Identification of DNA Sequences Specific for Vibrio vulnificus Biotype 2 Strains by Suppression Subtractive Hybridization

Vibrio vulnificus can be divided into three biotypes, and onlybiotype 2, which is further divided into serovars, containseel-virulent strains. We compared the genomic DNA of a biotype2 serovar E isolate (tester) with the genomic DNAs of threebiotype 1 strains by suppression subtractive hybridization andthen tested the distribution of the tester-specific DNA sequencesin a wide collection of bacterial strains. In this way we identifiedthree plasmid-borne DNA sequences that were specific for biotype2 strains irrespective of the serovar and three chromosomalDNA sequences that were specific for serovar E biotype 2 strains.These sequences have potential for use in the diagnosis of eelvibriosis caused by V. vulnificus and in the detection of biotype2 serovar E strains.

INTRODUCTION

Top

Introduction

Vibrio vulnificus is a gram-negative estuarine bacterium thatproduces diseases in humans and eels. In humans, this organismmay cause serious wound infections and septicemia with a highmortality rate, particularly in people with underlying conditionssuch as alcoholism hemochromatosis, and liver cirrhosis (12,25). In cultured eels, this species can cause the disease vibriosis,which was originally described in Japan in 1976 (20) and isone of the main causes of economic losses in brackish watereel culture in Europe (7, 16). The disease in its acute formis a primary septicemia characterized by external and internalhemorrhages affecting the major organs, such as the liver, kidney,spleen, and pancreas (6).

Strains of V. vulnificus have been subdivided into three biotypesbased primarily on differences in biochemical properties, suchas indole production and cellobiose fermentation, as well asthe epidemiological pattern and host range (10, 24). Biotype2 comprises the strains virulent for eels (5, 22, 24), whichcan be further classified into different serovars (9, 17). Onlyone of these serovars, serovar E, is clearly related to bothhighly virulent epizootics and human infections (3).

The mechanism of virulence of V. vulnificus biotype 2 in eelvibriosis remains unclear, although a few studies have beenconducted with serovar E strains (2, 4). We hypothesized thatthe biotype 2 strains may have genetic information, which isabsent from strains of other biotypes, that enables them toinfect and cause vibriosis in eels. In this case, a genome comparisonof biotypes to identify DNA sequences specific for the biotype2 strains may help workers discover the virulence determinantsof V. vulnificus for eels.

Suppression subtractive hybridization (SSH), a technique originallydeveloped to study gene expression in eukaryotes (15), has beensuccessfully used to identify strain- or species-specific DNAsequences in a variety of bacteria. In this technique, the DNAfragments derived from regions that are present in one strain,designated the tester, but absent in another strain, designatedthe driver, are not annealed when the genomic DNA fragmentsfrom the tester and driver are mixed, denatured, and reannealed.Such DNA fragments can then be amplified by PCR and cloned intoa suitable vector for sequence determination. Some genomic islandsimplicated in the virulence of a number of bacteria have beenidentified in this way (1, 11, 13, 14, 18, 23, 26).

The main objective of the present work was to identify V. vulnificusbiotype 2-specific DNA sequences by SSH, from which potentialvirulence genes for eels might be identified and probes usefulfor rapid diagnosis of eel vibriosis might be developed.

Genomic subtraction between V. vulnificus biotype 1 and biotype 2 strains.

Top

Genomic subtraction between v....

SSH was performed with a PCR-Select bacterial genome subtractionkit (Clontech, Palo Alto, CA). A serovar E strain, CECT4602,with high virulence for eels (5) was selected as the tester,and three clinical biotype 1 isolates, CS9133, YJ016, and ATCC27562, were selected as the drivers (Table 1). The sizes ofthe subtraction products amplified after PCR ranged from 300bp to 1,500 bp. These products were subsequently cloned intothe pGEM-T easy vector in Escherichia coli DH5 ${alpha}$ , and a totalof 85 recombinant clones were obtained. The specificity of theDNA fragment in each clone was checked further with the tester,the drivers, and four more biotype 2 serovar E strains by Southernhybridization. Thirty-three clones were found to contain thebiotype 2-specific DNA fragments. These clones were tested againby dot blot hybridization to exclude the clones that cross-hybridizedwith the randomly chosen clones. Finally, eight clones (designatedCT005, CT010, CT012, CT023, CT025, CT051, CT061, and CT067)that contained different biotype 2-specific DNA fragments wereidentified (the results of Southern hybridization with CT005are shown in Fig. 1).

View this table:
[in this window]
[in a new window]
TABLE 1. Sources, origins, and serovars of the V. vulnificus strains used in this study

View larger version (46K):
[in this window]
[in a new window]
FIG. 1. Southern hybridization to examine the specificity of a cloned sequence. The genomic DNAs were prepared from biotype 1 strains CS9133, ATCC 27562, and YJ016 (lanes A to C) and biotype 2 serovar E strains CECT4602, CECT4601, CECT4604, CECT4605, and ATCC 33147 (lanes D to H). They were digested with HindIII and then probed with ³²P-labeled plasmid DNA that was extracted from the subtractive clone CT005. Lane M contained a 1-kb Plus DNA ladder (Invitrogen).

Characterization of the biotype 2-specific sequences.

Top

Characterization of the biotype...

The nucleotide sequences of the biotype 2-specific DNA fragments(designated seq5, seq10, seq12, seq23, seq25, seq51, seq61,and seq67 for clones CT005, CT010, CT012, CT023, CT025, CT051,CT061, and CT067, respectively) were determined. DNA sequencesthat exhibited high levels of homology with the identified biotype2-specific sequences were then searched in the GenBank databaseby BLASTX. As shown in Table 2, four of these identified sequencesexhibited significant amino acid sequence homology with otherdatabase entries; seq5 and seq10 exhibited significant aminoacid sequence homology with a putative transposase of Vibrioanguillarum, and seq12 and seq67 exhibited significant aminoacid sequence homology with hypothetical proteins of Haemophilusinfluenzae and Vibrio parahaemolyticus, respectively.

View this table:
[in this window]
[in a new window]
TABLE 2. Features of the biotype 2-specific DNA sequences

The biotype 2 serovar E and serovar O3 strains harbor plasmidswith different molecular weights (8, 17, 19). We further determinedthe location of each identified biotype 2-specific sequencein the genome of CECT4602 by Southern hybridization using totalDNA and plasmid DNA, respectively, as the templates. The DNAsequence was considered to be located in the plasmid if theprobe derived from it hybridized with both the plasmid and totalDNA. In contrast, the DNA sequence was considered to be locatedin the chromosome if the probe derived from it hybridized withthe total DNA but not with the plasmid. seq5, seq10, seq12,seq25, and seq51 were found to be located in the plasmid, whileseq23, seq61, and seq67 were found in the chromosome (Table2). We then examined the plasmid profiles of eight serovar Estrains, including CECT4602, and one biotype 2 serovar A strainand found that all the strains harbored at least one plasmid.The restriction patterns of plasmids of serovar E strains weresimilar, while the restriction pattern of the serovar A strainwas significantly different (Fig. 2A). Nevertheless, all thebiotype 2 strains tested (both serovar E and serovar A) hada common HindIII-restricted DNA fragment that hybridized withthe probe derived from seq5, seq10, seq12, seq25, or seq51 (onlythe results for seq51 are shown in Fig. 2B).

View larger version (58K):
[in this window]
[in a new window]
FIG. 2. Southern hybridization of plasmids of various biotype 2 strains probed with a biotype 2-specific sequence. (A) The plasmid DNA extracted from each strain was digested with HindIII, fractionated by electrophoresis on a 0.8% agarose gel, and visualized by staining with ethidium bromide. (B) Southern hybridization of the digested plasmid DNAs shown in panel A probed with the ³²P-labeled PCR product amplified from seq51. Lanes A to I contained plasmid DNAs from biotype 2 strains CECT4601, CECT4602, CECT4604, CECT4605, ATCC 33147, CECT4603, CECT5198, CECT4607, and CECT4864. Most of these strains are serovar E strains; the only exception is CECT5198, which is a serovar A strain. The restriction fragment in panel A that was hybridized by the probe is indicated by an arrow.

Distribution of the biotype 2-specific sequences in V. vulnificus and other species.

Top

Distribution of the biotype...

The specificity of identified biotype 2-specific sequences wasfurther tested with 111 strains of V. vulnificus (Table 1) and37 strains of other species (listed below) by PCR using theprimer pairs derived from these sequences (Table 3). All ofthe V. vulnificus strains except five biotype 2 strains and21 biotype 1 strains that had been tested previously were firsttested for virulence in juvenile European eels (average weight,8 to 10 g) by a previously described method (5). As expected,all of the biotype 2 strains tested, irrespective of the serovar,were virulent for eels, while all biotype 1 and biotype 3 strainstested were avirulent.

View this table:
[in this window]
[in a new window]
TABLE 3. Primer sequences derived from the tester-specific sequences obtained from SSH

The strains of the other species tested are all fish pathogensor members of the normal fish microbiota. These strains belongedto the following species (one strain of each unless indicatedotherwise): Vibrio aesturianus, Vibrio alginolyticus, Vibrioanguillarum, Vibrio campbellii, Vibrio harveyi/Vibrio carchariae(two strains), Vibrio cholerae, Vibrio cincinnatiensis, Vibriodiazotrophicus, Vibrio fischeri, Vibrio fluvialis, Vibrio furnissii(two strains), Vibrio mediterranei, Vibrio mimicus, Vibrio mytilii,Vibrio natriegens, Vibrio nereis, Vibrio nigripulchritudo, Vibrioordalii, Vibrio orientalis, Vibrio parahaemolyticus, Vibrioproteolyticus, Vibrio salmonicida, Vibrio splendidus, Vibrioscophthalmi, Aeromonas allosaccharophila, Aeromonas encheleia,Aeromonas hydrophila, Aeromonas jandaei (two strains), Aeromonassobria, Edwarsiella tarda, Photobacterium damselae, Plesiomonasshigelloides, Pseudomonas sp., and Shewanella putrefaciens.

For PCRs, total DNA was isolated from an overnight bacterialculture and used as the template in a PCR. The reaction mixture(50 µl) contained 200 ng DNA, each deoxynucleoside triphosphateat a concentration of 6.25 mM, 75 mM MgCl₂, each primer at aconcentration of 0.2 µM, and 2.5 U of Taq polymerase (AmershamBiosciences, Buckinghamshire, England) in 1x PCR buffer (AmershamBiosciences). The reaction started with 5 min of denaturationat 94°C, which was followed by 25 cycles of 30 s of denaturationat 94°C, 1 min of annealing at 55 to 65°C, and 1 minof extension at 72°C. An additional extension at 72°Cfor 10 min completed the reaction. None of the sequences wasdetected in any other species tested, including V. anguillarumand V. parahaemolyticus, which were shown to contain sequenceshighly homologous to seq5 and seq10 and to seq67, respectively(Table 2). This was probably because the primers either werederived from sequences that were not shared by these speciesor contained high number of mismatches, despite having beenderived from the homologous regions. seq10, seq23, seq25, seq51,seq61, and seq67 were not detected in the biotype 1 and biotype3 strains (Table 4). seq5 and seq12 turned out to be not specificfor the biotype 2 strains; the former sequence was also detectedin three biotype 3 strains, and the latter sequence was presentin some biotype 1 strains and absent in several biotype 2 strains(data not shown).

View this table:
[in this window]
[in a new window]
TABLE 4. Summary of the PCR results for V. vulnificus strains tested with primer pairs derived from various biotype 2-specific DNA sequences

The plasmid-borne sequences seq10, seq25, and seq51 were biotype2 specific since all biotype 2 strains, irrespective of theserovar, gave a positive reaction with the primer pairs derivedfrom them (Table 4). This result suggests that the plasmidsmay be associated with virulence for eels. We are currentlyisolating derivatives of some biotype 2 strains that are curedof their plasmids, and we will test such strains for their virulencein eels. On the other hand, the primer pairs derived from seq23,seq61, and seq67, which were located on the chromosome, reactedonly with serovar E strains (Table 4).

In summary, by using SSH we identified the DNA sequences ofV. vulnificus that are specific for biotype 2 and biotype 2serovar E strains. These sequences are potentially useful fordeveloping a multiplex PCR method for the diagnosis of eel vibriosiscaused by V. vulnificus and for the detection of biotype 2 serovarE strains. The sequences common to biotype 2 strains appearedto be plasmid borne, suggesting that the virulence of this bacteriumin eels may be acquired by horizontal transfer of a virulenceplasmid.

Nucleotide sequence accession numbers.

Top

Nucleotide sequence accession...

The GenBank accession numbers for the biotype 2-specific sequencesare AY757304 (seq5), AY691407 (seq10), AY757305 (seq12), AY691410(seq23), AY691408 (seq25), AY691409 (seq51), AY691411 (seq61),and AY691412 (seq67).

ACKNOWLEDGMENTS

This work was supported in part by grant NSC 89-2320-B-006-018from the National Science Council, Taiwan, Republic of China,and by grant AGL2002-01291 from the Spanish Ministry of Scienceand Technology.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, College of Medicine, National Cheng-Kung University, Tainan 701, Taiwan, Republic of China. Phone: 886-6-2353535, ext. 5635. Fax: 886-6-2082705. E-mail: h061453{at}mail.ncku.edu.tw.

REFERENCES

Top