SXT-Related Integrating Conjugative Element in New World Vibrio cholerae

SXT-related integrating conjugative elements (ICEs) became prevalentin Asian Vibrio cholerae populations after V. cholerae O139emerged. Here, we describe an SXT-related ICE, ICEVchMex1, ina Mexican environmental V. cholerae isolate. Identificationof ICEVchMex1 represents the first description of an SXT-relatedICE in the Western Hemisphere. The significant differences betweenthe SXT and ICEVchMex1 genomes suggest that these ICEs haveevolved independently.

INTRODUCTION

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Introduction

SXT^MO10 is a Vibrio cholerae-derived integrating conjugativeelement (ICE) that encodes resistance to multiple antibiotics.Originally, SXT was found in the chromosome of the initial V.cholerae O139 isolates (17). This novel V. cholerae serogroupemerged in late 1992 on the Indian subcontinent as the firstnon-O1 serogroup to give rise to epidemic cholera (9). Priorto the emergence of this novel V. cholerae serogroup, SXT wasrarely if ever detected in V. cholerae O1 isolates (2); however,after the spread of V. cholerae O139 throughout much of Asia,SXT^MO10 or closely related ICEs have been found in most O1 andO139 clinical isolates from Asia (2). In addition, SXT-relatedICEs in African V. cholerae O1 isolates (10), in non-V. choleraevibrios (1), and in other ${gamma}$ -proteobacteria (12) have now beendescribed. All the V. cholerae-derived SXT-related ICEs reportedto date have been detected in isolates from Asia or Africa.

ICEs excise themselves from their host's chromosome, transferto a new host by conjugation, and then integrate into the chromosomeagain. Comparison of the complete DNA sequences of SXT^MO10 (99.5kb) and R391 (89 kb), an SXT-related ICE originally found inProvidencia rettgeri (6), revealed that both ICEs share a highlyconserved set of genes that code for their regulation, excision-integration,and conjugative transfer functions (3). Besides this conservedscaffold, these elements also harbor insertions, such as antibioticresistance genes, that confer element-specific properties (3).SXT^MO10 carries genes that mediate resistance to sulfamethoxazole,trimethoprim, chloramphenicol, and streptomycin. Some SXT-relatedICEs carry different antibiotic resistance genes (12). Furthermore,recent V. cholerae O139 isolates contain SXT-related elementsthat do not carry antibiotic resistance genes. Thus, it appearsthat selection for increasing resistance to antibiotics maynot be the only factor that explains the recent rapid spreadof SXT-related ICEs in vibrio populations.

We tested whether environmental V. cholerae isolates collectedduring 2001 and 2002 from the state of San Luis Potosi, Mexico,contained SXT-related ICEs. A PCR assay for detection of thecharacteristic SXT integrase gene, int, was used for this analysis(12). int is highly conserved in all SXT-related ICEs analyzedto date and is essential for SXT's integration and excision(7, 13). Eight out of 52 V. cholerae isolates from freshwater,sewage water, and freshwater or marine fishes were positivefor int. We chose one of these eight int⁺ strains at randomfor further analysis. Here we report functional and geneticcharacterization of an ICE, designated ICEVchMex1, found inV. cholerae 1-010118-075, isolated from sewage in 2001.

Classification of this isolate was difficult. We were unableto determine the serogroup of this isolate by use of a collectionof serotype-specific antisera (Public Health Laboratory, Schoolof Medicine, National Autonomous University [UNAM], Mexico).This strain did not agglutinate with anti- ${alpha}$ O139 antisera (kindlyprovided by G. B. Nair). This isolate was more typical of Vibriovulnificus than of V. cholerae, according to the API 20E V4.0(Biomerieux) set of biochemical tests. However, the DNA sequencesof recA, gyrB, and dnaE from this strain were from 96 to 100%identical to those from V. cholerae and only from 84 to 85%identical to those from V. vulnificus.

Functional characterization of ICEVchMex1.

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Functional characterization of...

V. cholerae 1-010118-075 was found to be resistant to ampicillin(Ap^r) but not to other antibiotics. We wondered whether theAp^r phenotype was mediated by a self-transmissible ICE, ICEVchMex1.To explore this possibility, we used V. cholerae 1-010118-075(Ap^r) as a donor strain in conjugation experiments, with Escherichiacoli CAG18439 (Tc^r) (7) used as a recipient strain. However,no Tc^r Ap^r exconjugants were isolated from mating experimentscarried out as previously described (7), suggesting either thatICEVchMex1 does not carry the Ap^r gene or that the int⁺ V. cholerae1-010118-075 strain does not harbor a functional ICE.

We introduced a spectinomycin resistance (Sp^r) marker adjacentto mex02, an ICEVchMex1 gene that was not likely to influenceICEVchMex1 transmissibility (see below), to further test whetherICEVchMex1 was transmissible by conjugation. When a V. cholerae1-010118-075 Sp^r derivative was used as a donor in mating experimentswith CAG18439 as the recipient, Sp^r Tc^r exconjugants were obtainedat low frequencies but only when the donor strain was treatedwith mitomycin C, an agent known to promote SXT transfer (5)(Table 1) . Thus, ICEVchMex1 is transmissible (albeit at lowfrequencies) by conjugation from V. cholerae to E. coli. LikeSXT^MO10 (13), ICEVchMex1 was found to integrate into the 5'end of prfC (data not shown).

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TABLE 1. ICEVchMex1 is self-transmissible and its transfer is inducible by mitomycin C

The transfer frequency of ICEVchMex1 from CAG18439 to otherE. coli strains was higher than that observed from V. choleraeto E. coli and did not require mitomycin C treatment of thedonor for its detection (Table 1). None of 50 Sp^r Tc^r E. coliexconjugants tested was Ap^r, indicating that the Ap^r phenotypewas not transferred by conjugation and thus likely not mediatedby ICEVchMex1. Therefore, the native ICEVchMex1 harbored byV. cholerae 1-010118-075 does not seem to carry antibiotic resistancegenes.

There was more than an 80-fold induction of ICEVchMex1 conjugativetransfer from CAG18439 when the donor strain was treated withmitomycin C, a compound that damages DNA and induces the SOSresponse (Table 1). Mitomycin C is thought to induce SXT^MO10transfer because it promotes the RecA-dependent cleavage ofSetR, the repressor of SXT^MO10 transfer (5). We found that mitomycinC induction of ICEVchMex1 transfer was recA dependent (Table1) and that the predicted ICEVchMex1 SetR sequence is identicalto that in SXT^MO10. Together these observations suggest thatmitomycin C activates ICEVchMex1 transfer and SXT^MO10 transferby the same mechanism.

Genetic characterization.

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Genetic characterization.

To begin to assess the similarity of the ICEVchMex1 genome tothe SXT^MO10 and R391 genomes, regions of ICEVchMex1 were amplifiedwith PCR and then sequenced (Table 2 and Fig. 1). The sequencesof several ICEVchMex1 genes that are part of the functionalcore of SXT-related elements were very similar to those in SXT^MO10.For example, the predicted sequences of the ICEVchMex1 Int,Xis, and TraI proteins shared 99%, 100%, and 93% identity, respectively,with the Int, Xis, and TraI proteins encoded by SXT^MO10.

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TABLE 2. DNA sequences of the primers used in this study and sizes of the amplicons in SXT^MO10, R391, and ICEVchMex1

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FIG. 1. Schematic linear representation of the SXT^MO10 and R391 conserved set of genes and of the insertions specific to ICEVchMex1. The black line represents the set of genes conserved in SXT^MO10 and R391. The three hotspots for the insertion of additional DNA are indicated with stars. Gray arrows represent the conserved genes in SXT^MO10, R391, and ICEVchMex1. The scale for the insertions is 1 cm per kb. The genes that are specific to ICEVchMex1 are shown as white arrows. SXT^MO10 genes that are not present in ICEVchMex1 are shown as black arrows. The location of the antibiotic gene cluster in SXT^MO10 is shown as a hatched box (not to scale). The locations of primers used for analysis (Table 2) are shown as arrowheads. ${Delta}$ IS, remnant of insertion sequence.

Some features of ICEVchMex1 appear to be more closely relatedto R391 than to SXT^MO10. R391 contains a cluster of three genesof unknown function between attL and xis that is not found inSXT^MO10 (3, 6). This cluster is also present in ICEVchMex1.Also, the sequence of the ICEVchMex1 exclusion determinant,eexR3, was 94% identical to that of eexR from R391 and only78% identical to that of eexS from SXT^MO10, suggesting thatICEVchMex1 belongs to the R391 exclusion group (15).

No insertions of antibiotic resistance genes were found in ICEVchMex1at sites in which SXT-related ICEs are known to harbor suchinsertions. SXT^MO10, SXT^ET (12), and SXT^LAOS (14) carry antibioticresistance determinants in a composite transposon-like structuredisrupting rumB (12), but the rumAB operon is intact in ICEVchMex1(Fig. 1). The mer operon in R391, which is found between traGand eex, is absent from ICEVchMex1. The R391 kanamycin resistancegene is carried by a transposon inserted between s026 and s027(Fig. 1), but no amplicon could be detected for ICEVchMex1 byuse of primers designed to amplify this region. In fact, additionalPCR and DNA sequence analyses established that the entire regionfrom s027 to s040 is missing in ICEVchMex1 and replaced by acluster of four open reading frames (ORFs) and a likely pseudogeneoriented in the same direction as s026 and traI (Fig. 1). Thesefour genes, hsdM, hsdS, fdp1, and hsdR, and the pseudogene, ${Psi}$ mrr, are similar in sequence to and have the same organizationas a cluster of genes in Shewanella frigidimarina NCIMB400 thatencode a putative type I restriction and modification system.Thus, even though the 17.8-kb region from s027 to s040 is sharedby SXT^MO10 and R391 (3), the true conserved core set of genesthat defines the family of SXT-related elements is shorter thanthe common set of genes shared by SXT^MO10 and R391. This ideais consistent with our previous functional studies of SXT^MO10,which revealed that the gene cluster from s027 to s040 is notrequired for SXT^MO10 mobility or maintenance (4).

Comparison of the SXT^MO10 and R391 DNA sequences suggested thatthere are three "hotspots" for the insertion of additional DNAsequences within intergenic regions from s043 to traL, fromtraA to s054, and from s073 to traF, which separate conservedgenes (3) (Fig. 1). The sequences found in these three regionsin ICEVchMex1 were unrelated to those found in SXT^MO10 and R391.In ICEVchMex1, the intergenic regions from s043 to traL andfrom traA to s054 each contain a unique ORF, mex01 and mex02,respectively, coding for putative proteins of unknown functions.The intergenic region from s073 to traF contains two ORFs, s075and s074, which are present in SXT^MO10, as well as three additionalORFs of unknown function, i.e., mex03, mex04, and mex05, andthe remnants of an insertion sequence (Fig. 1). These observationssuggest that the sites serving as hotspots for the insertionof additional DNA sequences into the core set of SXT genes areconserved in SXT-related ICEs.

Conclusions.

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Conclusions.

ICEVchMex1 is the first SXT-related ICE detected in the WesternHemisphere. Like SXT^MO10, ICEVchMex1 is self-transmissible andintegrates into prfC. ICEVchMex1 appears to share the core setof genes that mediate the transmission, maintenance, and regulationof SXT-related ICEs; however, it also carries unique and novelinsertions that illustrate the remarkable plasticity of thegenomes of SXT-related ICEs. The absence of antibiotic resistancedeterminants in ICEVchMex1 suggests that SXT-related ICEs likelyconfer advantages unrelated to resistance to antibiotics totheir hosts.

Molecular epidemiological studies suggest that SXT^MO10 and veryclosely related ICEs became widespread in Asian and AfricanV. cholerae populations only after the emergence of V. choleraeO139 in 1992 (2, 11). The major differences in the genomes ofICEVchMex1 and SXT^MO10 (Fig. 1) indicate that these two ICEshave evolved independently; clearly, ICEVchMex1 is not immediatelyderived from the Asian V. cholerae O139 SXT element. The acquisitionof antibiotic resistance determinants by an ICE such as ICEVchMex1,which lacks such genes, along with the presence of antibioticsin the environment, presumably has catalyzed the spread of SXT-relatedICEs in V. cholerae populations.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

The accession numbers of the ICEVchMex1 sequences describedherein were submitted to GenBank and have been assigned accessionnumbers DQ180349 (setR to int on the circular form), DQ180350(rumB to traD), DQ180351 (s043 to traL), DQ180352 (s073 to traF),and DQ180353 (traA to s054).

ACKNOWLEDGMENTS

This work was supported by grants from the NIH, the Howard HughesMedical Institute, and the NEMC GRASP Center. R.Q.-C. receivedsupport from SIHGO (20000202015) and CONACYT (ER026), Mexico.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Jaharis 425, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2730. Fax: (617) 636-2723. E-mail: matthew.waldor{at}tufts.edu.

REFERENCES

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