The Vibrio cholerae O1 chromosomal integron - CiteSeerX

Microbiology (2000), 146, 2605–2612

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The Vibrio cholerae O1 chromosomal integron Christopher A. Clark, Leanne Purins, Pranom Kaewrakon, Tony Focareta and Paul A. Manning† Author for correspondence : Paul A. Manning. Tel : j1 781 839 4000. Fax : j1 781 839 4500. e-mail : paul.manning!astrazeneca.com

Microbial Pathogenesis Unit, Department of Microbiology and Immunology, University of Adelaide, Adelaide, South Australia 5005

Until the discovery of the Vibrio cholerae repeat (VCR), the gene capture and expression systems termed integrons had been typically associated with antibiotic-resistance gene cassettes with usually less than five genes in an array. A method is described for the cloning of the ends of large cassette arrays. Conserved restriction sites within VCRs facilitated the mapping by Southern hybridization and cloning of the 5’ end of the VCR array, and using appropriate fragments it was possible to develop a physical map of the region of the V. cholerae chromosome. Sequence determination of the predicted beginning of this region revealed intI4, a member of the integron family of integrases. Comparison of these sequences from El Tor, Classical and serotype O134 V. cholerae strains identified the 3’ end of the attI site, thereby defining the class 4 integron in one of the V. cholerae chromosomes, and providing the first evidence for integron-like site-specific recombination within V. cholerae. Conduction assays demonstrated IntI1-mediated recombination between VCRs. Restriction mapping places the sequences of intI4 and 26 VCR gene cassettes in arrays within a 120 kb region of the V. cholerae O1 strain 569B genome. This region contains an estimated 150 VCR gene cassettes, dwarfing previously described arrays. Southern analysis of genomic DNA from strains of Vibrio anguillarum, Vibrio mimicus and a number of V. cholerae serotypes revealed fragments that hybridized with VCR-specific probes but showed a high degree of restriction fragment length polymorphism. These data facilitate the identification of part of a new class 5 integron from V. mimicus.

Keywords : gene capture, site-specific recombination, repetitive sequences, evolution

INTRODUCTION

We have previously described a chromosomal repetitive sequence element, designated VCR for Vibrio cholerae repeat, nine of which were associated with a locus determining mannose-fucose-resistant haemagglutination in V. cholerae O1 (Barker et al., 1994 ; Franzon et al., 1993). Like other repetitive chromosomal elements, such as REP (Higgins et al., 1982) and ERIC sequences (Hulton et al., 1991), the consensus of the 129 bp VCR has extensive, but imperfect, dyad symmetry. This can .................................................................................................................................................

† Present address : AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA. Abbreviations : 59-be, 59-base element ; 5’-CS, 5’ conserved sequence ; VCCI, Vibrio cholerae chromosomal integron ; VCR, Vibrio cholerae repeat. The GenBank accession numbers for the sequences determined in this work are AF025662, AF055586, X64097, AF179593 and AF179596. 0002-3966 # 2000 SGM

potentially form a stem–loop structure with a predicted free energy of k59n8 kcal mol−" (Barker et al., 1994 ; Hulton et al., 1991). Southern hybridization of the V. cholerae O1 strain 569B chromosome has suggested that there are between 90 and 100 copies of VCR (Barker et al., 1994). In contrast to other repetitive elements (REP) (Higgins et al., 1982), these were localized by PFGE to a single 200 kb region of the chromosome (Barker et al., 1994 ; Clark et al., 1997 ; Manning et al., 1999). Nucleotide sequencing of the original clone (Barker et al., 1994) revealed that each individual gene, with two minor exceptions, was oriented in the same direction and flanked by directly repeated copies of VCR. Both exceptions encoded small operons. This pattern is reminiscent of antibiotic-resistance gene cassette arrays of integrons, as recognized by Recchia & Hall (1995, 1997) who observed that the outer ends of the VCR consensus are related to the core and inverse core sites of 2605

C. A. CLARK and OTHERS

59-base elements (59-be) found in integron cassette arrays. Integron is a term applied to a genetic unit encoding an integrase gene intI, and immediately upstream, an attachment site attI with the core site, G7TTRRRY, defining its 3h end. IntI-mediated sitespecific recombination occurs at the nick site between GT residues of the core sites of attI and the 59-be in gene cassettes (reviewed by Collis & Hall, 1995 ; Recchia & Hall, 1995, 1997). The mobile elements termed gene cassettes, each of which carries a 59-be containing a core site, are defined as the region between two successive core nick sites within an array or as closed circles containing a single 59-be (Collis et al., 1993). We and others have recently reported the presence in V. cholerae of a new integrase (Clark et al., 1997 ; Manning et al., 1999 ; Mazel et al., 1998), homologous to the IntI family of integrases. It is divergently transcribed from the genes contained within the VCR region, in the manner of antibiotic-resistant integrons. The VCRassociated region was the first example of such a chromosomal locus containing all the components of an integron and was dubbed the Vibrio cholerae megaintegron (Clark et al., 1997), but will now be referred to as the Vibrio cholerae chromosomal integron (VCCI). The sequence of an intI4 was described along with the presence of VCR cassettes in Vibrio metschnikovii and the ability of a VCR cassette to be integrated into an intI1 integron (Mazel et al., 1998). In this study we present a general means of cloning the ends of this and similar arrays, and define the extent of the region. The extent of the region is defined and further evidence of its chromosomal variation is shown using both Southern blot and DNA sequence analysis in a variety of Vibrio spp. isolates. Together with evidence demonstrating that VCRs act as 59-bes in recombination assays, we show that the region functions as a new class of integron. METHODS Bacterial strains. The V. cholerae O1 strains used include 569B (Classical, Inaba), AA14073 (El Tor, Inaba), O17 (El Tor, Ogawa), H1 (El Tor, Ogawa), C5 (El Tor, Ogawa), C31 (El Tor, Ogawa), AA14073 (El Tor, Ogawa), BM69 (El Tor, Inaba). Non-O1 serotype V. cholerae strains include O41, O46, O48, O50, O110, O111, O128, O132, O133, O134, O137, O139, O140, O142, O150, O152 and O155 (M. J. Albert, ICDDR,B, Dhaka, Bangladesh). Other Vibrio species used included Vibrio mimicus strain V800, Vibrio parahaemolyticus strains NCTC 10884 (Kanagawa positive) and NCTC 10885 (Kanagawa negative), and Vibrio anguillarum strains ATCC 43305 and ATCC 43306. PCR. Amplification using Amplitaq DNA polymerase (Hoff-

man-La Roche) was carried out by standard protocols with the oligo-deoxynucleotide primers 343 (5h-GTGGTTDCGGTTGTTGTGTG-3h), 922 (5h-CCCCTTAGGCGGGCGTTA-3h), 923 (5h-CCCTCTTGAGGCGTTTGTTA-3h), 2355 (5h-TAACGCCCGCCTAAGGGGCT-3h) and 2583 (5h-GTACAACCGGTAACTTTCGTTCTAG-3h). DNA sequencing procedures. The nucleotide sequences from

strains 569B, O134, H1 and V800 were obtained using either Applied Biosystems 373 or 377 automated DNA sequencers. 2606

Dye-labelled terminators were used in the sequencing reactions. The accession numbers for the individual sequences are listed in the legend to Fig. 3. Southern hybridization. DNA from Vibrio strains was pre-

pared as described by Manning et al. (1986). Transfer of DNA from agarose gels to Hybond-Nj filters (Amersham) was performed as described by Southern (1975) with modifications described by Sambrook et al. (1989), except that two stringent washes were performed at 4 mC below Tm for all oligo probings. Detection was by enhanced chemiluminescence and autoradiography. Mating-out assay for site-specific recombination. Donor strains were constructed by introducing all three plasmids at once into Escherichia coli S17-1 by electroporation. These were mated with E. coli DH5α as previously described (Hansson et al., 1997) and plated on agar plates supplemented with nalidixic acid and chloramphenicol or nalidixic acid and kanamycin.

RESULTS Detection of VCR in Vibrio isolates by PCR and Southern hybridization

We examined 65 V. cholerae strains belonging to different O serotypes, by using VCR5, the VCR immediately downstream of mrhAB (Barker et al., 1994), as a probe in Southern hybridization. All strains revealed multiple reacting DNA fragments totalling in each instance more than 40 kb. A representative selection is shown in Fig. 1. The probe also hybridized to V. mimicus and V. anguillarum DNA, but no reaction was detected with V. parahaemolyticus even after longer exposures. A striking feature of these profiles generated by HindIII digestion is that each has a unique fingerprint.

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J K L

M N O P Q R

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Fig. 1. Hybridization of the VCR5 (2) probe to HindIII-digested chromosomal DNA of Vibrio isolates. Non-O1 serotype V. cholerae strains : lanes A, O128 ; B, O132 ; C, O133 ; D, O134 ; E, O137 ; F, O139 ; G, O140 ; H, O142 ; I, O150 ; J, O151 ; K, O152 ; L, O155. O1 V. cholerae strains : lanes M, H1 ; N, O17. Lane O, V. parahaemolyticus NCTC 100844 ; lane P, V. mimicus V800. V. anguillarum strains : lanes Q, ATCC 43305 ; lane R, ATCC 43306. Arrows indicate DNA molecular mass marker positions, sizes in kb.

Vibrio cholerae O1 chromosomal integron

However, closely related isolates such as the V. cholerae El Tor strains O17 and H1 have a number of bands in common (see Fig. 1). The hybridization pattern is somewhat reminiscent of a partial digest but this is a reflection on the ability of the probe to bind to different fragments determined by both the degree of homology to VCR5 and also the number of VCR copies on the particular fragment. The VCR-containing regions appear to be quite variable. Each of the strains in the remainder of the V. cholerae O serotype reference collection (obtained from M. J. Albert, ICDDR,B, Dhaka, Bangladesh) had at least six gene cassettes as determined by the number of discrete bands detected following PCR amplification with oligodeoxynucleotides 922 and 923, which are directed outwards from the conserved ends of VCR (data not shown). To confirm that the VCR elements are in fact all direct repeats, as with all 59-bes studied so far, single-primer PCR was performed with either oligo-deoxynucleotide 922 or 923 using DNA from a variety of O1 and non-O1 serotype strains. The inability to obtain a PCR product with the individual primers suggests that in each case the VCR sequences are present as direct repeats within the arrays.

(a) II

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Fig. 2. (a) Hybridization of VCR-specific probe 923 to KspIdigested chromosomal DNA of Vibrio isolates. O1 V. cholerae strains : lanes A, 569B ; B, AA14073 ; C, H1 ; D, O17 ; E, C5 ; F, C31 ; G, AA14073 ; H, BM69. Non-O1 V. cholerae strains : lanes I, O41 ; J, O46 ; K, O48 ; L, O50 ; M, O110 ; N, O111 ; O, 0128 ; P, 0133 ; Q, 0134 ; R, 0139. (b) The filter from (a) was stripped and reprobed with oligomers 2943 and 2944 (intI4 specific). Large arrows indicate DNA size markers, in kb. Small arrows indicate apparently conserved KspI restriction fragments. I and II denote the group of smaller and larger hybridizing fragments, respectively.

Identification of the 5’ restriction fragment flanking the VCR region by Southern hybridization

The conserved nature of the VCR sequence and the apparent organization into one contiguous region (Barker et al., 1994) provided the means of identifying sequences immediately flanking this region. The VCR sequences contain four 6 bp restriction sites : HincII, KspI, MluI and Bsu36I, which are conserved in 28, 26, 21 and 28, respectively, of the 32 VCR elements identified and sequenced. Considering that the mean distance between these VCRs is 0n7 kb, the size of these restriction fragments from within the array would be expected on average to be significantly smaller than those from the immediate region flanking the array. This is particularly true of KspI as it has a GjC content of 100 mol %, whereas that of the V. cholerae genome is 47–49 mol % (Baumann et al., 1984). Genomes with a similar GjC content to that of V. cholerae, such as E. coli, have a mean KspI restriction fragment size of greater than 20 kb. Thus, the 5h restriction fragment of the array could be expected to be large and carrying only the 5h part of the first VCR. Using each enzyme separately, the genomic DNA from a variety of V. cholerae non-O1, and El Tor and Classical O1 serotype strains was digested and probed with oligo-deoxynucleotide 923, which is derived from the 5h end of the VCR. In Fig. 1, hybridization of the probe to the 5h end of the VCR in chromosomal KspI digests reveals a series of bands, which we have divided into two groups of bands – one corresponding to fragments above 7 kb and the other to fragments below 5 kb. In the range 4n4–0n72 kb there is a ladder of more than 20 discrete bands in each track, but when higher percentage agarose gels were used, nu-

merous bands down to 0n3 kb were observed (data not shown), each probably corresponding to an individual VCR cassette. Throughout the size range there are a number of conserved restriction fragments among the O139 and all of the O1 serotype strains except for C31, which is known to have a major deletion in this region of the chromosome (Barker et al., 1994 ; Fig. 2). In the group of larger DNA fragments, probing with oligo-deoxynucleotide 923 revealed one or two KspI restriction fragments between 7n2 and 20 kb (Fig. 2). Since 923 binds 5h of the KspI site, only one large fragment was expected, corresponding to the 5h flanking fragment and the first cassette. Only strains O41, O46, O48, O111 and O134 showed the anticipated pattern. The remainder had a second smaller fragment of 7n2–8n5 kb, except for strain O128. The imperfect dyad symmetry of VCRs, particularly at the ends, suggested the possibility that the probe was binding to both ends. Even though this should have been excluded by stringent washes, the filter was stripped and reprobed with oligodeoxynucleotide 343, which binds 3h to KspI within the loop region potentially formed by the VCR. The absence, in most cases, of hybridization to the largest KspI fragment in each lane to probe 343 (data not shown) suggested that it contains the 5h end of the array. The second fragment, in most cases, reacted with the probe, indicating it is unlikely to contain the 5h end. A similar pattern was also seen for the Bsu36I Southern blot probed with 923, whereas the discrimination between fragment groups was less clear in several MluI and most HincII digests (data not shown). 2607


Cloning of the V. cholerae integrase gene intI4 and defining the class 4 integron

After identifying the potential 5h end of the VCR region, the presence of an integrase immediately upstream of the first was sought. Genomic DNAs of V. cholerae strains 569B and H1 were digested with KspI and fragments of approximately 20 kb were isolated by agarose-gel purification and further digested with 20 different six-base recognition restriction enzymes found within the polylinker of pBluescript KS. A bank for each strain was constructed from 20 separate chromosomal digests ligated to pBluescript KS cut with the corresponding enzyme. Screening each ligation by vector-anchored PCR amplification with oligo-deoxynucleotide 923 and pBluescript reverse primer revealed a 1 kb product in the ClaI ligations with both 569B and H1, with an additional 2 kb fragment for H1. Both fragments were cloned. Oligo-deoxynucleotide 2583 probe, derived from the 569B 1 kb clone sequence, hybridized to a cloned 2n24 kb HindIII fragment from a 2–3 kb 569B subgenomic library. After sequencing, an integrase identical to IntI4 (Mazel et al., 1998) of the IntI family was found in a transcriptionally divergent direction to the first VCR cassette in 569B and the first three cassettes in H1. This is characteristic of integrons. The two sequenced VCRs of H1 do not possess KspI sites, thus explaining the two amplicons as well as some of the potential 5h array end fragments still hybridizing with oligo-deoxynucleotide 343. Comparison of intI4 from 569B and H1 showed nucleotide sequence identity from the ClaI site within the gene to 218 nt upstream of the intI4 initiation site. The point of divergence appeared to be the 5h boundary of the first cassette of the array. PCR and sequence analyses of V. cholerae O134 and V. mimicus using primers 2583 and 2355 were conducted. The DNA of strain O134 was 98 % identical up to the same point of divergence in both sequences. The point of divergence from the 5h conserved sequence (5h-CS) in all three V. cholerae strains immediately follows 8 bp that conform to the core site consensus (see Fig. 4), thus defining the 3h end of the attI4 site and thereby defining the class 4 integron of V. cholerae. Southern hybridization to the intI4 probes, oligo-deoxynucleotides 2593 and 2594, shows that the strains surveyed for array 5h flanking fragments can be divided into two groups with a KspI fragment of either 20 kb or 13n5 kb in size (Fig. 2). Furthermore, in all but one instance the largest VCRhybridizing Bsu36I fragments contained the 5h end of the array (data not shown). The region containing the integrase and first gene cassette from V. mimicus strain V800 was cloned and sequenced. This revealed a level of nucleotide identity between V. mimicus and V. cholerae of 75 % and 65 % in the coding and non-coding regions, respectively, while there was no significant identity with other intI family members. The degree of variation in the coding region is sufficient to designate this as a fifth class of integrase, IntI5. Again the point at which homology to the previous V. cholerae DNA ends is at a core-like site and is tentatively designated the 3h end of attI5. 2608

Mapping the extent of the VCR region in 569B

In addition to the lipocalin-encoding gene vlpA, which we have cloned and sequenced (Barker & Manning, 1997), a second copy was identified on a 4n5 kb BamHI fragment, which contains an array of seven VCRs. Southern data, probing with oligo-deoxynucleotide 922, indicated the presence of three KpnI restriction fragments each with a similar size to the whole bacteriophage lambda genome. A 43 kb fragment was cloned into the cosmid vector c2XMCS (Reilly & Silva, 1993) from a KpnI genomic library and screened by PCR with oligo-deoxynucleotide 922 and 923 primers. The 1n3 kb and 6n2 kb KpnI\SacI ends of the insert were subcloned and sequenced (Fig. 3). The remainder of the cosmid insert had VCR-specific signals in a number of different digests in a Southern hybridization probed with 922, suggesting a continuous VCR cassette array (data not shown). Southern blots of 569B genomic DNA using both single and double restriction enzyme combinations were probed with oligo-deoxynucleotide 923, then stripped and reprobed with the probes indicated in Fig. 3. Combining these sources, a restriction map of the region showing all the KpnI fragments hybridizing with VCRs and the position of sequenced regions was constructed (Fig. 3). The first two KpnI fragments could not be linked. The two regions of the cassette array total 120 kb of VCR-containing DNA in the V. cholerae 569B chromosome. Analysis of gene cassettes

Nucleotide sequence analysis and examination of the encoded ORFs showed that the average cassette of 693 nt generally contained very little non-coding DNA in addition to the VCR (Fig. 3). Of the 28 cassettes with an identifiable ORF in the same orientation as the VCR, 20 had insufficient space for a promoter between the 5h end of the VCR and the translational initiation site. Typical of 59-bes (Reilly & Silva, 1993), most ORFs finish within 20 nt of a VCR, and in fact, many use the TAA of the inverse core as the stop codon. We have given ORFs within cassettes that do not have clear database homologues the name Vco for Vibrio cassette ORF. The associated numbers are the same as previously published ORF numbers where possible ; otherwise they have been numbered in the order in which they appeared in the NCBI database. Subsequently identified vco homologues with greater than 95 % identity are numbered in the order of discovery ; for example, vco13.2. An unusual feature is the presence of ORFs transcribed in the opposite direction to that of the array such as the gene pairs vco3AB, vco21AB and vco27AB, as well as vco24B which is convergent with vco24A (Fig. 3). All of these ORFs have sufficient space for a promoter between the proposed start and the VCR upstream of it. IS1360 is a new IS element with 77 % nucleotide identity with IS5 (accession no. X13668) and was found inserted into the first complete cassette on the cosmid clone C169. IS1359 apparently disrupts ORF23, the gene after the last cassette (Fig. 3), and is a new member of the IS3 family


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Fig. 3. A KpnI (K) restriction map of the VCR region of 569B. K* indicates that the distance between these KpnI sites is unknown. The positions of sequenced regions are indicated by shaded boxes each labelled underneath with a capital letter in bold. Cloned BamHI (B) and KpnI–SacI (S) fragments are indicated within these regions. The sequences of both KpnI–SacI and both BamHI fragments were extended 5 ’, both 5’ and 3 ’, respectively, by PCR walking to the next VCR. The KspI (Ks) and PstI (P) sites at either end are the first of these sites 5’ and 3’ of the cassette array. The underlined segment indicates the cloned KpnI fragment in cosmid C169. The thick lines above the diagram show the extent of regions which contain VCR sequences. The genetic organization of each of the sequenced regions is shown below and is preceded by its corresponding capital letter in bold. Shaded boxes represent ORFs within cassettes. Black triangles represent VCRs. White boxes represent non-cassette ORFs, striped boxes indicate IS element ORFs. Arrows below indicate the polarity of transcription. The lines and * above the schematic of the sequenced region represent PCR-amplified and oligo probes, respectively, used in mapping. Accession numbers are AF025662, AF055586, X64097, AF179593 and AF179596 for the region A, B, C, D and E sequences, respectively.

with 64 % identity at the nucleotide level to the functional IS911 of Shigella dysenteriae. IS1359 appears to mark the end of the chromosomal VCR array, as the last 4n8 kb of the cosmid is devoid of VCR elements. mccF1, a new cassette-associated ORF of 344 residues, has 55 % identity at the nucleotide level over its full length with the E. coli microcin C7 immunity protein MccF (Gonzalez-Pastor et al., 1995). A second homologue was found to lie on a 7n3 kb BamHI fragment within the second KpnI fragment by probing with mccF1 (data not shown). The vco13 cassette is yet another with homologues : the vco20 cassette is 67 % identical. The vlpA and vlpa2 genes were nearly identical. DISCUSSION

V. cholerae is a diverse species, as shown by the more than 165 different O serotypes, which also represent temporal, geographical and ecological diversity. PCR and Southern analysis of all these strains (shown here) and work by others on a limited subset (Shangkuan et al., 1997) has shown that VCR elements are ubiquitous throughout the species, suggesting that their presence predates the speciation of V. cholerae. This is further supported by Southern hybridization studies in V. mimicus and V. anguillarum, as well as earlier in V. metschnikovii (Mazel et al., 1998). However, we were

unable to detect VCR in strains of V. parahaemolyticus, although the presence of at least three VCR cassettes detected by PCR amplification has been reported (Mazel et al., 1998). While horizontal transfer may have influenced the spread of VCR arrays, the likelihood of a single vertical ancestral VCR contained in a progenitor of these species is consistent with phylogenetic trees of the genus Vibrio based on rRNA sequences (KitaTsukamoto et al., 1993 ; Aznar et al., 1994). Furthermore, the 25 % divergence between intI4 and the partial intI5 sequences is similar to that seen between the coding regions of phl (lecithinase), vmhA (haemolysin) and vmc (metalloprotease) of V. mimicus and their homologues in V. cholerae (Kang et al., 1998 ; Kim et al., 1997). Thus, it is very likely that residency in the genome of VCRassociated integrons at least predates the separation of the two species. The HindIII and KspI restriction polymorphisms that were detected imply that the VCR array in Vibrio is plastic and potentially provides a rapid means of demonstrating whether strains are closely related. The biotype El Tor strains are considered to be a clonal group for some loci (Byun et al., 1999). However, no two strains had identical Southern blot profiles, although most did have many fragments in common. The absence of common fragments in strain C31 confirms the previous suggestions that it has undergone a major 2609


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Fig. 4. Comparison of the VCCIs of strains 569B and N16961. The physical map of the VCCI of Classical strain 569B generated in this paper is aligned with the DNA sequence contigs of El Tor strain N16961 sequenced by The Institute for Genomic Research (http ://www.tigr.org). The hatching patterns are the same as those in Fig. 3.

deletion within the region of the chromosome encoding the mrhAB locus (Barker et al., 1994 ; Van Dongen et al., 1987). Restriction enzyme sites within the VCR elements also provided a means of identifying the 5h-CS of the region in many strains. The approach presented here for cloning the 5h end of the array may be generally applicable to cloning the ends of other large arrays of highly conserved repetitive elements. Cosmid C169 appears to contain the 3h end of the array since 8n8 kb of VCR-free DNA is located downstream of its last VCR. Furthermore, the sequence up to the end of IS1359 of region E from 569B (Fig. 3) is nearly identical to a similar region devoid of VCR on the chromosome of the V. cholerae El Tor strain N16961 that has been sequenced by The Institute for Genomic Research (see http : \\www.tigr.org) (Fig. 4). The sequence in 569B after IS1359 is not homologous to that of N16961, and preliminary PCR analysis indicates that El Tor and Classical strains differ at this point, suggesting that IS1359 may be a recombinational hotspot. The location of IS elements at the 3h ends of arrays is quite typical of other classes of integrons (Hall, 1995). The size of the integron array here is defined as approximately 120 kb, consistent with a previous estimate in which strain 569B was estimated by densitometry to have 90–100 copies of VCR (Barker et al., 1994), suggesting that there could be in excess of 150 gene cassettes, vastly greater than the few genes detected in the average antibiotic-resistance integron. Recent data from the incomplete genome sequence of V. cholerae El Tor strain N16961 have identified 147 VCR elements within a 147 kb chromosomal integron (see http :\\www.tigr.org) confirming that this complexity is not restricted to Classical strains. We have compared the organization of the sequenced regions of the 569B VCCI and the genes within them with the available data for N16961 (Fig. 4). It is immediately apparent that the spacing between the regions differs and that region B does not exist, at least in 2610

the same organization seen in 569B. In addition, there are more copies of IS1359, three in each chromosome of N16961 but only two associated with the VCCI in 569B. Also there are two copies of vlpA in the VCCI in 569B, but four copies in N16961, only one of which is identical and in the same position as 569B, namely that in region C. The additional copies all have a few mismatches within the gene (12, 13 and 13 bp). Furthermore, although both strains have two copies of mccF, the spacing differs. These data suggest that there is continual microevolution occurring within VCCI leading to gain, loss or duplication of genes, presumably all mediated by the specific integrase. The comparison of strains 569B, H1 and O134 implies that the 5h-CS satisfies all of the definitional requirements for the fourth class of integron and also provides the first direct evidence that attI core-site-specific recombination typical of integrons has occurred in V. cholerae. Recently, it has been shown that a VCR cassette can be incorporated into the attI site of the class 1 integron on R388 (Mazel et al., 1998). Using a cointegration assay, we have been able to demonstrate that the VCR functions as a 59-be and that it can act as a substrate for site-specific recombination using IntI1 (unpublished data). The presence of IntI1 led to a 350fold higher recombination rate, which was confirmed by sequence analysis to correspond to site-specific recombination events that are consistent with typical recombination involving a 59-be core, as has been shown previously for a 59-be (Collis et al., 1993 ; Martinez & de La Cruz, 1990). There are three main differences between the VCCI and the plasmid-borne antibiotic-resistant integrons : size, mobility and homogeneity of the 59-be. The expression of the antibiotic-resistance cassettes within integrons derived solely from the Pant promoters located in the 5hCS (Lesesque et al., 1994) seems to be one of the limitations on their size unless most of the array is silent. However, this arrangement would be inadequate within the VCCI. This is of particular concern since some 59bes may act as weak terminators (Collis & Hall, 1995) as


has been shown for VCRs within region C (A. Barker, S. G. Williams & P. A. Manning, unpublished). This implies therefore that either large regions of the VCCI are silent or there are numerous internal promoters. While the presence of promoters within VCRs is an intriguing possibility, this activity, as yet, has not been demonstrated. An alternative is that promoters are provided by some of the cassettes with enough space 5h of the start codon to encode a promoter, as has been shown for mrhAB (Barker et al., 1994). Clearly, the presence of four cassettes, with ORFs in the opposite orientation with respect to the array, and which has not been seen in other integrons, further indicates the necessity for endogenous promoters, and each of these cassettes has sufficient coding capacity. This ad hoc arrangement might well explain the need for multiple copies of genes, such as in the case of the promoterless vlpA, which is present in up to five copies in some strains (P. Kaewrakon, P. A. Manning and T. Focareta, unpublished) It has been suggested (Mazel et al., 1998) that the high degree of homology of VCR elements indicates that each of the previously variant 59-be represents a single member of similar vast arrays found in many different bacterial genera. It is indeed likely that there are other chromosomal integrons and that they are an important means of gene capture and chromosomal building. However, there are a number of factors that may contribute to drift in 59-bes outside these integrons. For example, the permanent residency of the integron provides a stable genetic environment for co-evolution with the host not likely to be duplicated, as in the mobile plasmid-associated integrons. To date, there appears to be a significant difference in the types of genes acquired by the VCCI, with the possible exception of mccF1 should it prove to act as a microcin-immunity protein. This difference is likely to reflect the antibiotic-resistance bias in sampling of the plasmid-based integrons examined, rather than any fundamental differences between the two systems. The pool for gene capture is likely to be the converse. That is, the VCCI samples genes from episomes passing through. For example, the large plasmid carrying mccF (Gonzalez-Pastor et al., 1995) might be the ancestral source for mccF1. In this way the VCCI captures useful genes and eliminates the burden of maintaining the episomes. It is tempting to speculate that the chromosomal integrons, at least within the genus Vibrio, act as a large gene pool for horizontal gene transfer between Vibrios. This could potentially occur via natural transformation of VCR cassette circles or by plasmid-integron intermediates such as that found recently in an El Tor isolate (Fablo et al., 1999). ACKNOWLEDGEMENTS The authors wish to acknowledge the financial support of the National Health and Medical Research Council of Australia and Ruth Hall for many invigorating discussions. We would

also like to thank Chris Cursaro for his outstanding technical assistance over many years.

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Received 5 January 2000 ; revised 29 June 2000 ; accepted 6 July 2000.