Distribution of IS1358 and linkage to rfb-related genes in Vibrio anguillarum

Kathy E. Jedani1, Uwe H. Stroeher2 and Paul A. Manning3

Microbial Pathogenesis Unit, Department of Microbiology and Immunology, The University of Adelaide, Adelaide, SA 5005, Australia1
Mikrobiologie II, Universität Tübingen, D-72076 Tübingen, Germany2
Microbiology and Molecular Biology, AstraZeneca R&D Boston, 128 Sidney Street, Cambridge, MA 02139-4239, USA3

Author for correspondence: Paul A. Manning. Tel: +1 617 576 3900. Fax: +1 617 576 3030. e-mail: paul.manning{at}astrazeneca.com


   ABSTRACT
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INTRODUCTION
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DISCUSSION
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The insertion sequence IS1358 is linked to the rfb regions of both Vibrio cholerae O1 and O139, and its location was suggestive of a role in generating new combinations of rfb genes. This provoked an examination of the distribution and localization of IS1358 in Vibrio anguillarum. IS1358 was widely distributed in a number of V. anguillarum serogroups. In particular, when cosmid clones of V. anguillarum O1 were screened with IS1358 and subsequently subcloned and sequenced, it was found that rfb-like genes were linked to this region. Furthermore, when the previously identified genes virA and virB from V. anguillarum O1, now known to be involved in LPS biosynthesis, were used as probes, it was discovered that they too are present on the same large EcoRI fragment as IS1358. This clearly indicated that IS1358 was linked to the rfb region of V. anguillarum O1. Further analysis of the location of IS1358 in other serotypes indicated that V. anguillarum O2 also has IS1358 associated with rfb-like genes. In V. anguillarum O2 there is more than one copy of IS1358, suggesting that this element is a site for recombination, gene duplication or that it may be capable of transposition. Following this latter premise, IS1358 elements from a variety of V. anguillarum strains have been cloned and sequenced. Only those strains with multiple copies of IS1358 produce a full-length putative transposase, as shown by protein overexpression, further strengthening the argument that the element is transposing within these strains.

Keywords: O-antigen, polysaccharide, insertion sequences, transposase, evolution

The GenBank accession numbers for the IS1358 sequences are U93587U93597.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Lipopolysaccharide (LPS) is the most abundant molecule on the cell surface of Gram-negative bacteria. It is a protective barrier to antibiotics and hydrophobic agents and can be used as the basis for serological differentiation within species. It is important for virulence in pathogenic bacteria such as Vibrio cholerae (Manning et al., 1986 ), Vibrio anguillarum (Amor & Mutharia, 1995 ), Yersinia enterocolitica (Zhang et al., 1997 ) and Shigella flexneri (Okada et al., 1991a , b ; Phalipon et al., 1995 ; Van den Bosch et al., 1997 ).

For some time this laboratory has been studying the rfb gene locus involved in O-antigen biosynthesis in V. cholerae. The genes have been cloned and sequenced for the O1 serotype (Manning et al., 1986 , 1995 ; Morona et al., 1991 , 1995a ; Stroeher et al., 1992 , 1995a ) and a defective insertion-sequence-like element, IS1358d1, has been identified (Stroeher et al., 1995a ). Interestingly, this element was also found linked to the rfb operon of the recently emerged V. cholerae serogroup O139 (Stroeher et al., 1995a ). IS1358 contains a putative transposase (tnpA) and has all the features of a transposable element (Stroeher et al., 1995a ). Xiang et al. (1994) located an homologous element in the rfb cluster of Salmonella enterica (serovar Typhimurium) serogroup D2, and proposed a role in genetic rearrangement of polysaccharide synthesis genes. More recently, a similar element was found in the type 3 capsule locus of Streptococcus pneumoniae representing the first example of this type in a Gram-positive pathogen (Yother et al., 1997 ). IS1358 also shows strong homology to ISAs1, a known mobile genetic element which is associated with the expression of a paracrystalline surface protein array (A-layer) in Aeromonas salmonicida (Gustafson et al., 1994 ).

Accumulated evidence suggests that V. cholerae O139 arose from a strain of the V. cholerae O1 El Tor serotype and that IS1358 may have been involved in the rearrangement (Bik et al., 1995 ; Comstock et al., 1996 ; Johnson et al., 1994 ; Stroeher et al., 1995a ). The most dramatic difference between these serogroups is the O-antigen chains found on the LPS molecules. How IS1358 facilitated these rearrangements is unknown but the most likely mechanism involves transposition and subsequent homologous recombination.

Amor & Mutharia (1995) recently cloned and expressed the rfb (O-antigen biosynthesis) genes from the O2 serotype of V. anguillarum in Escherichia coli but until recently the V. anguillarum O1 rfb gene cluster had not been identified (K. E. Jedani and others, unpublished). However, two genes, virAB, were isolated by Norqvist & Wolf-Watz (1993) that were shown to be associated with LPS in the O1 serogroup. It is therefore possible that these genes are part of the rfb gene cluster.

Due to the apparent linkage of these IS1358-like elements with bacterial surface layers and O-antigen genes, we decided to investigate the distribution and location of these elements in a number of different Vibrio spp.


   METHODS
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INTRODUCTION
METHODS
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Bacterial strains and culture conditions.
Bacterial strains used in this study are listed in Table 1. Vibrio anguillarum strains were cultured in Tryptic Soy Broth (TSB) or on agar (TSA) (Difco) at 25 °C. V. cholerae strains were grown in either brain heart infusion broth (Difco) or Luria broth as previously described (Morona et al., 1991 ). Cultures were grown with shaking at 37 °C. All other strains were routinely grown in nutrient broth (NB) or on agar (NA). Antibiotics were used when appropriate at the following concentrations: ampicillin, 100 µg ml-1; chloramphenicol, 25 µg ml-1 (Sigma/Boehringer Mannheim).


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Table 1. List of bacterial strains used in this study

 
DNA isolation and analysis.
Whole genomic DNA of V. anguillarum and V. cholerae strains was prepared according to the method described previously (Manning et al., 1986 ). The method was modified for the extraction of DNA from Vibrio strains. Proteinase-K-treated cells were incubated in 0·1 M Tris/HCl (pH 8·0) saturated phenol for 1 h at 4 °C prior to three sequential phenol extractions. Genomic DNA was precipitated by the addition of 4 vols ice-cold 100% ethanol. Plasmid DNA was prepared using the alkaline lysis procedure. Restriction enzymes were used with the buffers supplied by the manufacturers (Amersham, Boehringer Mannheim and New England Biolabs). Procedures, including agarose gel electrophoresis, were performed as described by Sambrook et al. (1989) . Chromosomal DNA was incubated at 37 °C with various enzymes for 24–36 h.

Cosmid bank construction.
Genomic DNA from V. anguillarum strain 85-3954-2 was partially digested with Sau3AI. Fragments of approximately 40 kb were then cloned into the cosmid vector pPM2101 (Sharma et al., 1989 ). This material was packaged into {lambda} phage heads using the PACKAGENE system (Promega) and transfected into E. coli DH5{alpha} cells. The cosmid bank was subsequently screened using probes derived from IS1358 and virB.

Southern hybridization.
Unidirectional transfer of DNA from agarose gels to nitrocellulose filters (Schleicher & Schuell) was performed as described by Southern (1975) and modified according to Sambrook et al. (1989) . Stringency washes were as follows: twice in 2xSSC/0·1% SDS at 37 °C for 5 min, followed by two washes in 0·2xSSC/0·1% SDS at 65 °C for 15 min. Southern blots using oligonucleotides as probes were washed three times in 5xSSC at room temperature. Detection was done colorimetrically (75 mg nitro blue tetrazolium ml-1 in 70% dimethylformamide and 50 mg 5-bromo-4-chloro-3-indolyl phosphate ml-1 in 100% dimethylformamide). Labelling of probes was carried out by random priming using the DNA Labelling and Detection kit (Boehringer Mannheim) according to the manufacturer’s instructions. PCR labelling of probes involved normal PCR conditions except 0·1 mM Dig-11-dUTP was added to the reaction. Probes were precipitated with 4 M LiCl and 100% ethanol. The pellet was washed with 70% ethanol, dried and resuspended in 20 µl water. Oligonucleotide probes were labelled by Terminal Transferase and Dig-11-dUTP (Boehringer Mannheim).

PCR and inverse PCR.
PCR amplification using Amplitaq DNA polymerase (Hoffman-La Roche) was performed using standard protocols. The oligonucleotide primers used were: 773, 5'-CA(G/C)GGAAACCGCATGAA-3', which is complementary to the 17 bp inverted repeat of IS1358 in the 5' to 3' direction; 1065, 5'-TTCATGCGGTTTCC(C/G)TG-3', which is complementary to the 17 bp inverted repeat of IS1358 in the 3' to 5' direction; and 2194, 5'-TCTCTGTTGCTACAGCCG-3'. This primer is complementary to IS1358 at base number 984–1002 in the 3' to 5' direction. This primer was used in PCR, sequencing and as an internal probe for IS1358 in Southern hybridization. For inverse PCR, the method used was described by Ochman et al. (1988) , with modifications. Chromosomal DNA was digested with appropriate enzymes as described above (see DNA isolation and analysis). Digested DNA was ethanol precipitated to remove salts and enzyme. T4 DNA ligase (Progen) was used to circularize digested chromosomal DNA at 4 °C for 24 h. T4 ligase and buffer were removed from the mixture by ethanol precipitation, and DNA was resuspended in 20 µl water. PCR was performed using standard conditions, with an elongation time ranging from 2 to 5 min. The annealing temperature used in successful PCR reactions was 50 °C. In the PCR reaction mix, 1/10 of the purified DNA was used, and 100 pmol oligonucleotide.

Sequencing analysis.
Sequencing of the IS1358 elements was performed using an AB377 or AB373A Automated DNA Sequencer with a Stretch upgrade using dye terminator and dye primer sequencing protocols (Applied Biosystems). Sequence data obtained were analysed using DNASIS (Hitachi-LKB Software), BLASTN and BLASTX on the NCBI server. Oligonucleotides 2091, 5'-GAT GAA TCC GGC AGC GTT-3', 2092, 5'-GCT ACA GCC GAT TCT TGG-3', and 2193, 5'-GCT GGG ATG GCA TTA TCG-3', were used to complete the sequence of the IS1358 elements.

In vitro cloning.
For sequencing, DNA fragments were cloned into suitable vectors. PCR products were cloned into pGEM-T using the protocol supplied by the manufacturer with slight alterations (Promega). The ligations were conducted at room temperature for 16–24 h. Restriction fragments of cosmids were ligated to pBC KS(-) (Stratagene) cut with suitable restriction enzymes at 4 °C for 16 h. E. coli DH5{alpha} cultures were made competent by CaCl2 treatment.

T7 expression system.
The temperature-inducible T7 RNA polymerase expression system of Tabor & Richardson (1985) was used for the expression of proteins. L-[35S]Methionine (37 TBq mmol-1; Amersham) was incorporated into the reactions for labelling protein. Proteins were visualized by autoradiography following SDS-PAGE.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Distribution of IS1358 in Vibrio species
To determine if IS1358 was more widespread, a number of members of the Vibrionaceae were tested for the presence of the element using colony blot hybridization (probed with the complete V. cholerae O139 IS1358 element). The strains that were tested belonged to species of V. anguillarum, Vibrio fluvialis, Vibrio mimicus and Vibrio parahaemolyticus. The only strains that were found to be positive were those that belonged to some serogroups of V. anguillarum.

Based upon the colony hybridization data, Southern hybridization was performed to confirm the colony blot data and to determine the copy number of IS1358-like elements in each of the positive serogroups (Fig. 1). This was confirmed and demonstrated that IS1358-like elements are not present in the type strains representing serogroups O5, O6, O8 and O10 (Table 2). Of the other strains tested, serogroups O1, O3, O4 and O11 have a single copy of IS1358. Strain ATCC 43311 (serogroup O7) was found to contain at least two copies of IS1358 and the two remaining serogroups, O2 (ATCC 43306) and O9 (ATCC 43313), had more than six elements based on Southern hybridization using full-length IS1358 as a probe. Using an internal oligonucleotide probe (2194), the O2 and O9 serogroups have at least nine copies of IS1358 (Table 2). Further Southern hybridizations using different restriction enzymes confirmed the number to be accurate (data not shown).



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Fig. 1. Southern hybridization analysis using a PCR-derived probe of IS1358 from V. anguillarum strain 85-3954-2. Chromosomal DNAs from the various V. anguillarum strains shown were digested with HindIII. Lanes: 1, 85-3954-2 (O1); 2, 85-3954-1 (O1); 3, 86/3674 (O1); 4, 89/3748 (O11); 5, ATCC 43305 (O1); 6, ATCC 43306 (O2); 7, ATCC 43307 (O3); 8, ATCC 43308 (O4); 9, ATCC 43309 (O5); 10, ATCC 43310 (O6); 11, ATCC 43311 (O7); 12, ATCC 43312 (O8); 13, ATCC 43313 (O9); 14, ATCC 43314 (O10). Size markers are EcoRI-generated fragments of bacteriophage SPP1 DNA.

 

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Table 2. Distribution of IS1358 in V. anguillarum

 
PCR amplification of IS1358
The data indicated that 10 out of the 14 strains tested contained an IS1358 element. Using an oligonucleotide (773) that recognizes the 17 bp inverted repeats at the 5' and 3' ends of previously sequenced IS1358 elements (Stroeher et al., 1995a ), we attempted to PCR-amplify the equivalent elements from the V. anguillarum strains. Using chromosomal DNA from each of the 10 strains, we successfully amplified elements from all strains except ATCC 43308 (O4) and 89/3748 (O11). Thus the inverted repeat sequence, which is present in the oligonucleotides to amplify the element, is either missing or divergent in these particular IS1358 elements. Alternatively, the IS1358 elements in these strains are only partial copies. An internal IS1358 oligonucleotide probe (2194) does not hybridize to any of the bands previously observed in Fig. 1 in serogroups O4 (ATCC 43308) or O11 (89/3748) (data not shown). This indicates that these strains contain only partial copies. In strains with multiple copies only those elements with the conserved inverted repeats will be amplified; however, the individual copy/copies that have been amplified have yet to be determined.

Cloning, sequencing and analysis of different IS1358 elements
The PCR-amplified IS1358 elements from the different V. anguillarum serogroups were purified, cloned and sequenced using universal and internal primers. Multiple independent clones of each element were sequenced to reduce the likelihood of sequencing PCR errors and regions of ambiguity were clarified by resequencing. These data are summarized in Fig. 2 and Table 3.



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Fig. 2. Genetic organization of the IS1358 elements found in V. cholerae O1 and O139 and V. anguillarum O1, O2 and O3. The sequence above the triangle represents the inverted repeat flanking IS1358. The large triangle below the V. cholerae O139 IS1385 is the sequence of the 31 bp repeat which occurs twice; the underlined portion represents the same sequence as the inverted flanking repeats. The positions of oligonucleotides 773, 1065 and 2194 are indicated as is the conserved HindIII site. The UAG/amber stop codon is shown in the V. anguillarum O3 IS1358 element. Vertical lines represent the stop codons of the various truncated ORFs.

 

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Table 3. Sequence similarity of IS1358 in different strains and serogroups

 
Sequence analysis revealed that some serogroups contained elements with uninterrupted TnpA ORFs whilst others encoded elements with three ORFs. The O1 and O3 strains contained single elements that encoded three small ORFs, a similar organization to that observed in V. cholerae O1 (Stroeher et al., 1995a ). Elements from O2, O7 and O9 strains, which all contain multiple copies of IS1358, encoded single uninterrupted ORFs of 1133, 1109 and 1133 bp, respectively. V. cholerae O139 also encodes an IS1358 with a single ORF (1127 bp) which has been proposed to encode a functional transposase of ~42 kDa and hence may be a mobile genetic element (Stroeher et al., 1995a ).

Protein expression from the IS1358 elements of V. anguillarum
The T7 RNA polymerase expression system was employed to confirm the predicted sizes of the encoded proteins. A protein corresponding to the predicted size of ~42 kDa could be detected in O7, O9 and O2 strains (Fig. 3). A similar protein band was identified in the O3 serotype, but the sequenced IS1358 gene encoded an incomplete ORF. However, closer inspection of the sequence revealed an amber stop codon (UAG) at base number 663 which was not found in other interrupted IS1358 elements (Fig. 2). This stop codon may have been suppressed in E. coli strain DH5, which was used in the overexpression, thereby resulting in a product. Proteins produced by the smaller ORFs encoded by the other serogroups were not detected, indicating that they are either rapidly degraded or are not translated as reported previously for V. cholerae O1 (Stroeher et al., 1995a ). The expression data indicate proteins of the expected size (42 kDa), which suggests that the uninterrupted ORFs of IS1358 in V. anguillarum O2, O7 and O9 may be functional.



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Fig. 3. Detection of IS1358-encoded proteins. Proteins were expressed by using the T7 promoter/RNA polymerase system (Tabor & Richardson, 1985 ) and labelled with L-[35S]methionine. Samples were electrophoresed in SDS on a 15% polyacrylamide gel, after which the gel was dried and proteins were detected by autoradiography. Lanes: 1, ATCC 43305 (O1); 2, 86/3674 (O1); 3, ATCC 43307 (O3); 4, ATCC 43311 (O7); 5, ATCC 43313 (O9); 6, ATCC 43306 (O2); 7, 85-3954-1 (O1); 8, 85-3954-2 (O1); 9, pGEM5zf+ negative control. Molecular size markers (Pharmacia-LKB) are indicated as follows: 94 kDa (phosphorylase B), 67 kDa (bovine serum albumin), 43 kDa (ovalbumin), 30 kDa (carbonic anhydrase).

 
IS1358 and virAB
Due to the prevalence of IS1358 in the V. anguillarum strains we were interested in the location of these elements in the chromosome, in particular in the possible linkage to rfb or polysaccharide genes.

Initially, we constructed Sau3A cosmid gene libraries of the V. anguillarum O1 serotype using strain 85-3954-2. Colony blot hybridization was performed using a DNA probe of IS1358 to isolate cosmid clones that contained IS1358 and surrounding DNA. Approximately 1000 cosmid clones were screened and we identified five hybridizing to IS1358. These were confirmed by PCR using oligonucleotide 773 to the inverted repeats of IS1358. Four out of the five clones contained the complete element. PCR reactions on all five cosmid clones allowed amplification of a 1·1 kb fragment consistent with the size of virB. This was confirmed by sequencing the PCR fragments. Southern hybridization was performed to determine the linkage of IS1358 to virB using IS1358 and virB probes on EcoRI-digested chromosomal and cosmid DNA. This demonstrated that the genes were located on the same large (~20 kb) EcoRI fragment (data not shown), again indicating the close proximity of IS1358 with an rfb/polysaccharide gene locus as observed in V. cholerae O1 and O139. To examine what other genes involved in polysaccharide biosynthesis were contained within the cosmids, restriction fragments were cloned and the resulting data were processed through BLASTX. Various subclones showed significant homology to previously published polysaccharide/rfb genes from a number of organisms (data not shown). A physical map of the V. anguillarum O1 chromosome around IS1358 and virB was constructed using Southern hybridization (Fig. 4).



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Fig. 4. Chromosomal map of the putative rfb operon in V. anguillarum 85-3954-2 (O1). The map was derived by Southern hybridization using a number of subclones and initial sequencing data. The map shows linkage of IS1358 to the putative rfb genes and to previously identified genes virA and virB (Norqvist & Wolf-Watz, 1993 ). Restriction enzyme sites are indicated above the line. The arrows indicate the direction of transcription. wbh is the accepted prefix for the rfb genes of V. anguillarum O1.

 
rfb-like genes are located between IS1358 elements in V. anguillarum O2
The multiple copies of IS1358 found in serogroups O2 and O9 and the single uninterrupted ORFs noted for elements sequenced from these strains suggest that IS1358 may be mobile. Since elements of the same class as IS1358, such as ISAs1 from A. salmonicida, have been shown to be mobile and to be involved in genetic rearrangement (Gustafson et al., 1994 ), it is conceivable that IS1358 is also involved in rfb/polysaccharide gene rearrangements as has been proposed for the H-repeat in the Sal. enterica D2 rfb region (Xiang et al., 1994 ).

To determine if there is any relationship in the O2 and O9 serogroups between IS1358 and rfb/polysaccharide genes we investigated the genes located between some of the elements. PCR was used with an oligonucleotide (1065) complementary to the inverted repeats of IS1358, allowing DNA between the elements to be amplified. When chromosomal DNA from the strains that contained multiple copies of IS1358, ATCC 43306 (O2) and ATCC 43313 (O9), was used in the PCR reactions, it was only possible to amplify three bands of 2·2, 1·8 and 0·72 kb from ATCC 43306 (data not shown).

The amplified DNA was cloned and the sequences were analysed using BLASTX. Similarities to capsule and Rfb proteins were found on the 1·8 kb PCR product. Two genes were identified; one showed homology to bplA from the LPS biosynthesis operon of Bordetella pertussis (64% identity at DNA and amino acid level) (Allen & Maskell, 1996 ), and the other was homologous to a Vi polysaccharide biosynthesis protein, VipA from Salmonella typhi (66% identity at DNA level and 63% at amino acid level) (Hashimoto et al., 1993 ; Waxin et al., 1993 ). These two genes were designated ORF 1 (bplA) and ORF 2 (vipA), and are both thought to encode dehydrogenase enzymes involved in polysaccharide biosynthesis (Fig. 5). ORF 1, which shows significant homology to bplA, is a complete ORF whereas ORF 2 is interrupted by the IS1358 element located downstream (Fig. 5). This suggests that the IS1358 element may have transposed into this gene or complex recombination events occurred resulting in the interruption to ORF 2 and the possible introduction of ORF 1. It is conceivable that the introduction of a new gene of similar function (and disruption of the old gene) could alter the antigenic properties of O-antigen. However, it is more likely that this recombination/transposition event was one step in many in the formation of this putative rfb/polysaccharide biosynthesis region.



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Fig. 5. Map representing the DNA located around two IS1358 elements of V. anguillarum O2 derived by Southern hybridization, inverse PCR and sequencing data from various subclones. The genes within this region are indicated on the map. ORF 1 and ORF 2 correspond to the homologues of bplA and vipA, respectively. ORF 2 is truncated due to the adjacent IS1358 element. The rol (ORF 3) and rfaD (ORF 4) homologues are also represented. Arrows indicate the direction of transcription. The putative JUMPstart sequence is represented by the sequence above the large triangle above the figure. Boxed nucleotides indicate an identical match to the JUMPstart consensus sequence (Hobbs & Reeves, 1994 ). Underlined nucleotides show homology to some JUMPstart sequences.

 
Inverse PCR was used to characterize further the DNA flanking these closely linked IS1358 elements. PCR using oligonucleotide 1065 was performed on ligated chromosomal DNA that had previously been digested with NdeI. NdeI was used as it cuts inside the 1·8 kb fragment but not within IS1358. Southern hybridization using ORF 1 as a probe indicated that a fragment of approximately 3·0 kb should be generated by the PCR amplification if NdeI was used to cut the chromosome (data not shown). This single product was cloned and sequenced. Subsequent BLASTX searches revealed the presence of ORF 1 (bplA) and another ORF with homology to cld/rol genes (37% identical, 70% similar at amino acid level) from E. coli and other pathogenic bacteria (Morona et al., 1995b ) where cld/rol regulates the chain length of the O-antigen. Another ORF (ORF 4), located next to the rol homologue (ORF 3), is transcribed in the opposite direction to ORF 3 and has homology to rfaD from V. cholerae O1 (60% identical at amino acid level) (Fig. 5). The RfaD protein of V. cholerae is thought to be involved in core biosynthesis and is required for LPS production (Stroeher et al., 1995b ). Between the divergently transcribed ORFs 3 and 4 is a putative JUMPstart sequence (Hobbs & Reeves, 1994 ; 5'-TAGGAGCGGATACCTAAGGGCGGTAGCGTGC-3') (Fig. 5) often found at the beginning of polysaccharide biosynthesis associated operons. Attempts to clone the DNA further downstream of ORF 2/IS1358 proved to be unsuccessful.

The other products that were amplified from the O2 serogroup of V. anguillarum showed no significant homology to any sequences currently in the database. It is important to note that IS1358 has again been associated with rfb/polysaccharide genes in the O2 serotype of V. anguillarum, which supports the suggestion that IS1358 is commonly associated with these genetic loci.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
IS1358 has been linked to rfb/capsular loci in several V. cholerae serotypes (Manning et al., 1995 ; Stroeher et al., 1995a ; Bik et al., 1996 ). Since IS1358 has been implicated in the genetic rearrangements of rfb loci in V. cholerae (Stroeher et al., 1995a ), we were interested to determine its distribution, location and linkage in other Vibrio species. Although a number of species were examined, IS1358 was detected in only some V. cholerae and V. anguillarum serogroups and these species appear to be very closely related within Vibrio (Stroeher et al., 1994 ).

Analysis of the IS1358 elements from different V. anguillarum serogroups revealed them to be virtually identical with only minor single base pair substitutions. Within V. anguillarum O1 the elements were identical, with the exception of strain 86/3674, which was slightly more divergent (99·8%).

For IS1358 to be involved in rearrangements of rfb loci by a process involving transposition it would require a functional transposase. In this study, only strains with multiple copies of IS1358 produced a protein of the correct size (42 kDa), suggesting that the presence of multiple copies indicates the active nature of IS1358 within serogroups O2, O7 and O9. In some cases, the organization of IS1358 within the chromosome is reminiscent of complex transposon-like structures in which two insertion sequences flank the potentially mobile regions. Thus excision or transposition of this region would allow the genes trapped between the insertion elements to move. However, in the O9 serotype, the copies are widely spread throughout the chromosome. Experiments to show transposition of single copies of IS1358 have been unsuccessful in our laboratory. However, recently, Mahillon & Chandler (1998) reported that IS1358 was able to undergo simple insertion into a plasmid in E. coli producing 10 bp direct repeats.

An important finding of this study was that once again IS1358 was associated with rfb/polysaccharide genes. Furthermore, in the V. anguillarum O2 serotype, homologues to rol as well as rfaD and a putative JUMPstart sequence were found associated with this region. The rol homologue appears to be functional as it contains the essential proline/glycine-rich domain at the carboxy-terminal end of the protein (Becker et al., 1995 ). Complementation of the published Shig. flexneri rol mutant (Morona et al., 1995b ) with ORF 3 showed 10–15% restoration of function as shown by silver staining (data not shown). Thus the organization of this putative rfb region in V. anguillarum O2 is reminiscent of that observed in V. anguillarum O1 and V. cholerae O1 and O139. All of these loci are linked to rfaD with an adjacent JUMPstart sequence and then the LPS biosynthesis genes. Recently, the rfb locus from the V. anguillarum O2 serogroup was cloned and expressed by Amor & Mutharia (1995) . A published chromosomal map of this region was examined to see if the V. anguillarum O2 genes sequenced in our study were located in this rfb locus; however, a number of discrepancies could not be resolved. This may be explained by strain differences, or indicate the presence of other rfb-like genes in the O2 chromosome that may contribute to LPS (O-antigen) or capsule biosynthesis.

In conclusion, we demonstrated that IS1358 is widely distributed in V. anguillarum and commonly linked to genes associated with polysaccharide biosynthesis. This clearly implies that it has the potential to mediate transposition and shuffling of polysaccharide genes and generate new combinations of genes which could produce new O-antigen and/or capsule specificities within the Vibrionaceae.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Jeremy Carson and Peter Hanna for kindly supplying the Vibrio anguillarum strains, and John Albert for providing the type Vibrio cholerae strains. K.E.J. was the recipient of an Australian Postgraduate Research Award. This study was supported by the Australian Research Council and the Diarrhoeal Diseases Global Vaccines Programme of WHO. We would also like to thank Craig Daniels for critically reading this paper.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Allen, A. & Maskell, D. (1996). The identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis. Mol Microbiol 19, 37-52.[Medline]

Amor, P. A. & Mutharia, L. M. (1995). Cloning and expression of rfb genes from Vibrio anguillarum serotype O2 in Escherichia coli: evidence for cross-reactive epitopes. Infect Immun 63, 3537-3542.

Becker, A., Niehaus, K. & Puhler, A. (1995). Low molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterised by a periplasmic N-terminal domain and a missing C-terminal domain.Mol Microbiol 16, 191-203.[Medline]

Bik, E. M., Bunschoten, A. E., Gouw, R. D. & Mooi, F. R. (1995). Genesis of the novel epidemic Vibrio cholerae O139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis.EMBO J 14, 209-216.[Abstract]

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Received 20 April 1999; revised 29 October 1999; accepted 4 November 1999.