Comparative Analysis of Mobilizable Genomic Islands

Aurélie Daccord, Daniela Ceccarelli,* Sébastien Rodrigue, Vincent Burrus

Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada

Mobilizable genomic islands (MGIs) are small genomic islands of less than 35 kbp containing an integrase gene and a sequencethat resembles the origin of transfer (oriT) of an integrating conjugative element (ICE). MGIs have been shown to site-specifi-cally integrate and excise from the chromosome of bacterial hosts and hijack the conjugative machinery of a coresident ICE todisseminate. To date, MGIs have been described in three strains belonging to three different Vibrio species. In this study, we re-port the discovery of 11 additional putative MGIs found in various species of Vibrio, Alteromonas, Pseudoalteromonas, andMethylophaga. We designed an MGI capture system that allowed us to relocate chromosomal MGIs onto a low-copy-numberplasmid and facilitate their isolation and sequencing. Comparative genomics and phylogenetic analyses of these mobile geneticelements revealed their mosaic structure and their evolution through recombination and acquisition of exogenous DNA. MGIswere found to belong to a larger family of genomic islands (GIs) sharing a similar integrase gene and often integrated into thesame integration site yet exhibiting a different mechanism of regulation of excision and mobilization. We found that MGIs canexcise only when an ICE of the SXT/R391 family is coresident in the same cell, while GIs still excise regardless.

The SXT/R391 family of integrating conjugative elements(ICEs) is a well-studied family of self-transmissible mobile ge-

netic elements responsible for the dissemination of antibiotic re-sistance genes in clinical and environmental vibrios and relatedgammaproteobacteria, such as Providencia rettgeri, Shewanellaputrefaciens, and Photobacterium damselae (1–3). This family cur-rently includes more than 30 members (4, 5) that share a set of 52conserved genes and the same chromosomal integration site, the5= end of prfC, which encodes peptide chain release factor 3. EachICE of this family also contains variable DNA inserted in specificpositions of the backbone (hot spots) and encoding various func-tions, such as resistance to antibiotics or heavy metals.

Mobilizable genomic islands (MGIs) are small genomic islands(approximately 20 kpb) that rely on an unusual mechanism totransfer at high frequency from one cell to another (6). MGIs carrya conserved sequence that mimics the origin of transfer (oriT) ofSXT/R391 ICEs and utilize both the DNA processing and conju-gative apparatus encoded by these ICEs to transfer to a new recip-ient cell. To date, three MGIs have been described, each in a dif-ferent Vibrio species: MGIVvuTai1 and MGIVflInd1 in the clinicalisolates Vibrio vulnificus YJ016 and Vibrio fluvialis H-08942, re-spectively, and MGIVchUSA1 in the environmental isolate Vibriocholerae RC385 (Table 1). All three MGIs share the same integra-tion site, i.e., the 3= end of a gene encoding a putative stress-induced protein named yicC in Escherichia coli (6). Besides theoriTSXT-like sequence, the three MGIs also share four conservedgenes: intMGI, which encodes a tyrosine recombinase responsiblefor MGI excision and integration; rdfM, which encodes a recom-bination directionality factor necessary for excision; and two pu-tative genes of unknown function (cds4 and cds8) (6, 19).MGIVchUSA1 encodes two different putative toxin-antitoxin sys-tems related to the HipA/B system of E. coli, a system that has beenshown to promote bacterial multidrug tolerance (20). These tox-in-antitoxin systems could also improve the stability of thisMGIVchUSA1 by acting as postsegregational killing systems (21).MGIVvuTai1 encodes a type I restriction modification system thatcould protect its host from bacteriophage infection (22). Potentialselective advantages conferred by MGIVflInd1 are unknown, as itswhole sequence is not available to date (6).

Unlike the vast majority of genomic islands (GIs) for which themechanisms of dissemination remain unclear, the regulation ofMGI excision, transfer, and integration has recently been eluci-dated (6, 19). In the absence of an SXT/R391 ICE, an MGI inte-grated into the chromosome of its host does not excise. If a cores-ident SXT/R391 ICE is present, the ICE-encoded transcriptionalactivator SetCD triggers the expression of both rdfM and intMGI topromote MGI site-specific excision (19). Once the MGI is excisedas a circular covalently closed molecule, oriTMGI is recognized bythe ICE-encoded relaxase to translocate the MGI into the recipientcell as a single-stranded DNA molecule through the mating bridgeencoded by the ICE (6). Mobilization of the MGI to the recipientcell is independent of the cotransfer of the ICE (6, 19). Further-more, MGI integration into the 3= end of yicC has been shown tobe mediated by IntMGI, which is expressed at low levels in a SetCD-independent manner in the recipient cell (19). Besides promotinggenomic plasticity through their own mobilization, MGIs can alsomobilize chromosomal DNA located 5= of their integration site.Chromosomal DNA mobilization is driven by the SXT/R391 ICEthrough the recognition of the oriTSXT-like sequence on the MGI.This process can lead to the mobilization of at least 1 Mbp ofchromosomal DNA (6).

Here, we report the characterization of 11 additional putativeMGIs in the genome of various gammaproteobacteria. Compara-tive genomics and phylogenetic analyses of these elements re-vealed their mosaic structure and their evolution through recom-bination and acquisition of exogenous DNA. MGIs were found to

Received 15 October 2012 Accepted 22 November 2012

Published ahead of print 30 November 2012

Address correspondence to Vincent Burrus, Vincent.Burrus@USherbrooke.ca.

* Present address: Daniela Ceccarelli, Maryland Pathogen Research Institute,University of Maryland, College Park, Maryland, USA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01985-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.01985-12

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belong to a larger family of GIs sharing a similar integrase gene andoften integrated into the same integration site, yet exhibiting adifferent mechanism of regulation of excision and mobilization.

MATERIALS AND METHODSBacterial strains. The bacterial strains and plasmids used in this study aredescribed in Table 1. The strains were routinely grown in Luria-Bertani(LB) broth at 37°C in an orbital shaker/incubator and were maintained at�80°C in LB broth containing 15% (vol/vol) glycerol. Alteromonasmacleodii Deep ecotype was grown in marine broth (Difco, BD). Antibi-otics were used at the following concentrations: ampicillin (Ap), 100 �g/ml; chloramphenicol (Cm), 20 �g/ml; erythromycin (Em), 200 �g/ml;kanamycin (Kn), 50 �g/ml; spectinomycin (Sp), 50 �g/ml; tetracycline(Tc), 12 �g/ml.

Bacterial conjugation. Conjugation assays were used to transferMGIVflInd1 and MGIVchMoz6 into AD175 (intermediate) and AD157(recipient). Mating assays were carried out by mixing equal volumes of7-h-grown cultures of donor, intermediate, and recipient strains. The cellswere harvested by centrifugation for 5 min at 5,500 � g and resuspendedin a 1/20 volume of LB broth. Cell suspensions were poured onto LB agarplates and incubated at 37°C for 15 h. The cells were then resuspended in1 ml of LB medium, and serial dilutions were plated onto the appropriateselective media to determine the number of donors, intermediates, recip-ients, and exconjugants. Exconjugants were purified and tested by PCRfor the presence of the MGI.

Construction of plasmids and strains. The plasmids used in thisstudy are described in Table 1. Plasmid pVB201 was constructed bycloning the MGI attB site from V. cholerae N16961 (attBVchN16961) intothe HindIII restriction site of pBeloBac11 (New England BioLabs).attBVchN16961 was amplified using the primer pair attB-F/attB-R (see Ta-ble S1 in the supplemental material) and genomic DNA of V. choleraeN16961 as a template. Deletion of yicC in AD175 and AD157 was con-structed by using the one-step chromosomal gene inactivation technique(23) using primer pair attB-WF/attB-WR (see Table S1) and pVI36 as a

template. Deletion of dapA was introduced in MG1655 by P1vir transduc-tion of �dapA::(erm-pir) from E. coli �2163 into E. coli AD171 as therecipient.

Molecular biology techniques. All the enzymes were used accordingto the manufacturer’s instructions (New England BioLabs). Plasmid DNAwas prepared with a QIAprep spin miniprep or QIAfilter plasmid midikit(Qiagen), and chromosomal DNA was prepared with a Wizard genomicDNA purification kit (Promega) as described in the manufacturer’s in-structions. Colony blotting was performed as previously described (6).PCR assays were carried out in 50-�l PCR mixtures with 1 U of Taq DNApolymerase (New England BioLabs). The PCR conditions were as follows:(i) 3 min at 94°C; (ii) 30 cycles of 30 s at 94°C, 30 s at a suitable annealingtemperature, and 30 s to 60 s at 72°C; and (iii) 5 min at 72°C. Whenneeded, PCR products were purified using a QIAquick PCR purificationkit (Qiagen) according to the manufacturer’s instructions. E. coli wastransformed by electroporation according to Dower et al. (24), using aBio-Rad GenePulser Xcell apparatus set at 25 �F, 200 �, and 1.8 kV.

Nested PCR experiments on MGIs were performed as previously de-scribed (6). For the related GIs in Shewanella sp. W3-18-1 and Vibriomimicus VM573, the same method was used with primers attPSpu-L1/attPSpu-R1 and attPSpu-L2/attPSpu-R2 and attPVmi-L1/attPVmi-R1and attPVmi-L2/attPVmi-R2, respectively (see Table S1 in the supple-mental material).

Sequencing. MGIVflInd1 (GenBank accession no. KC117176) andMGIVchMoz6 (GenBank accession no. KC117175) were captured inpVB201, and the resulting plasmids were grown in E. coli. The DNA wasextracted, and Illumina sequencing libraries were prepared as describedpreviously (25) with the following modifications. The DNA was shearedwith 1 unit of double-stranded DNA (dsDNA) shearase (ZymoResearch)at 37°C for 60 min and purified using AMPureXP beads (sheared DNA-to-beads [vol/vol] ratio of 0.7) before proceeding to Illumina adapterligation. DNA inserts had an average size of approximately 500 bp. Librar-ies were barcoded and mixed with additional samples and sequenced onan HiSeq2000 instrument at the McGill University and Genome Quebec

TABLE 1 Strains and plasmids used in this study

Strain or plasmid Relevant genotype or phenotypea Reference

StrainsEscherichia coli K12

CAG18439 MG1655 lacZU118 lacI42::Tn10 (Tcr) 7AD157 CAG18439 �yicC (Tcr) This studyAD171 MG1655 �yicC prfC::R997 pVB201 (Apr Cmr) This studyAD175 MG1655 �yicC �dapA::(erm-pir) prfC::R997 pVB201 (Emr Apr Cmr) This study�2163 (F�) RP4-2-Tc::Mu �dapA::(erm-pir) (Knr Emr) 8

Alteromonas macleodii Deep ecotype MGIAmaMed1, environmental, 2002, Mediterranean Sea 9Shewanella sp. W3-18-1 ICESpuPO1 GISpuPO1, environmental, Pacific Ocean 10Vibrio cholerae

N16961 O1 El Tor 11RC385 MGIVchUSA1, O135, environmental, 2005, USA 123AMOZ ICEVchMoz2 MGIVchMoz2, environmental, 2002, Mozambique 137AMOZ ICEVchMoz3 MGIVchMoz3, environmental, 2002, Mozambique 138AMOZ ICEVchMoz4 MGIVchMoz4, environmental, 2002, Mozambique 1316AMOZ ICEVchMoz6 MGIVchMoz6, environmental, 2003, Mozambique 13

Vibrio fluvialis H-08942 ICEVflInd1 MGIVflInd1, clinical, 2002, India 14Vibrio mimicus VM573 GIVmiUSA1, clinical, 1990, United States 15Vibrio parahaemolyticus 21AMOZ ICEVpaMoz1 MGIVpaMoz1, environmental, 2002, Mozambique 13Vibrio vulnificus YJ016 MGIVvuTai1, clinical, 1992, Taiwan 16

PlasmidspBeloBac11 Cmr 17pVB201 pBeloBac11::attBVchN16961 (Cmr) This studypVI36 aad7 (Spr), PCR template for one-step chromosomal gene inactivation 18

Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Emr, erythromycin resistant; Knr, kanamycin resistant; Spr, spectinomycin resistant; Tcr, tetracycline resistant.

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Innovation Centre. Paired-end reads of 100 bp were generated and de-multiplexed using Novobarcode. MGIs were assembled using the RochegsAssembler version 2.5 software. Gene detection and annotations werecarried out using BLAST software (26) and databases available fromNCBI.

Bioinformatics analyses. Phylogenetic trees were generated using thePhyML version 3.0 program (27) with the LG substitution model forthe protein sequence-based tree and the HKY85 substitution model forthe DNA sequence-based tree. Tree topologies were optimized by PhyMLusing both the nearest-neighbor interchange (NNI) and subtree pruningand regrafting (SPR) methods, and the starting tree was estimated usingBioNJ. Branch support of the phylogenies was estimated using nonpara-metric bootstrap (100 replicates). Phylogenetic analyses were computedfrom reliable amino acid or DNA alignments built by MUSCLE multiplesequence alignment software (28). Removal of poorly aligned regionsfrom amino acid alignments was carried out by trimAl version 1.2 soft-ware using the automated heuristic approach (29) prior to phylogeneticanalyses. Phylogenetic trees were viewed using iTOL version 2 (30).

RESULTS AND DISCUSSIONFive environmental Vibrio strains from Mozambique harborputative MGIs. To estimate the prevalence of MGIs in vibrios andother bacterial species, we screened by Southern blotting hybrid-ization a large collection of V. cholerae (O1 and non-O1, non-O139), Vibrio parahaemolyticus, V. vulnificus, V. fluvialis, Vibriometschnikovii, P. rettgeri, Providencia stuartii, Providencia alcalifa-ciens, E. coli, Pseudomonas aeruginosa, Salmonella, Aeromonascaviae, and Aeromonas hydrophila. Among 149 strains analyzedwere clinical and environmental strains isolated between 1992 and2004 in Italy, Vietnam, India, Bangladesh, Taiwan, Mexico, An-gola, Mozambique, Rwanda, Somalia, Swaziland, and Zimbabwe.Using intMGI as a probe, five additional related MGIs were identi-fied in Mozambican environmental strains of V. parahaemolyticus

(21AMOZ) and V. cholerae (3AMOZ, 7AMOZ, 8AMOZ, and16AMOZ) (Table 2).

Based on PCR amplification and DNA sequencing, we foundthat these strains also contain the oriTSXT-like sequence, cds4, cds8,and rdfM and are integrated into the 3= end of the same orthologueof E. coli’s yicC. These results suggest that these strains harborMGI-like elements. Interestingly, all of them also contain an ICEof the SXT/R391 family (Table 1), suggesting that the putativeMGIs could be mobilized at relatively high frequency.

Design of an MGI capture system and MGI sequencing. Theidentification of five putative MGIs in environmental vibrios iso-lated from the African continent was a great opportunity to take alook at the diversity and variability of MGI structures and se-quences. However, since MGIs usually are integrated into thechromosome, excise at low frequency, and do not seem to repli-cate autonomously, extraction of MGI DNA using standard plas-mid preparation protocols was not achievable. To avoid this prob-lem, we devised a strategy allowing us to capture complete MGIson a low-copy-number vector (pVB201) more suitable for DNAextraction and sequencing. pVB201 is a derivative of pBeloBac11(17), a single-copy E. coli plasmid that allows the cloning andstable maintenance of large DNA fragments. pBeloBac11 wasmodified to include a 400-bp fragment that encompasses the MGIattachment site (attB). This modification enabled IntMGI-cata-lyzed site-specific recombination between attB on pVB201 andattP on an excised and transferred MGI to drive MGI capture andretransfer to a recipient strain (Fig. 1). This strategy was derivedfrom a similar approach developed by Wozniak et al. (4) to cap-ture SXT/R391 ICEs, yet it employs a triparental mating due to thelack of known selectable markers on the putative MGIs to be re-covered. Conjugation was carried out between a Vibrio MGI�

TABLE 2 Properties of MGI-like elements

Name Host strain Type, yr, and site of isolation% identity tointMGIVchUSA1

SXT/R391ICE

GenBankaccession no. Reference

MGIVchUSA1 V. cholerae RC385 Environmental, 2005,United States

100 ZP_06943583.1 12

MGIVvuTai1 V. vulnificus YJ016 Clinical, 1992, Taiwan 98.8 NP_933070.1 16MGIPspArc1 Pseudoalteromonas sp. BSi20311 Environmental, 2009, Arctic Sea 98.3 ZP_09227437.1 31MGIVflInd1 V. fluvialis H-08942 Clinical, 2002, India 97.4 ICEVflInd1 KC117176 14MGIVchHai4 V. cholerae HE45 Gray Water, External Clinic,

2010, Haiti96.9 EJH66094.1 32

GIVchHai3 V. cholerae HE-09 Environmental, 2010, Haiti 96.9 EGS63339.1 32MGIAmaMed1 A. macleodii Deep ecotype Environmental, 2002,

Mediterranean Sea90.9 YP_004427371.1 9

MGIVchMoz6 V. cholerae 16AMOZ Environmental, 2003,Mozambique

88.5 ICEVchMoz6 KC117175 13

MGIVchHai1 V. cholerae HE39 Environmental, 2010, Haiti 87.8 EGR03948.1 32MGIAspKor1 Alteromonas sp. SN2 Environmental, 2007, Korea 83.5 YP_004469681.1 33MGIMamKor1 Methylophaga aminisulfidivorans MP Environmental, 2007, Korea 83.1 ZP_08537443.1 34MGIVchHai5 V. cholerae HC-43B1 Clinical, 2010, Haiti 82.3 ALDP00000000.1 32MGIVchHai2 V. cholerae HE48 Environmental, 2010, Haiti 76.3 EGR10536.1 32MGIVchMoz2 V. cholerae 3AMOZ Environmental, 2002,

MozambiqueICEVchMoz2 13

MGIVchMoz3 V. cholerae 7AMOZ Environmental, 2002,Mozambique

ICEVchMoz3 13

MGIVchMoz4 V. cholerae 8AMOZ Environmental, 2002,Mozambique

ICEVchMoz4 13

MGIVpaMoz1 V. parahaemolyticus 21AMOZ Environmental, 2002,Mozambique

ICEVpaMoz1 13

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ICE� donor strain and an E. coli intermediate recipient strain(AD175) lacking dapA as well as yicC (and thus chromosomalattB) but harboring pVB201 and R997, the latter being an ICE ofthe SXT/R391 family conferring resistance to ampicillin. The pro-cedure yielded exconjugant cells containing the transferred MGIintegrated into pVB201. The resulting plasmid containing theMGI becomes mobilizable by R997 and can thus be transferredinto the final E. coli recipient strain also lacking yicC. Exconjugantcolonies containing the transferred MGI integrated into pVB201were selected using the Cmr antibiotic resistance marker presenton the plasmid as well as the Tcr marker of the final E. coli recipientstrain (AD157). The �dapA mutation ensured the absence ofAD175 exconjugants on media not supplemented with diamino-pimelic acid (DAP), a phenomenon commonly observed in ourinitial attempts that we attributed to the Hfr-like mobilization ofthe Tcr marker from AD157 back into AD175 mediated by R997(data not shown). Using this capture system, MGIVflInd1 of V.fluvialis H-08942 and MGIVchMoz6 of V. cholerae 16AMOZ re-located into pVB201, with the help of the coresident ICEsICEVflInd1 (4, 14) and ICEVchMoz6 (13), respectively. The re-sulting plasmids were completely sequenced and assembled. An-notation of the sequences led to the identification of 18 open read-ing frames (ORFs) in the 23,229-bp MGIVflInd1 and 17 ORFs inthe 19,740-bp MGIVchMoz6.

The MGI family expands and is not restricted to Vibriostrains. In addition to the MGIs we identified in our strain collec-tion and sequenced, BLAST analysis of IntMGIVchUSA1 against theGenBank database (release 191.0) revealed the presence of 8 newMGIs in different environmental strains of V. cholerae (HE-39,HE-45, HE-48, and HC-43B1), in Alteromonas macleodii Deepecotype, in Alteromonas sp. SN2, in Pseudoalteromonas sp.

BSi20311, and in Methylophaga aminisulfidivorans MP (see Ta-ble 2 for strain details).

All of these new MGIs share the same conserved features thatcharacterize the family, are integrated into the same site, and ex-hibit variable DNA regions (Fig. 2A). Interestingly, out of 12 se-

quenced MGIs, as many as 9 different structures emerge based onthe gene content and orientation in each variable region. Only twostructures appear in more than one MGI. The first pattern is ob-served for MGIVflInd1 and MGIVchHai4 found in the Indian V.fluvialis H-08942 and in the Haitian V. cholerae HE45 isolates,respectively. The second conserved pattern is observed forMGIVchMoz6, MGIVchHai2, and MGIVchHai5 found in threeenvironmental V. cholerae strains, respectively, isolated from en-vironmental or clinical sources in Mozambique and Haiti. There-fore, the structure of MGIs gives us crucial clues on strain circu-lation. Indeed, MGIVflInd1 and MGIVchHai4, isolated in twoopposite parts of the globe (India and Haiti, respectively) have thesame exact gene content and orientation in each variable region. Itis also the case for MGIVchMoz6, isolated in Mozambique, andMGIVchHai2 and MGIVchHai5, isolated in Haiti.

Although the overall genetic diversity of MGIs is rather limited,strains isolated from the same geographical area harbor MGIsexhibiting rather dissimilar variable regions. Out of five Haitian V.cholerae strains collected in 2010 (32), three contain differentMGIs (MGIVchHai1, MGIVchHai2, and MGIVchHai4), and afourth contains an atypical GI, GIVchHai3, which lacks cds8,rdfM, as well as part of oriTMGI (repeats R1, R2, and R3 are miss-ing) and is likely no longer mobilizable by SXT/R391 ICEs (Fig. 2Aand B, Table 2). This diversity in a small group of strains isolatedduring the same period and in the same area suggests frequentexchange of MGIs between strains or frequent recombinationevents between MGIs or with other mobile genetic elements in theenvironment.

Our results support the idea that the MGI family is not re-stricted to vibrios but still appears to be limited to gammaproteo-bacteria, and more precisely to marine bacteria. Indeed, the mostdivergent elements of the family, MGIAmaMed1, MGIAspKor1,and MGIPspArc1, were all found in marine strains of the generaAlteromonas and Pseudoalteromonas (Mediterranean, Yellow, andArctic seas) (see Fig. 2A and Table 2).

MGI genetic content. The sizes of MGIs vary from 18,500 bp to32,493 bp. Based on the analysis of 13 fully and 4 partially se-quenced MGIs, we observe that with the exception of the atypicalGIVchHai3, the core structure of MGIs appears to be restricted tooriTMGI and 4 conserved genes, intMGI, cds4, cds8, and rdfM. Wepreviously showed that IntMGI mediates the site-specific integra-tion and excision of MGIs into and from the chromosome of theirhost (6, 19). RdfM is required only for the excision process (19).To date, the functions of cds4 and cds8 remain unknown. Com-parison of the different oriTMGI sequences revealed the conserva-tion of the imperfect direct and inverted repeats that have beenpreviously identified in oriTSXT (18) (see Fig. S1 in the supplementalmaterial). Such structures likely play a role in the specific recognitionof the oriT by the putative relaxase TraI and/or auxiliary proteinsduring conjugative transfer initiation. This observation suggests thatthe oriTs of these newly identified MGIs are functional, which is ac-tually supported by the result of our MGI capture approach describedabove for MGIVflInd1 and for MGIVchMoz6. Interestingly, thisalignment also highlights two additional regions almost perfectlyconserved among SXT and MGIs (see Fig. S1, positions 53 to 67 and78 to 90). Given their conservation, these regions might also play animportant role in transfer initiation for both ICEs and MGIs.

Three variable regions can be observed in the sequenced MGIs,and to date each MGI exhibits the three regions, with differentgenetic contents from one MGI to the other (Fig. 2A). The genetic

FIG 1 MGI capture system. Conjugation between a donor strain bearing anICE and an MGI with an intermediate strain (AD175) bearing R997 (1) butlacking attB and dapA yields exconjugants that contain the transferred MGIintegrated into pVB201. These exconjugants cannot be directly selected be-cause of the lack of a selective marker on the MGI; yet the resulting plasmidbecomes mobilizable by R997 and transfer to a �attB recipient strain (2),yielding exconjugants harboring pVB201::MGI (3) that can be selected usingthe chloramphenicol marker present on pVB201 and the ability of the recipi-ent strain (AD157) to grow in the presence of tetracycline and in the absence ofdiaminopimelic acid (dap).

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content of each region is described in Table S2 in the supplementalmaterial. The first variable region is inserted between intMGI andoriT. In this locus, MGIs often contain cds15, cds1, cds2, and cds3.MGIAmaMed1 and MGIMamKor1 harbor a putative toxin-anti-toxin system (orf3/orf2) also found in ICEs R391 and ICEVch-Mex1 (4). This system is related to the E. coli toxin-antitoxinsystem hipA-hipB, which mediates multidrug tolerance (20). orf3-orf2 could also promote MGI maintenance in a manner similar tothat of the MosAT system recently identified in the ICE SXT (35).

The second variable region, located between cds4 and cds8,appears to be the most variable one. In all but one MGI, putativeR/M systems are found in this region. Two of them are type Ihost-specific defense (hsd) systems. The hsdRMS1 system is foundin MGIVvuTai1, MGIVflInd1, MGIVchHai4, MGIVchHai1,MGIAmaMed1, and MGIAspKor1. The hsdRMS2 system is foundin MGIPspArc1 only. At the same locus, MGIMamKor1,MGIVchMoz6, MGIVchHai2, and MGIVchHai5 harbor a putative

type III R/M system which is composed of two genes encoding amethyltransferase (met) and a restriction endonuclease (res). R/Msystems allow bacteria to distinguish invading foreign DNA fromtheir own genome, conferring resistance to infection by bacterio-phages. The prevalence of R/M systems in MGIs could be stronglyselected by the pressure exerted by bacteriophages in aquatic en-vironments (36, 37) and confer a selective benefit to strains har-boring such elements. Interestingly, MGIVchHai1 also harbors invariable region 2 a transposon-like structure flanked by multipleputative transposases and encoding a putative diguanylate cy-clase/phosphodiesterase. These enzymes are involved in the syn-thesis and degradation of the second messenger c-di-GMP, whichis known to increase biofilm formation and decrease motility in V.cholerae (38, 39). c-di-GMP is also considered an important playerin the transition of V. cholerae from a host to persistence in theenvironment (38, 40). Interestingly, this is not the first report of adiguanylate cyclase/phosphodiesterase encoded on a mobile ge-

FIG 2 General structures of MGIs and phylogenetic analyses of the core genes. (A) Genetic structure of 12 sequenced MGIs; (B) atypical structure of GIVchHai3,from Vibrio cholerae H-09; (C) genetic content of GISpuPO1 and GIVmiUSA1 from Shewanella sp. W3-18-1 and Vibrio mimicus VM573, respectively. Colors ofthe predicted open reading frames indicate the following putative functions: pink and red, DNA replication, recombination, and repair; purple, diguanylatecyclase/phosphodiesterase; blue, transcription; green, restriction modification systems; orange, putative HipA-like toxin; gray, unknown function. See Table S2in the supplemental material for predictions of putative function of genes identified in variable regions.

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netic element. Two diguanylate cyclases have been previously de-scribed in ICEs of the SXT/R391 family (41). The presence of sucha gene in MGIVchHai1 could alter the pathway regulating thetransition from free-living, motile to biofilm lifestyle of its host,which may provide a potential benefit in specific environmentalconditions.

The third variable region is located between rdfM and attR (Fig.2A). The most common features observed in this region are twodistinct toxin-antitoxin systems (cds10-cds22 and cds13-cds14), allrelated to E. coli hipA-hipB.

Phylogenetic analysis of the MGI backbone. In order to ex-plore the evolution of the conserved backbone, we constructedphylogenetic trees based on nucleotide sequences of the differentcore regions, including the one containing oriT (Fig. 3). UnlikeSXT/R391 ICEs, MGIs have a small core set of conserved se-quences, allowing us to construct phylogenetic trees for each re-gion.

Interestingly, each region generated distinct branching pat-terns, highlighting tree incongruences. Although some MGIsalways cluster together, as it is the case for MGIVchHai1,MGIVchHai4, and MGIVflInd1, others tend to cluster in verydifferent groups depending on the analyzed region. The most strik-ing example is MGIVchMoz6, which has an int gene almost identical

to the one of MGIVflInd1 but clusters with MGIVchHai2 for oriT-cds4 and cds8-rdfM regions (Fig. 3). Similar observations can also bemade for MGIVchHai5, which clusters with MGIVchHai2 for int andoriT-cds4 yet has a more distantly related cds8-rdfM region. Such treeincongruences likely reflect a model of evolution of MGIs by inter-MGI recombination events.

A closer look at the distribution of variable genes among theMGIs also suggests evidence of recombination in the evolutionalhistory of MGIs. The vast majority of the variable genes are alwaysfound in more than one MGI. For example MGIVchUSA1,MGIVflInd1, MGIPspArc1, and MGIVchMoz6 have the same ge-netic content in variable region 1 (Fig. 2A); however, the sameMGIs exhibit very different genetic contents in variable regions 2and 3. The genetic variation between the variable DNA of MGIsindicate that these elements are mosaics and again supports theidea that intra-MGI recombination is an important driver of theirevolution in a manner similar to that of the SXT/R391 ICEs (4). Itwas previously shown that when two SXT/R391 ICEs are presentin a cell, they can form tandem arrays by integrating into the samesite (42) and recombine to form hybrids using either the hostprotein RecA or a distant relative of the � Red recombinationsystem that they encode (43, 44). This phenomenon enhancestheir diversity and drives their evolution. MGIs do not seem tocode for putative homologous recombination functions. How-ever, RecA could be responsible for MGI recombination, and theinvolvement of the ICE-encoded recombination system is alsolikely. In the future, it would be interesting to study the ability ofMGIs to form tandems and verify whether some MGIs excludeothers, as it was reported for the SXT/R391 ICEs (45, 46).

MGIs are part of a larger family of GIs found in various gen-era. The strong conservation of int among MGIs is an interestingtool to search genomic databases for additional related GIs. Todate, proteins that share at least 80% identity with IntMGIVchUSA1

have systematically revealed an MGI. However, using this methodto screen for new MGIs, we also found many putative integrasesrelated to IntMGI but falling below the 80% threshold. We won-dered whether the genes coding for these integrases were part ofGIs that could share an evolution history with MGIs. The 31closest integrases (sharing between 69% and 41% identity withIntMGIVchUSA1; see Table S3 in the supplemental material) wereincluded in our analysis. We found that they were encoded by GIsoften integrated into the same integration site as the MGIs, as 25GIs out of 31 (81%) are integrated into a gene coding for aputative stress-induced protein related to yicC of E. coli. We con-structed phylogenetic trees based on the translation products ofthese putative int genes or on the nucleotide sequence of rpoB foreach of these strains and all the strains bearing an MGI (Fig. 4).Nucleotide sequence of rpoB was chosen to build the tree to pro-vide a better resolution at the species level, while the amino acidsequence of int genes was favored due to the well-known rapiddivergence of the nucleotide sequence of genes encoding tyrosinerecombinases (47, 48). We found that MGIs form a distinct clusterbased on the integrases, even when they originate from the mostdistant species based on rpoB phylogeny (e.g., MGIPspArc1 andMGIVchHai2). This observation suggests frequent loss and hori-zontal acquisition of MGI by these strains. These results were ex-pected considering the mode of transmission of MGIs and therelative high transfer frequency that we previously observed forMGIVflInd1 (6). Interestingly, only MGIs harbor an oriT se-quence and cds4 as well as cds8 and rdfM. None of the GIs encod-

FIG 3 Phylogenetic analysis of the MGI backbone. Phylogenetic trees basedon the nucleotide sequences of the indicated core genes or region. Bootstrapvalues are indicated when over 80%. The individual scale bars represent ge-netic distances.

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ing a related integrase that clusters outside the MGI clade harbor asimilar structure. This strongly suggests that the related GIs arenot mobilizable by ICEs of the SXT/R391 family. However, asMGIs and their related GIs share the same integration site, MGIsmight form tandem arrays with these related GIs. Then recombi-nation events could lead to the acquisition of new genes by MGIs.As such, these related GIs could constitute a significant pool ofadaptive genes available for transfer by SXT/R391 ICEs.

Excision of putative MGIs and related GIs. Besides the con-servation of oriT in all the putative MGIs identified to date, an-other way to confirm their functionality as mobile elements is to

verify that they can excise in the presence of an SXT/R391 ICE. Wetested the 5 Mozambican MGI� strains by nested PCR to amplifyattP sequences resulting from MGIs’ excision (Fig. 5A). Two dif-ferent sets of primers were used due to the polymorphism of theregion located on the left side of attR (see Table S1 in the supple-mental material). We found that each one of these strains, whichall contain an SXT/R391 ICE, yielded a PCR product of expectedsize (Fig. 5A, lanes 1 to 5 and 7). Sequence analysis of two of theamplicons confirmed the excision event (see Fig. S2 in the supple-mental material), indicating that these MGIs are able to form cir-cular intermediates. Surprisingly, MGIAmaMed1, from A. ma-

FIG 4 Phylogenetic analysis of MGIs and related GIs. (A) Tree based on protein sequences of Int from SXT, MGIs, and 31 related GIs; (B) phylogenetic tree basedon the nucleotide sequences of the rpoB gene from the corresponding strains. Bootstrap values are indicated when over 80%. The individual scale bars representgenetic distances. Refer to Table S3 in the supplemental material for GI and strain details.

FIG 5 Excision of MGIs and related GIs. (A) Ethidium bromide-stained 1.5% agarose gels of PCR products amplified by nested PCR assays for detection of theexcision of the MGIs and related GIs. Lanes: M, 2-log DNA ladder; 1, MGIVpaMoz1; 2, MGIVchMoz2; 3, MGIVchMoz3; 4, MGIVchMoz4; 5, MGIVchMoz6; 6and 7, MGIAmaMed1; 8, GIVmiUSA1 from Vibrio mimicus VM573; 9, GISpuPO1 from Shewanella sp. W3-18-1. Lane 7, coresident R391 was present in the cells.(B) Model of excision/integration of MGIAmaMed1 by site-specific recombination (solid crossover) and by homologous recombination in the absence of R391(dotted crossover). Refer to Fig. S3 in the supplemental material for details about the alignment of MGIAmaMed1 attachment sites.

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cleodii Deep ecotype, an ICE-free strain, was able to excise,yielding a weak PCR product of the expected size (Fig. 5A, lane 6).This result was in contradiction with our previous observationsthat demonstrated the requirement for SetCD, the main activatorof SXT/R391 ICEs, to trigger MGI excision (6, 19). However, se-quence analysis of this specific amplicon revealed that excisionoccurred by homologous rather than site-specific recombination(Fig. 5B). In fact, unlike other MGIs, MGIAmaMed1 harbors a180-bp repeated region on its internal left side and on the rightside of its integration site (highlighted in orange in Fig. S3 in thesupplemental material); this repeated region seems to be longenough to allow homologous recombination to occur. In order toverify whether despite this particular mechanism of excisionMGIAmaMed1 was still able to respond to induction by SetCD,we introduced R391, the other prototypical member of the SXT/R391 family originally from P. rettgeri, into A. macleodii Deepecotype and repeated the same PCR experiment on an R391�

exconjugant. In this case, a strong band corresponding to the am-plicon of the expected size was detected, confirming thatMGIAmaMed1’s excision is still triggered by ICEs of the SXT/R391 family (Fig. 5A, lane 7). The anomalous structure ofMGIAmaMed1, which seems to be flanked by an attL and an attPsite, suggests that a related GI, or MGI, was integrated on the rightside of MGIAmaMed1 in an ancestor of the strain A. macleodiiDeep ecotype and was likely lost and supports the idea that two ormore MGIs are able to integrate into the same site and form tan-dem arrays like SXT/R391 ICEs.

We wondered whether the GIs related to MGIs were also ableto excise by site-specific recombination from the chromosome oftheir host. We tested the ability of GISpuPO1 and GIVmiUSA1from Shewanella sp. W3-18-1 and V. mimicus VM573, respec-tively (Fig. 2C), to form a circular extrachromosomal molecule byamplifying the resulting attP locus by nested PCR (Fig. 5A, lanes 8and 9). Both GIs yielded PCR products of expected sizes, confirm-ing the excision event. Interestingly Shewanella sp. W3-18-1 bearsICESpuPO1, an SXT/R391 ICE, whereas V. mimicus VM573 doesnot harbor any SXT/R391 ICE. This finding suggests that, unlikeMGIs, the mechanism of excision of these related GIs does notrequire activation by the SXT/R391 ICE regulator SetCD.

Concluding remarks. The mechanisms of acquisition ofgenomic islands are often unknown. There are only few examplesof GIs for which the transmission mechanism has been revealed.Nonreplicating Bacteroides units (NBUs) and Tn4555 have beenshown to be excised and mobilized in trans by Bacteroides conju-gative transposons (49–51). The high-pathogenicity island (HPI)found in many Enterobacteriaceae is linked to ICEs and can bemobilized in cis by them (52). In Staphylococcus, transduction hasbeen found to be the mechanism of transfer of Staphylococcusaureus pathogenicity islands (SaPIs). SaPIs are mobilized byhelper phages that also induce their excision (53). As for MGIs,their mechanism of mobilization is now well understood (6, 19),and this study gives a better understanding on their characteristicsand origins.

Genomic islands are often studied from a strain-centric per-spective, and extended studies about families of genomic islandsare less frequent. We believe such studies have their importancebecause they provide a better understanding of the mechanismsinvolved in the transmission and evolution of genomic islands andthe role they play on bacterial genome plasticity.

ACKNOWLEDGMENTS

We are grateful to Steve Vo and Nicolas Carraro for their technical assis-tance and Dominick Matteau for his contribution to the preparation ofIllumina sequencing libraries. We thank Mauro M. Colombo (Universitàdi Roma Sapienza, Rome, Italy), Francisco Rodriguez-Valera (Universi-dad Miguel Hernandez, Spain), Fabiano Thompson (Federal Universityof Rio de Janeiro, Brazil), and Alexander S. Beliaev (Pacific NorthwestNational Laboratory, WA) for the kind gift of strains.

This work was supported in part by a Discovery Grant from the Nat-ural Sciences and Engineering Council of Canada. D.C. was supported bya fellowship from Cenci Bolognetti, Institut Pasteur Foundation, Italy,and part of the work was funded by a grant from PRIN 2007, Italy. V.B.holds a Canada Research Chair in molecular biology, impact and evolu-tion of bacterial mobile elements, and is a member of the FRSQ-fundedCentre de Recherche Clinique Étienne-Le Bel. The funders had no role instudy design, data collection and analysis, decision to publish, or prepa-ration of the manuscript.

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