Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore 1192601
Author for correspondence: K. Y. Leung. Tel: +65 8747835. Fax: +65 7792486. e-mail: dbslky{at}nus.edu.sg
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ABSTRACT |
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Abbreviations: R/M, restriction/modification;; SSH, suppression subtractive hybridization
The GenBank accession numbers for the sequences determined in this work are given in Table 5.
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INTRODUCTION |
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A. hydrophila strains PPD134/91 and PPD35/85 were isolated from our local environment and used in our previous studies. A. hydrophila PPD134/91 was defined as virulent whereas PPD35/85 was defined as avirulent on the basis of their differences in LD50 values in tilapia and blue gourami (Leung et al., 1995b ). PPD134/91 can survive and proliferate in tilapia serum and phagocytes (Leung et al., 1995a
, b
), and it can internalize and induce severe morphological changes in EPC (epithelioma papillosum of carp) cells (Tan et al., 1998
). On the other hand, A. hydrophila PPD35/85 is serum-sensitive, unable to invade or produce cytotoxic effects in EPC cells, and is incapable of surviving in phagocytes after opsonization.
It is important to determine whether there are any special virulence genes present in A. hydrophila PPD134/91 but not in PPD35/85. Subtractive hybridization has been used to identify sequences that are present in one genome but absent in another (Straus & Ausubel, 1990 ; Mahairas et al., 1996
). The analysis of the differences between two complex genomes holds promise for the discovery of unknown virulence-associated factors and probes useful for genetic studies (Lisitsyn et al., 1993
; Quinn et al., 1997
). Traditional subtractive hybridization methods involved several rounds of hybridization and physical separation of single-stranded and double-stranded DNA. Recently a new technique called suppression subtractive hybridization (SSH) overcame some of these limitations. The step of suppression PCR can prevent undesirable amplification while enrichment of target molecules proceeds (Diatchenko et al., 1996
; Gurskaya et al., 1996
).
The objective of this study was to identify genetic differences between virulent and avirulent strains of A. hydrophila. Sixty-nine subtracted genomic regions unique to PPD134/91 were identified. Among them, 22 fragments (21 ORFs) were present in most of the virulent strains. These presumptive virulence genes were identified, sequenced, and their biological functions analysed. This will form a foundation for further studies in elucidating how A. hydrophila causes disease in humans and fish.
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METHODS |
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Suppression subtractive hybridization (SSH).
Bacterial genome subtraction was performed following the user manual of the PCR-Select Bacterial Genome Subtraction Kit (Clontech). Briefly, the tester (PPD134/91) and driver (PPD35/85) genomic DNAs were digested with RsaI. The tester DNA was then subdivided into two portions, each of which was ligated with a different adaptor provided by the subtraction kit. Two hybridizations were performed. In the first, an excess of driver was added to each adaptor-ligated tester sample. The samples were then heat-denatured and allowed to anneal. In the second hybridization, the two primary hybridization samples were mixed together without denaturing. The entire population of molecules was then subjected to PCR to amplify the tester-specific sequences. The PCR amplification product was cloned into pT-Adv and transformed into E. coli TOP10F' competent cells according to the manual of the Advantage PCR Cloning Kit (Clontech). Positive clones were screened on LB medium supplemented with X-Gal (Sigma), IPTG (Sigma) and ampicillin.
Southern hybridization.
Southern blots were performed to identify the subtractive clones that contained PPD134/91 unique fragments. At the same time, similar probes were used to screen genomic DNA of several virulent and avirulent strains of A. hydrophila. Nick-translated RsaI fragments of recombinant plasmids were used as probes to hybridize with RsaI digests of PPD134/91, PPD35/85, and other bacterial genomic DNA. Transfer of DNA to nylon membrane (GeneScreen, NEM Research Products) and hybridization conditions were in accordance with standard methods (Sambrook et al., 1989 ). Probe DNAs were labelled by nick-translation with biotin-14-dATP (BioNick Labelling System, Gibco-BRL) and visualized with strepavidinalkaline phosphate conjugate (BluGene Nonradioactive Nucleic Acid Detection System, Gibco-BRL).
DNA sequencing.
DNA sequencing was carried out on an Applied Biosystems PRISM 377 automated DNA sequencer by the dye termination method. The ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit was used (Applied Biosystems). The sequences were edited by using the manufacturers software. Sequence assembly and further editing were done with DNASIS DNA analysis software. TBLASTN and FASTA sequence homology analyses were performed by using the National Centre for Biotechnology Information BLAST network service.
Genome walking.
Unknown genomic DNA sequences adjacent to the subtracted DNA fragments (F3, 33, 44, 46, 52, 101, 106, 108, 109 and 121) were identified following the user manual of the Universal GenomeWalker Kit (Clontech). GenomeWalker libraries using five restriction enzymes (DraI, EcoRV, PvuII, ScaI and StuI) were constructed and two PCR amplifications were performed for the DNA walking. Specific primers were synthesized by Gibco-BRL for primary (P) and nested (N) PCR as well as for right (R) and left (L) sides of each subtracted fragment. PCR was performed using an Advantage Tth Polymerase Mix purchased from Clontech and following two-step cycle parameters: 7 cycles of 25 s at 94 °C, 3 min at 72 °C, 32 cycles of 25 s at 94 °C and 3 min at 67 °C. The oligonucleotides for genome walking were as follows: F3-LP, 5'-GCATCCCCGTTTCGCATTATC-3', F3-LN, 5'-CCGTTTCGCATTATCTGAAC-3', F3-RP, 5'-GATAATGCGAAACGGGGATGC-3', F3-RN, 5'-CGGGGATGCCACGGCATCC-3'; F33-LP, 5'-ATTTGATGCGCTTTTGTCCC-3', F33-LN, 5'-CGCTTTTGTCCCATTGACAG-3', F33-RP, 5'-TCGTTGCTTTTGGGTTACCAAG-3', F33-RN, 5'-GGGTTACCAAGATACTACGTTC-3'; F44-LP, 5'-ACACTCCCACGTCGTTTTAC-3', F44-LN, 5'-GCTGTTTTACTCATAGCTAC-3', F44-RP, 5'-GCTTGCATCGTTATGCGTCTGT-3', F44-RN, 5'-GCGTCTGTAGCTATCAATGTG-3'; F46-RP, 5'-GCAGGTGGGGAAATCGATGAAC-3', F46-RN, 5'-TCGATGAACTCAAGGCGGT-3'; F52-LP, 5'-TGGAGTGATGTCGCGTGCGG-3', F52-RP, 5'-GTAATCCCCAAAACCCG-3'; F101-LP, 5'-CATCATCGTCAGAAAATGCG-3', F101-LN, 5'-AGAAAATGCGTTATTTCTAC-3', F101-RP, 5'-CTAAGATTGTGTCTGCGAGTG-3', F101-RN, 5'-GTCTGCGAGTGATAAACAAAAG-3'; F106-LP, 5'-TGC TCT CAT TGC TGG GGG GC-3', F106-LN, 5'-GCTGGGGGGCACCTTGTCCAC-3', F106-RP, 5'-ACATCTAGCGCACGAGATAAAT-3', F106-RN, 5'-CACGAGATAAATCAGGCCCAAGC-3'; F108-LP, 5'-AGACAAGCAGAATAACGCCCCG-3', F108-LN, 5'-AATAACGCCCCGAAATATAACCG-3', F108-RP, 5'-CAGCGGATTGGCGAAGGTATT-3', F108-RN, 5'-TTGGCGAAGGTATTTATGTTG-3'; F109-LP, 5'-TCGTCCTTATTTCGGGTAGGGATCAAGCGG-3', F109-RP, 5'-CTCCTTGGAAGGTAGACCCCGAACTCTACT-3'; F121-LP, 5'-GCCCATAGCATCCACATCGG-3', F121-LN, 5'-TCCACATCGGCCGTATATTC-3', F121-RP, 5'-GATTACCAGAGGTTTGGCCAAT-3', F121-RN, 5'-GAGGTTTGGCCAATATTGCCC-3'.
Nucleotide sequence accession numbers.
Sixteen ORFs, which derived from 23 subtracted fragments, had homologues with known sequences in GenBank and were assigned with 16 accession numbers (Table 5). Seven of these (F3, 33, 46, 52, 101, 106 and 121) represented complete ORFs via genome walking while nine of them represented partial ORFs.
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RESULTS |
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In the first group (tester only), subtracted fragments only hybridized to the tester genome digest. There were 69 clones (60% of the total population studied) in this group. Examples of this group included F33, 51, 60 and 66 (Fig. 1). In F51, 60 (Fig. 1
) and 111 (data not shown), double bands were detected on the tester genomic DNA.
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Inserts in the last group (false positive) hybridized to both tester and driver, and had the same hybridization patterns (F10 and 40; Fig. 1). These clones were false positive, which is a result of the inefficiency of subtraction hybridization. Our Southern analysis indicated that we achieved a high level of enrichment of PPD134/91-specific DNA whereby 60% of our subtracted clones were present only in the tester but not in the driver (Table 2
). In the tester-only group, 75% of the subtracted fragments were less than 0·69 kb.
Identification of common virulence genes in A. hydrophila
Southern hybridization was carried out to survey the distribution of these 69 PPD134/91-specific fragments among eight virulent and seven avirulent strains of A. hydrophila. Fig. 2 and Tables 3
and 4
show the hybridization results. The fraction of hybridization was calculated for both virulent and avirulent strains (x/8 for virulent and y/7 for avirulent strains, where x and y are the number of hybridized strains).
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Subtracted fragments that were tester-specific (i.e. hybridized only to PPD134/91 plus at most two other strains) were classified as group 2 (Table 4, x
3 and y
2, n=47). In F101 and 121, among all the virulent and avirulent strains, only PPD134/91 and PPD64/90 (for F121 only) hybridized (Fig. 2
).
Sequence analysis and gene walking
The 69 subtracted fragments were sequenced and their DNA sequences were subjected to homology search in PIR/GenBank. Of the 69 subtracted fragments, 46 (66·7%) demonstrated no significant matches with entries in the databases and potentially represented new and novel virulence determinants in A. hydrophila (data not shown). In the remaining 33·3% of the clones, 23 DNA fragments representing 16 ORFs showed high homology to known proteins of other bacteria and four identical pairs were found (Table 5).
From the 16 ORFs that had homologues, five belong to group 1 (F2/3, 52, 106, 108 and 88/109; Table 3), 11 to group 2 (30, 33, 36/121, 44/86, 46, 50, 51, 60/91/101/119, 82, 110 and 126; Table 4
). Full sequences were determined by genome walking for F3, 33, 44, 46, 52, 101, 106 and 121 for detailed analysis (Table 5
). Table 5
shows subtracted and full-length fragments that showed high homology to known sequences in GenBank.
Five ORFs in group 1 were found to correspond to known bacterial virulence genes of A. hydrophila. These include haemolysin (hlyA), protease (oligopeptidase), histone-like protein (HU-2), outer-membrane protein (Omp) and multidrug-resistance gene. In group 2, five groups of genes were found to represent the heterogeneity of motile aeromonads. These include genes for the synthesis of O-antigen of LPS (phosphomannomutase, rhamnosyl transferase, O-acetyltransferase, O-antigen methyltransferase, GDP-mannose 4,6-dehydratase and mannosyltransferase B), type II restriction/modification system (modification methyltransferase of PstI and BsuBI), CII of bacteriophage P4, histidine protein kinase and DNA regulatory protein (repA).
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DISCUSSION |
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Surveying the distribution of virulence genes in A. hydrophila
Two strains were chosen for the SSH protocol: A. hydrophila PPD134/91 as the tester strain and PPD35/85 as the driver strain. In order to confirm that subtracted fragments were unique not only in the tester strain but also among other virulent strains, Southern hybridization analysis was used to probe eight virulent and seven avirulent strains of A. hydrophila (Tables 3 and 4
). Two groups of clones were derived based on the hybridization patterns. Presumptive universal virulence genes (21 ORFs including five predicted proteins) were classified in group 1. Genes encoding heterogeneity or strain variations (44 ORFs including 11 predicted proteins) are in group 2 and these also include some known virulence genes. Genes in group 2 may have undergone extensive modifications such that they are not even conserved among different strains of the same species. Many of these genes are described for the first time in motile aeromonads. Studying the distribution of these gene fragments provides insight to their relative importance.
Group 1 (common virulence genes)
Haemolysin (F108).
F108 is highly homologous to the hlyA gene of an A. hydrophila strain, A6 (Wong et al., 1998 ), and a ß-haemolysin gene of A. salmonicida (Hirono & Aoki, 1993
). Haemolysin is one of the virulence factors produced by motile aeromonads and has been cloned from different strains of aeromonads (Howard et al., 1996
; Wong et al., 1998
). A. hydrophila secretes at least two types of haemolysins. One of them is aerolysin (AerA) which can oligomerize on erythrocyte cell membranes, form channels, and lead to cell lysis (Wilmsen et al., 1990
). The second is a non-channel-forming haemolysin (HlyA) and is proposed to be a Vibrio cholerae-HlyA-like haemolysin (Wong et al., 1998
). These two types of haemolytic toxins are low in homology and believed to be distinct. It was suggested that these two haemolytic toxins contribute to virulence in A. hydrophila and are widespread within virulent strains of motile aeromonads (Vadivelu et al., 1995
; Hirono et al., 1992
). Therefore, it is not a surprise to detect the absence of the hlyA gene in four of our avirulent strains including PPD35/85. In addition, our hybridization results confirm that hlyA is present in most of our virulent strains (7/8).
Histone-like protein (F52).
F52 is homologous to HU-2 genes of Aeromonas proteolytica (Giladi et al., 1992 ), Salmonella typhimurium (Higgins et al., 1988
) and E. coli (Kano et al., 1987
). These are histone-like proteins of prokaryotes (such as HU proteins and integration host factors) that are small, basic, heat-stable and bind to single- and double-stranded DNA (Drlica & Rouviere-Yaniv, 1987
). Although the biological functions of histone-like proteins are not fully understood, they are known to alter DNA recognition by changing DNA dynamic flexibility and accessibility (Flashner & Gralla, 1988
). The resulting alterations in DNA structure and topology affect several cellular processes, including initiation of DNA replication, DNA partitioning and cell division, and transposition of bacteriophage Mu (Huisman et al., 1989
; Jaffe et al., 1997
). In addition to the physiological functions, secreted histone-like protein has been demonstrated to have a potential role in the pathogenesis of Streptococcus-induced tissue inflammation (Stinson et al., 1998
). The biological function of the HU-2 gene in A. hydrophila will be elucidated in future experiments.
Protease (oligopeptidase A) (F88, 109).
A number of extracellular proteases of A. hydrophila (metallopeptidases and serine peptidases) have been described (Howard et al., 1996 ) and correlations have been made between the production of proteases and virulence (Leung & Stevenson, 1988b
). Extracellular proteases may aid the organism in overcoming initial host defences such as resistance to serum killing, and provide amino acids for cell proliferation (Leung & Stevenson, 1988b
). Furthermore, proteases are needed for the maturation of exotoxins such as aerolysin (Howard & Buckley, 1985
). For F88 and 109, the predicted protein showed similarity to the OpdA sequence of S. typhimurium, Haemophilus influenzae and E. coli (Conlin & Miller, 1992
; Conlin et al., 1992
; Fleischmann et al., 1995
). Oligopeptidase A is the major soluble enzyme in E. coli which is able to hydrolyse free lipoprotein signal peptide in vitro (Novak et al., 1986
). Fragments F88 and F109, encoding the putative OpdA protein, were present in all the virulent strains of A. hydrophila we tested and may be involved in peptide processing of virulence factors which in turn affect pathogenicity.
Omp (F2 and 3).
A homologue of the outer-membrane protein OmpA was identified in our tester strain. The OmpA protein is one of the major outer-membrane proteins of a wide range of Gram-negative bacteria such as A. salmonicida (Costello et al., 1996 ), Shigella dysenteriae (Braun & Cole, 1982
) and E. coli (Beck & Bremer, 1980
). Major physiological functions include maintenance of the structural integrity and morphology of the cells and porin activity, as well as a role in conjugation and bacteriophage binding. A role in bacterial virulence has been implicated in increased serum resistance as in E. coli (Weiser & Gotschlich, 1991
) and Neisseria gonorrhoeae (Rice et al., 1986
).
Drug-resistance gene (F106).
Bacteria have developed a number of mechanisms to protect them from environmental toxins and antibiotics, including degradation and inactivation of drugs by enzymic modifications, alteration of the drug target, and the production of multidrug transporters (Lewis, 1994 ; Nikaido, 1994
). The predicted protein of F106 showed similarity to multidrug-resistance protein 2 of Bacillus subtilis (P39843, E= 1e-07) (Ahmed et al., 1995
). It is possible that this multidrug transporter in A. hydrophila PPD134/91 and other virulent strains is used for the transport of other antibiotics or toxic substances. This presumptive virulence factor will be studied in future experiments.
Group 2 (PPD134/91-specific sequences)
O-antigen/polysaccharide (F33, 36/121, 44/86, 46, 51, 110 and 126).
LPS is a major component of the outer membrane of Gram-negative bacteria. It consists of three regions: the lipid A, the core oligosaccharide and the O-antigen (Schnaitman & Klena, 1993 ). Serotypes are distinguished on the basis of O-antigens, and heterogeneity is known to exist among motile aeromonads (MacInnes et al., 1979
; Leblanc et al., 1981
). The unique set of O-antigen modification enzymes (such as rfb) carried by various strains of bacteria contributes to the diversity of O-antigen. A. hydrophila PPD134/91 and PPD35/85 belong to different serogroups (Leung et al., 1995b
). This is probably the reason for the absence of some O-antigen modification enzymes in A. hydrophila PPD35/85. These include mannosyltransferase B (F33), GDP-mannose 4,6-dehydratase (F36, 121), phosophomannomutase (F44, 86), rhamnosyltransferase (F46), O-acetyltransferase (F51), glycosyltransferase (F110) and O-antigen methyltransferase (F126).
The O-antigen is thought to be important in the pathogenesis of many bacteria. It is involved in serum resistance and protecting bacteria from phagocytosis (Stinavage et al., 1989 ). The O-antigen of A. hydrophila is also an important adhesin (Merino et al., 1996
). When aeromonads were devoid of O-antigen, their LD50 value increased by 100-fold (Merino et al., 1991
). Further studies will be conducted to investigate the differences between the LPSs of these two strains and their roles in pathogenicity.
Restriction/modification system (F60, 91, 101 and 119).
The ORF encoded by F60, 91, 101 and 119 showed high homology match to site-specific DNA-methyltransferase BsuBI of B. subtilis (Xu et al., 1992 ) and Rhizobium leguminosarum (Rochepeau et al., 1997
). These enzymes belong to a type II restriction/modification (R/M) system that functions to destroy foreign DNA (Xu et al., 1992
). Thus, it is reasonable to postulate the existence of a similar type II R/M system in A. hydrophila PPD134/91. This is the first report of a R/M system in A. hydrophila. Its presence may be the reason why previous attempts to introduce plasmids and transposons into the tester strain (PPD134/91) failed (data not shown). Understanding and crippling this R/M system will allow easier genetic manipulation of the bacteria.
Conclusions
Elucidating the genetic differences between virulent and avirulent strains of A. hydrophila provides insight into the types of virulence genes. In our previous studies, many virulence factors present in PPD134/91 but not in PPD35/85 were characterized (Leung et al., 1995a , b
; Tan et al., 1998
). Attempts are made to correlate some of these phenotypes with the presumptive virulence genes we obtained in this study. Features such as resistance to serum and to phagocyte-mediated killing in PPD134/91 can be explained by the detection of Omp and O-antigen-modification enzymes. The previous failure to introduce transposons into PPD134/91 has also been explained by the detection of a type II R/M system. Furthermore, the presence of haemolysin, histone-like protein, multidrug-resistance protein and other proteins is also important in virulence. Elucidation of other virulence characteristics such as adhesion and invasion awaits further study of the novel genes and examination of all the subtracted clones. Although the subtraction was successful, the library isolated is by no means complete. This was shown by the presence of only four pairs of identical clones (2/3, 44/86, 60/101, 91/119). New rounds of SSH will be carried out to complete the mapping of genetic differences between virulent and avirulent strains. Strains PPD64/90 and L36 drew particular attention. Although avirulent, they contained many tester-specific fragments (Tables 3
and 4
). There might exist some regulatory mechanism or other key virulence factors that silenced their expression.
It is hoped that the identification of critical genetic differences between virulent and avirulent A. hydrophila can provide insights into the pathogenic mechanisms, thus supplying the groundwork for the development of new therapies for A. hydrophila infections in both fish and humans. On the other hand, the presence of HlyA and a bacteriophage protein CII among the subtracted fragments may signify the presence of pathogenicity island(s) in A. hydrophila (Hacker et al., 1997 ). Experiments are currently under way to address this possibility.
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ACKNOWLEDGEMENTS |
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Received 31 August 1999;
revised 2 December 1999;
accepted 11 December 1999.