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
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ABSTRACT |
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Keywords: O-antigen, polysaccharide, insertion sequences, transposase, evolution
The GenBank accession numbers for the IS1358 sequences are U93587U93597.
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INTRODUCTION |
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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.
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METHODS |
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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
phage heads using the PACKAGENE system (Promega) and transfected into E. coli DH5
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 manufacturers 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 9841002 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 1624 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 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.
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RESULTS |
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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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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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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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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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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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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.
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DISCUSSION |
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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 1015% 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 April 1999;
revised 29 October 1999;
accepted 4 November 1999.