBan1,
ICEVchBan5. Line 4: R391; Line 5: ICEPmiUSA; Line 6: ICESpuPO1; Line 7: ICEPdaSpa1. (D) Hotspot 4, which is present between traN and s063. Line
1: SXT, ICEVchInd4. Line 2: ICEVchInd5, ICEVchBan5; Line 3: ICESpuPO1, ICEPmiUSA; Line 4: R391, ICEVchMoz10, ICEVchBan9, ICEVflInd. Line
5: ICEPdaSpa1; Line 6: ICEPalBan1; Line 7: ICEVchMex1. (E) Hotspot 5, which is present between s026 and traI. Line 1: SXT, ICEVchInd4, ICEPdaSpa1,
R391, ICEVchMoz10, ICEVchBan9; Line 2: ICEPmiUSA; Line 3: ICESpuPO1, ICEPalBan1, ICEVflInd1; Line 4: ICEVchInd5, ICEVchBan5; Line 5: ICEVchMex1.
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deletion of s063, which is also found in pIP1202, reduced the
transfer frequency of SXT by ,100-fold, nearly the same amount
as deleting the entire region (Figure 4B). Complementation
analyses revealed that the absence of s063 accounted for the
transfer defect of SXTDs063 (data not shown). Even though
SXTDs063 was still capable of transfer, in our view, the drastic
reduction in the transfer frequency of this mutant warrants
inclusion of s063 into the minimum functional SXT ICE genome
(shown in Figure 4C). Other deletions in this region, including
deletions of bet,exo,s067,s068 and s070, which have orthologues in
IncA/C plasmids, resulted in #10-fold reductions in transfer
frequency. We therefore did not include these genes in the
minimal functional core SXT/R391 genome (Figure 4C).
The findings from our experiments testing the transfer
frequencies of SXT derivatives harboring core gene deletions
(shown in Figure 4B), coupled with our previous work demon-
strating the requirements for the predicted SXT tra genes in the
element’s transfer [32], suggest a minimal functional SXT/R391
ICE structure as shown in Figure 4C. This minimum element is
,29.7 kb and consists of 25 genes. Genes with related functions,
which in some cases encode proteins that likely form large
functional complexes (such as the conjugation apparatus), are
grouped together in the minimal genome. At the left end of the
minimum ICE genomes are xis and int, the integration/excision
module of SXT/R391 ICEs. In the minimal ICE genome, the
ICE oriT and mobI, which encodes a protein required for SXT
transfer [39], are no longer separated from the other genes (traIDJ)
that are also thought to play roles in the DNA processing events
required for conjugative DNA transfer. The genes required for
formation of the conjugation machinery, including the pilus, and
mating pair formation and stabilization [32,39] are divided
between three clusters (denoted mpf1-3 in Figure 4C). Finally, at
the right end of the minimal functional genome are the genes that
regulate ICE transfer (setC/D and setR). Thus, the minimal
Figure 4. Comparison of the SXT/R391 core genome with the genome of pIP1202 and defining the minimal functional SXT/R391
gene set. (A) Alignment of the conserved core genes of SXT/R391 ICEs with the genome of the IncA/C conjugative plasmid pIP1202 from Yersinia
pestis. The top line shows the same core ICE genes shown in Figure 2A. ORFs are color coded as follows: DNA processing, yellow; mating pair
formation, orange; DNA recombination and repair, green; integration/excision, red; replication, purple; regulation, gray; entry exclusion, blue;
homologous genes of unknown function, black; genes without corresponding counterparts in ICEs and pIP1202, white. Numbers shown in the
middle represent % identity between the orthologous proteins encoded by SXT and pIP1202 [GenBank:NC_009141]. The positions of the hotspots in
SXT/R391 ICEs are marked by downward pointing arrowheads. For pIP1202, the size of the sequences (which include IncA/C backbone DNA as well as
variable DNA) found at these locations as well as resistance markers are indicated by upward pointing arrowheads. aphA,aadA and strAB confer
resistance to aminoglycosides. sul1 and sul2 confer resistance to sulfonamides. cat,bla
SHV-1
,tetAR,qacED1 and merRTPCADE confer resistance to
chloramphenicol, b-lactams, tetracyclines, quaternary ammonium compounds and mercury ions, respectively. Detailed descriptions of the conserved
backbone of the IncA/C conjugative plasmids have been published elsewhere [48,50]. Regions that were deleted from SXT to investigate the function
of genes of unknown function (see panel B) are indicated with straight lines. Dotted lines indicate that the deletion included DNA in the adjacent
hotspots. (B) Influence of deletion of genes of unknown function on the frequency of SXT transfer. The mean values and standard deviations from
three independent experiments are shown. * indicates that the frequency of transfer was below the detection level (,10
28
). Deletion mutants SXTDa,
SXTDk and SXTDl, transferred at frequencies that were not significantly different from that of wild-type SXT (data not shown). (C) Proposed minimal
set of genes necessary for a functional SXT/R391 ICE. int, integration/excision module; mob, DNA processing module; mpf, mating pair formation
modules; reg, regulation module.
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functional SXT/R391 ICE is relatively small and organized into 3
discrete functional modules that mediate excision/integration,
conjugation, and regulation.
Even though deletion of 27 out of 52 SXT/R391 ICE core
genes proved to have little or no effect on SXT transfer frequency,
and hence these genes were not included in Figure 4C, it is
reasonable to presume that these genes encode functions that
enhance ICE fitness given their conservation. For example, the
presence of highly conserved bet and exo genes in all SXT/R391
ICEs suggests that there has been selection pressure to maintain
this ICE-encoded recombination system that promotes ICE
diversity by facilitating inter ICE recombination (G Garriss, MK
Waldor, V Burrus, in press). A key challenge for future studies will
be to determine how core genes of unknown function promote
ICE fitness.
Variations in the similarity of core genes
To identify genes in the SXT/R391 core genome that may be
subject to different selection pressures, we compared the percent
identity of each ICE’s core genes to the corresponding SXT gene
(Figure 5). Most of the ICEs’ core genes exhibited 94% to 98%
identity on the nucleotide level to SXT’s core genes. There was no
discernable difference in the degree of conservation of most core
genes that were or were not part of the minimal ICE, suggesting
that there are equal selective pressures on essential and non-
essential genes. However, we identified 8 genes (s026,traI,orfZ,
s073,traF,eex,s086, and setR) that exhibit significantly different
degrees of conservation (Figure 5 and Figure S1). Three of these
showed unusually high conservation, while the other 5 had below
average conservation. Two of the highly conserved genes, setR and
s086, are found at the extreme 39end of the elements. The
conservation of setR may reflect the key role of this gene in
controlling SXT gene expression. S086 may also play a role in
regulating SXT transfer [52]. The other highly conserved gene,
orfZ, is found between bet and exo and has no known function.
s026 and s073 are the most divergent of all the genes in the
backbone. s026 encodes a hypothetical protein with homologues in
many gram negative organisms. Although S026 is predicted to
contain a conserved domain, COG2378, which has a putative role
in transcription regulation, this protein is not required for SXT
transfer [32]. The significant divergence of s026 along with its lack
of essentiality suggests that this gene could become a pseudogene.
A similar argument could be made for s073, which encodes a
hypothetical protein that is also not required for ICE transfer.
However, this argument does not hold for traI or traF, two genes
which are essential for ICE transfer. Although the reasons which
account for the different degrees of conservation of these 8 core
genes are hard to ascertain at this point, the data in Figure 5
suggests that individual core genes are subject to different
evolutionary pressures.
Figure 5. Variations in the nucleotide conservation of core ICE genes. The nucleotide sequence of each core gene from each ICE was
compared to the corresponding sequence in SXT using pairwise BLASTn analyses to determine the percent identities. The average values for all of the
ICEs, excluding SXT and ICEVchInd4, are shown in the inset.
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Comparisons of core gene phylogenies
We created phylogenetic trees for each core gene based on their
respective nucleotide sequences to further explore the evolution of
the conserved backbone of SXT/R391 ICEs. Since we found such
a high degree of conservation for most of the core genes, the
bootstrap values for most of these trees were relatively low. Thus,
we concentrated on the most polymorphic genes found in Figure 5,
s026,s073,traI, and eex, for phylogenetic analyses. As shown in
Figure 6A, the trees for s026,traI and s073 exhibit 3 distinct
branching patterns. The lack of similarity in these phylogenetic
trees suggests that either individual core genes have evolved
independently or that high degrees of recombination mask their
common evolutionary history. The latter hypothesis seems more
likely since experimental findings have revealed that SXT/R391
ICEs can co-exist in a host chromosome in tandem [26] and
recombination between tandem elements can yield novel hybrid
ICEs with considerable frequency [53] (G Garriss, MK Waldor, V
Burrus, in press). Also, as noted above, the distributions of variable
genes among the ICEs shown in Figure 2 also supports the idea
that inter-ICE recombination is commonplace.
Unlike most core genes, the trees for traG and eex were similar.
In these two trees, the ICEs segregate into two evolutionarily
distinct groups (Figure 6B), confirming and extending previous
observations that revealed that there are two groups of eex and traG
sequences in SXT/R391 ICEs [54]. These two groups correspond
to the two functional SXT/R391 ICE exclusion groups.
Interactions between traG and eex of the same group mediate
ICE exclusion [55]. Thus, the identical 2 clusters of traG and eex
Figure 6. Phylogenetic analysis of several core ICE genes. Nucleotide sequences of the indicated core genes were used to generate the
phylogenetic trees shown. Bootstrap values are indicated at branch points. The individual scale bars represent genetic distances and reflect the
number of substitutions per residue.
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sequences observed in their respective trees reveals the co-
evolution of the traG/eex functional unit. The two groups of eex
sequences can also be observed in Figure 5 where the bifurcating
pattern reveals the 2 exclusion groups. This pattern is difficult to
discern for traG, perhaps because of the large size of this multi-
functional gene.
ICEVchBan8, an SXT-like ICE that lacks Int
sxt
The sequence of ICEVchBan8, which was derived from a non-
O1, non-O139 V. cholerae strain, is incomplete but it appears to
contain 49 out of 52 SXT/R391 core genes. However, since this
strain lacks Int
sxt
it was not included in our comparative analyses
above. It is not known if ICEVchBan8 is capable of excision or
transmission; however, it contains a P4-like integrase and a
putative xis. It is tempting to speculate that the genome of
ICEVchBan8 provides an illustration of how acquisition (presum-
ably via recombination) of a new integration/excision module may
generate a novel ICE family.
Perspectives
Comparative analysis of the genomes of the 13 SXT/R391
ICEs studied here has greatly refined our understanding of this
group of mobile genetic elements. These elements, which have
been isolated from 4 continents and the depths of the Pacific
Ocean, all have an identical genetic structure, consisting of the
same syntenous set of 52 conserved core genes that are interrupted
by clusters of diverse variable genes. All the elements have
insertions of variable DNA segments in the same five intergenic
hotspots that interrupt the conserved backbone. Furthermore,
some of the elements have additional insertions outside the
hotspots; however, in all cases the acquisition of variable DNA has
not compromised the integrity of the core genes required for ICE
mobility. Functional analyses revealed that less than half of the
conserved genes are necessary for ICE transmissibility and the
contributions of the 27 core genes of unknown function to ICE
fitness remains an open question. Finally, several observations
presented here suggest that recombination between SXT/R391
ICEs has been a major force in shaping the genomes of this
widespread family of mobile elements.
Although comparisons of the 13 ICE genomes analyzed here
strongly suggest that these mobile elements have undergone
extensive recombination during their evolutionary histories, there
is a remarkable degree of similarity among the SXT/R391 ICEs.
All of these ICEs consist of the same syntenous and nearly identical
52 genes. In contrast, other families of closely related mobile
elements, such as lambdoid or T4-like phages for example, exhibit
greater diversity [56,57]. Since the elements that we sequenced
were isolated from several different host species and from diverse
locations, the great degree of similarity of the SXT/R391 ICE
family does not likely reflect bias in the elements that we
sequenced. It is possible that this family of mobile elements is a
relatively recent creation of evolution and has yet to undergo
significant diversification.
To date, relatively few formal comparative genomic analyses of
other ICE families have been reported. Mohd-Zain et al [11]
identified several diverse ICEs and genomic islands that shared a
largely syntenous set of core genes with ICEHin1056, an ICE
originally identified in Haemophilus influenzae. However, even
though these elements share a similar genomic organization, they
exhibit far greater variability in the sites of insertion of variable
DNA and in the degree of conservation in their core genes
compared to SXT/R391 ICEs. Thus, although this group of
elements appears to share a common ancestor, they seem to have
diverged earlier in evolutionary history than the SXT/R391 ICEs.
However, when comparative genomic analyses were restricted to
ICEHin1056-related ICEs found in only two Haemophilus sp., Juhas
et al found that, like the SXT/R391 family of ICEs, these 7
ICEHin1056-related ICEs share greater than 90% similarity at the
DNA level in their nearly syntenous set of core genes [12]. It will
be interesting to learn the extent of conservation of genetic
structure and DNA sequence in additional ICE families to obtain a
wider perspective on ICE evolution.
Comparative genomic studies of bacteriophages have led to the
idea that the full range of phage sequences are part of common but
extremely diverse gene pool [58,59]. The SXT/R391 ICE
genomes suggest that there may be an even larger network of
phylogenetic relationships linking sequences found in all types of
mobile genetic elements including phages, plasmids, ICEs and
transposons. The genomes of SXT/R391 ICEs appear to be
amalgams of genes commonly associated with other types of
mobile elements. Many of the ICE core genes are usually
associated with phages, such as int,bet,exo and setR, or with
plasmids, such as the tra genes. Additionally, the SXT/R391 ICEs
and IncA/C plasmids clearly have a common ancestor, as we
found that the entire set of SXT/R391 tra genes are also present in
IncA/C plasmids. Thus, the genes present in all types of mobile
genetic elements appear to contribute to a common gene pool
from which novel variants of particular elements (such as
ICEVchBan8) or perhaps even novel types of mobile genetic
elements can arise.
Materials and Methods
ICE Sequencing
ICEPalBan1, ICEVchMex1, ICEVchInd4, ICEVchInd5 and
ICEVchBan5 were isolated using the plasmid capture system
described in Figure 1. The SXT chromosomal attachment
sequence, attB, was introduced into the modified F plasmid
pXX704 [34] to create pIceCap. This plasmid was then
introduced into a DprfC derivative of the Tc
R
E. coli strain
CAG18439. Exconjugants derived from matings between this
strain and those harboring the 5 ICEs listed above resulted in
strains carrying a pIceCap::ICE plasmid. Once captured, the
plasmids were isolated using the Qiagen plasmid midi kit for low-
copy plasmids (Qiagen). Isolated pIceCap::ICE plasmids were
then sequenced.
ICEVflInd genome was determined by sequencing several
overlapping cosmids that encompassed this ICE’s genome. Briefly,
genomic DNA from a Vibrio fluvialis strain carrying ICEVflInd was
prepared using the GNome DNA kit (QBIOgene). Sau3A1
restricted genomic DNA was used to create a SuperCos1
(Stratagene)-based cosmid library according the manufacture’s
instructions. The library was subsequently screened for cosmids
containing ICE-specific sequences using PCR with primers to
conserved core ICE sequences. Four cosmids containing overlap-
ping ICEVflInd sequences were identified and sequenced.
The genomes of 6 ICEs were sequenced by the Sanger random
shotgun method [60]. Briefly, small insert plasmid libraries (2–
3 kb) were constructed by random nebulization and cloning of
pIceCap::ICE DNA or of cosmid DNA for ICEVflInd. In the
initial random sequencing phase, 8–12 fold sequence coverage was
achieved. The sequences of either pIceCap or pSuperCos were
subtracted and the remaining sequences were assembled using the
Celera Assembler [61]. An initial set of open reading frames
(ORFs) that likely encode proteins was identified using GLIM-
MER [62], and those shorter than 90 base pairs (bp) as well as
some of those with overlaps eliminated.
Comparative ICE Genomics
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Bioinformatics
Nucleotide and amino acid conservation were assessed with the
appropriate BLAST algorithms. ICEs were aligned using clustalW
with default settings [63]. MAUVE [37] and LAGAN [38] were
used to identify core genes in Figure 2. To map the boundaries of
the hotspots, sequence comparisons were made using MAUVE
and then manually compared to find boundaries between
conserved and variable DNA as shown in Figure 3.
Phylogenetic trees were generated from alignments of nucleotide
sequences using the neighbor-joining method as implemented by
ClustalX software, version 2.011 [64]. The reliability of each tree
was subjected to a bootstrap test with 1000 replications. Trees
were edited using FigTree 1.22 (http://tree.bio.ed.ac.uk/software/
figtree/).
Generation and testing of SXT deletion mutants
CAG81439 harboring SXT was used as the host strain to create
the SXT deletion mutants shown in Figure 3; the deletions were
constructed using one-step gene inactivation as previously
described [44,65]. The primers used to create the deletion mutants
are available upon request. Matings were conducted as previously
described [16,44] using deletion mutants and a Kn
R
E. coli
recipient, CAG18420. Exconjugants were selected on LB agar
plates containing chloramphenicol, 20mg/ml (for SXT selection)
and kanamycin, 50 mg/ml. The frequency of exconjugant
formation was calculated by dividing the number of exconjugants
by the number of donors.
Supporting Information
Figure S1 Variations in the conservation of individual core ICE
genes. The percent identity of the nucleotide sequence of each
core gene and oriT versus the corresponding sequence in SXT was
calculated for all ICEs studied. The average values for each gene
(as shown in the inset of Figure 5) were then used in one-way
ANOVA comparisons to determine genes that exhibit significantly
more or less conservation compared to other core genes. p-values
of one-way ANOVA comparisons of each core ICE gene are
shown. The grid represents all pair-wise comparisons, and the
color indicates the level of significance as follows: red: p,.001,
orange: p,.01, and yellow: p,.05. Genes that exhibited a p-
value,.05 when compared with at least 50% of all other core
genes are discussed in the text.
Found at: doi:10.1371/journal.pgen.1000786.s001 (0.56 MB TIF)
Table S1 Contents of the hotspots.
Found at: doi:10.1371/journal.pgen.1000786.s002 (0.14 MB
DOC)
Acknowledgments
We thank Brigid Davis and Fre´de´rique Le Roux for helpful comments on
the manuscript. We thank Robert Hall for facilitating the work described
here and Yoshiharu Yamaichi for suggesting the design of pIceCap.
Author Contributions
Conceived and designed the experiments: RAFW VB MKW. Performed
the experiments: RAFW DEF MS DC GG CD VB. Analyzed the data:
RAFW MS DC VB MKW. Contributed reagents/materials/analysis tools:
MMC. Wrote the paper: RAFW VB MKW.
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