Genetic Basis for Variability in Arabinose Fermentation in Vibrio parahaemolyticus

System. Appl. Microbiol. 15, 420-426 (1992) © Gustav Fischer Verlag, StuttgartlNew York

Genetic Basis for Variability in Arabinose Fermentation in Vibrio parahaemolyticus SUMITRA MURALIDHAR 1 and MARY

J. VOLL"

Department of Microbiology, University of Maryland, College Park, MD 20742, USA 1 Laboratory of Viral Diseases, NIAID, NIH, 12441 Parklawn Dr., Rockville, MD 20852, USA

Received January 8, 1992

Summary Ability to ferment arabinose is a variable characteristic in the species Vibrio parahaemolyticus. To study the genetic basis of this variability, the arabinose fermentation genes from an arabinose utilizing strain were cloned into the plasmid vector pBR322. Complementation studies employing plasmids deleted in segments of the cloned DNA established the presence of genes analogous to the araA, araB, araC, and araD genes of E. coli. The order of the ara genes in V. parahaemolyticus is different than that in Escherichia coli or Bacillus subtilis. Three radioactively labelled probes spanning the ara structural gene region were hybridized under stringent conditions with HindIlI digested genomic DNA from arabinose positive and negative strains of V. parahaemolyticus and other Vibrio species. No DNA homologous to the probes was found in the arabinose negative V. parahaemolyticus strains indicating that absence rather than mutational inactivation of gene expression was responsible for the Ara- phenotype. Little or no hybridization of the ara probes to arabinose positive Vibrio fluvialis and Vibrio hollisae strains was detected.

Key words: V. parahaemolyticus - Arabinose fermentation - Ara genes - Variable characteristic - Gene cloning - DNA:DNA hybridization

Introduction Ability to ferment arabinose is an important taxonomic characteristic in the genus Vibrio (Kelley et ai., 1991). Historically arabinose was one of three sugars whose fermentation formed the basis of the Heiberg classification scheme for Vibrio and Vibrio-like organisms (Heiberg, 1936; Smith and Goodner, 1965). While most Vibrio species are either arabinose positive or arabinose negative, the ability to ferment arabinose is variable in V. parahaemolyticus, a halophilic estuarine bacterium that is a causative agent of seafood borne gastroenteritis (Baumann et ai., 1984). Kelley et ai. (1991) reported that 80% of V. parahaemolyticus strains from clinical specimens are arabinose positive. In a recent numerical taxonomy study of vibrios isolated primarily from coastal areas of the United States, 50 per cent of 68 strains identified as V.

* Corresponding author

parahaemolyticus were arabinose positive (West et ai., 1986). The genetics of arabinose fermentation has been characterized in E. coli (Lee, 1980), Salmonella typhimurium (Horwitz et ai., 1981) and Bacillus subtilis (Sa-Nogueira and De Lencastre, 1989). Arabinose catabolism is encoded in three genes, araA, araB, and araD which encode for L-arabinose isomerase, L-ribulokinase, and L-ribulose5-phosphate 4-epimerase, respectively. The three genes form an operon regulated by the product of the araC gene. Iuchi and Tanaka (1976) studied L-arabinose isomerase in V. parahaemolyticus and found that this enzyme, like the L-arabinose isomerase of E. coli, was inducible by arabinose and subject to catabolite repression. This paper reports the cloning of the arabinose genes from an arabinose positive strain of V. parahaemolyticus and the utilization of the cloned genes to investigate the genetic basis of variation in arabinose fermentation ability in V. parahaemolyticus strains.

Arabinose Fermentation Genes of Vibrio parahaemolyticus

Materials and Methods Bacterial strains and plasmids. E. coli strains are listed in Table 1. V. parahaemolyticus strains are listed in Table 3. Strains A127, SN40, M23/77, W424, 915/77, 887/77, SN36, A7, and D51 were isolated in India from various sources (human cases, human carriers, prawns, fly, water) and are of different serotypes except for M23/77 and A7 which share the 05: K17 serotype (McNicol et al., 1983). The type strain ATCC 17802 (serotype 01: K1) (Fujino et al., 1974) and S162-71 (serotype 04: K34) (Guerry and Colwell, 1977) were isolated from fish in Japan and strain 7E10 (serotype 05: K17) (Isaki et al., 1983) was isolated in Africa from a healthy carrier. All V. para-haemolyticus strains were Kanagawa phenomenon negative (KP) except for A7 and D51. The plasmid vector pBR322 was purchased from Sigma Chemical Co. and the plasmid vector pUC19 was a gift from Dr. D. Stein, University of Maryland, College Park. Media and chemicals. E. coli strains were grown on LuriaBertani broth and agar (Schleif and Wensink, 1981), MacConkey agar base (Difco) supplemented with 0.5% (w/v) arabinose (MacConkey-arabinose agar) or M9 agar (Maniatis et al., 1982) with 0.5% (w/v) arabinose as the carbon source (M9-arabinose) supplemented as appropriate with 20 Ilg/ml amino acids and 50 Ilg/ ml ampicillin. Vibrio strains were grown on tryptic-soy broth (Difco) and MacConkey-arabinose agar both supplemented with 1% NaCI (w/v). L-arabinose, antibiotics, IPTG, RNase and lysozyme were purchased from Sigma Chemical Co., T4 DNA ligase and a nick translation kit from Bethesda Research Laboratories (BRL), restriction enzymes from BRL and International Biotechnologies Inc. and 32P-deoxycytosine triphosphate from ICN Biochemicals. Isolation of chromosomal and plasmid DNA. The technique of Marmur (1963) was used for the isolation and purification of chromosomal DNA. Plasmid DNA was isolated by the alkaline lysis procedure of Birnboim and Doly (1979) and was purified by cesium chloride-ethidium bromide density centrifugation or by RNase treatment, chloroform amyl-alcohol (24: 1) extraction and ethanol precipitation. Table 1. E. coli strains used in cloning and complementation Strain

Genotype

E. coli LA3

F+ ilara(OC)719

SourcelReference

Greenfield et al.,

proA2 lac Yl thi galK2 xyl-S mtl-l rpsL20 hsdS20 F+ araB24 proA2 lacYl galK2 xyl-5 mtl-l supE44 thi rpsL20 hsdS20

Greenfield et al.,

E. coli NL20-046

araA

N.L. Lee

E. coli NL20-039

araB24

N.L. Lee

E. coli NL30-133

araD

N.L. Lee

E.coli AD92

araD139 galU galK il(araABIOC-leu) lac(IPOZY)X74 hsr hsm+

Casabadan and Cohen, 1980

E. coli LA7

E. coli JM105

thi rpsL endA sbcB15 hsdR4 il(lac-proAB) [F' traD36 proAB lacIq ZilM15]

1978

1978

421

Restriction enzyme digestion, agarose gel elctrophoresis, and isolation of restriction fragments. Digestion of DNA was performed under the conditions recommended by the supplier of the restriction enzyme using 5 to 10 [1g chromosomal DNA in a total reaction volume of 50-70 [11 or 3 to 5 [1g plasmid DNA in a total reaction volume of 20-25 [1l. Samples of restricted DNA were analyzed on 0.8% agarose gels in Tris-borate buffer, pH 8.2 or Tris-acetate buffer, pH 8.0 for 4 to 6 h at 6 V/cm and stained with ethidium bromide. To isolate restriction fragments, agarose blocks containing the desired fragment were cut our of the gel, placed in dialysis bags with a small amount of electrophoresis buffer, and the DNA was electroeluted and purified by phenolchloroform extraction and ethanol precipitation. Construction of recombinant plasmids. The ara gene region of V. parahaemolyticus was cloned by partially digesting the genomic DNA of strain 7E10 with HindIII and fractionating the digest on a sucrose density gradient. DNA from a gradient fraction containing fragments in the 8-20 kb size range was ligated with HindIII digested pBR322 DNA in the presence of T4 DNA ligase and the ligation mixture was transformed into Ara - E. coli strains. Ara + transformants were selected on MacConkey arabinose agar supplemented with ampicillin. Plasmid pVM101 was isolated from an Ara + transformant of E. coli LA7. Derivatives of pVM101 with deletions of the V. parahaemolyticus insert DNA were obtained by cleaving plasmid DNA with an appropriate restriction endonuclease and allowing self ligation of the larger fragment to occur in the presence of T4 DNA ligase. Ligation mixtures were transformed into E. coli strains and transformants carrying plasmids of the expected size were identified. Plasmids pVM102, pVM103 and pVM104 are pVM101 derivatives deleted in a 2.5 kb Sail fragment, a 2.5 kb Xbal fragment and a 4.3 Clal fragment, respectively. Plasmid pVM108 is a derivative of pVMI04 deleted in a 0.95 kb HindIII fragment. To construct plasmids pVM105 and pVM106, the Sail and Xbal fragments of pVMlOl were cloned into the Sail and Xbal sites, respectively, of the vector plasmid, pUC19. All of the derived plasmids were verified by restriction enzyme analysis. Complementation analysis. Recombinant plasmids containing V. parahaemolyticus arabinose genes were transformed into a set of E. coli strains mutant in different regions of the ara operon. The ability of the plasmids to complement ara mutations was assayed by plating transformants on LB agar or MacConkeyarabinose agar both supplemented with ampicillin. In either case about 10 Ampr transformed colonies were tested for ability to utilize arabinose as a carbon source by streaking for growth on M9-arabinose agar supplemented with ampicillin. In complementation studies using the pUC19 vector plasmids, transformants were selected on MacConkey-arabinose ampicillin plates supplemented with 0.1 mM IPTG/ml. Southern hybridization. Restriction fragments isolated from pVM101 were labelled with a-32 p_dCTP using a nick translation kit. Bacterial DNAs were digested with HindIII, electrophoresed on O.8f/o agarose gels, and the DNA bands were transferred to Gene Screen Plus nylon hybridization membranes (New England Nuclear; NEN) by the method of Southern (1975). Membrane bound DNA was hybridized with probe DNA under stringent conditions according to the protocol supplied by NEN.

Results Yanisch-Perron et al., 1985

Cloning and characterization of the ara genes of V. parahaemolyticus Plasmid pVM101 (Fig. 1) is a recombinant plasmid isolated in this study which contains a HindIII fragment of V. parahaemolyticum DNA inserted into the HindIII site of

422

S. Muralidhar and M. J. Voll Xba I Cia I

Xbal

Xho I

Hind III

pVM101 12.2 kb

8

Hind III

9 Sail

Fig. 1. Restriction map of plasmid pVMI01. The light line represents pBR322 DNA and the heavy line represents Vibrio DNA. Numbers on the inside of the map give distances in kilobases starting at the ClaI site on pBR322.

pBR322. The plasmid complemented E. coli AD92, a strain mutant in all of the genes of the arabinose operon, indicating that it has a full complement of genes capable of mediating arabinose fermentation. The size of the V. parahaemolyticus insert DNA was determined to be 7.8 kb by restriction analysis. Derivative plasmids pVM102, pVM103, pVM104, pVM10S, pVM106 and pVM108 (Fig. 2) were tested for their ability to complement E. coli strains mutant in either the araA, araB, araC or araD genes (Table 2). On the basis of positive complementation results, genes corresponding to the E. coli araA and araB genes could be assigned to defined regions of the cloned V. parahaemolyticus DNA. Complementation of an araA mutation by pVMI02 indicates an araA gene is located to the left of the san site on the insert DNA, and complementation of the araA mutation by pVM106 further locates the gene to the XbaI insert DNA fragment. Complementation of araB mutations by pVM104 and pVM108 locates an araB gene to the region

between the ClaI site at 4.3 kb and the HindIII site at 6.9 kb. As expected from these results the araB mutation in strain LA7 is complemented by pVM103. The reason why the araB mutation in NL20-039 is not likewise complemented by pVM103 is uncertain. Complementation of an araC mutation by pVM103 but not pVM104 suggests that an araC gene is located in the left most ClaI-HindIII fragment of the insert DNA. Complementation of an araD mutation was only obtained with the original pVM10l plasmid and suggests that at least part of a araD gene is located in the 1.6 kb ClaI-San fragment of the insert. From the complementation analysis, the probable order of the V. parahaemolyticus ara genes is, starting at the left end of the insert, araC, araA, araD, araB. Base sequence analysis of the cloned V. parahaemolyticus DNA has shown this to be the correct order (N. L. Lee, pers. commun.).

Hybridization of ara gene fragments with HindIII digested chromosomal DNA of arabinose positive and arabinose negative Vibrio strains Three restriction fragments spanning the ara structural gene segment of pVM10l were isolated and used as hybridization probes. The probes were the 2.5 XbaI fragment, the 1.6 kb XbaI-SalI fragment and the 2.5 kb SalI fragment. The probes were nicktranslated using 32p_dCTP and hybridized with HindIII digested genomic DNA of six arabinose fermenting and five non-arabinose fermenting strains of V. parahaemolyticus, and with strains of the arabinose fermenting species, V. f/uvialis, V. hollisae and Aeromonas hydrophila, and of the arabinose non-fermenting species, V. cholerae, V. mimicus and V. alginolyticus. Fig. 3 shows the results obtained using V. parahaemolyticus DNAs and the XbaI-Sali fragment as probe. A summary of the hybridization results is given in Table 3. All three probes hybridized with genomic DNA from arabinose positive V. parahaemolyticus strains but not with genomic DNA from arabinose negative V. parahaemolyticus strains. The Sail fragment should hybridize to two DNA fragments of the arabinose positive strains because the SalI fragment contains a V. parahaemolyticus specific HindIII site. All genomic DNAs from the arabinose positive strains gave one hybridized fragment which showed the same electrophoretic mobility in all of the DNAs. Ad-

Table 2. Gene complementation of Ara-

Host strain

E. coli strains by rec~mbinant plasmids which

contain segments of the arabinose region of V. parahaemolyticus

Plasmid 'spVMIOl S'spVMI02 S'spVMI03 S'spVMI04 S'spVMI08 S'spVMI05 S'spVMI06

LA3

NL20-046

LA7

NL20-039

NL30-133

(araOC)

(araA)

(araB)

(araB)

(araD)

+a + +

+ + +

+

+

+

ND ND ND

ND

+ + +

+ +

+

ND ND

ND ND

a +, complementation; -, no complementation; ND, not determined.

ND ND ND

Arabinose Fermentation Genes of Vibrio parahaemolytieus

A. pBR322

I

B. pVM101

A

I

I

A

D. pVM103

E. pVM104

F. pVM108

I

G. pUC19

K S

Jnlc

KS

I I

I I H H

I I

K S

I

I I

dlH

X

Xb

X

I

I

(!;x

A

P

I

I

P

Xb

I

I

MCS

xjlc

I

dlH

Sc

I

p

I

I

I

KS

X

P

I

xic

dlH

A

A

I

I

P

A

C. pVM102

4.4 kb

I

A

423

I H H

I

S

12.2 kb

I

A

II~

~X:~

~x?lc

c~

I

I I

X

KS

S

I

I

S

I

9.7kb

I

A

7.9 kb

I

H H

S

A

~

I

I

S

9.7 kb

A

7.0 kb

A

2.68 kb Sc

H. pVM105

I

I

Sc

I. pVM106

I

Sc

I

Xb

i

Xb

S

i

I

H

I

H

I

S

I

5.2 kb

Sc

5.2 kb

Sc

SCALE 1cm=1kb Fig. 2. Restriction maps of recombinant plasmids derived from pVMIOl. Size of the plasmids is shown at right. Plasmids A through F are linearized at the Aval site of pBR322 and plasmids G through I at the Seal site of pUC19. The thin line is pBR322 DNA, the open line is pUC19 DNA, and the thick line is Vibrio DNA. Cloned segments are aligned under the corresponding segments in pVMIOl. Peaked segments represent deleted portions of the DNA. Restriction enzyme sites are: A, Aval; C. Clal; H, HindIII; K, Kpnl; S, San; Sc, Seal; X, Xhol; Xb, Xbal. The multiple cloning site (MCS) in pUC19 includes San and Xbal restriction sites.

ditionally some of these DNAs gave a second and more faintly hybridized fragment of higher mobility. The XbaI-Sail fragment (Fig. 3) hybridized to a single DNA band which showed the same mobility in the six Ara + genomic DNAs, and the same was observed for the XbaI fragment probe. The Sail and XbaI-Sall fragment probes did not hybridize with HindIII digested DNA of the other Vibrio species or Aeromonas hydrophila. Some weak hybridization of the XbaI probe with DNA of the Ara + V. fluvialis and V. hollisae strains was noted.

Discussion Complementation studies indicate that fermentation of arabinose in V. parahaemolyticus occurs by the same metabolic pathway as characterized in other bacteria which have been studied. The clustering of the ara structural genes and the presence of an araC gene imply an operon structure. The order of the ara genes in E. coli and S. typhimurium is araC araB araA araD (Lee et al., 1980; Horwitz et al., 1981). Lei et al. (1985) studied genes of

424

S. Muralidhar and M. J. Voll

A8CDE FGHI J

arabinose fermentation in Erwinia carotivora, a species whose DNA is 50% homologous to that of E. coli. They were able to locate araA, araB, and araC genes relative to one another. The orientation of these genes was the same as in the other two enteric species except there was a 3.5 kb distance between araC and araB. In B. subtilis the structural gene order is araD araB araA and the araC gene is located in a different region of the chromosome (SaNogueira et al., 1988; 1989). The order of the ara genes found in V. parahaemolyticus, araC araA araD araB, is different from that found in the three gram negative species and also from that found in gram positive B. sub-

tilis.

Three radiolabelled probes consecutively spanning the

V. parahaemolyticus ara structural gene segment were

hybridized under stringent conditions to the genomic DNA of arabinose positive and negative strains of V. parahaemolyticus. The V. parahaemolyticus strains were isolated from diverse environmental and clinical samples and most belong to different serotypes. Each probe hybridized with the genomic DNA of the Ara + strains, while no probe hybridized with the DNA of the Ara - strains. We conclude that in the Ara - strains the Ara - phenotype is due to the absence of ara genes as opposed to inactivation of the genes by mutation(s) in a structural or regulatory gene. The five negative strains were diverse either in serotype or source, and although four were isolated in India the fifth was isolated in Japan. This suggests that the Ara- phenotype in V. parahaemolyticus is in general due to absence of the genes for arabinose fermentation. Study of the population genetics of bacteria has concentrated on investigation of polymorphisms arising from allelic forms of the same gene in a given species (Young,

Fig. 3. An autoradiograph of a Southern blot of HindIII digested genomic DNAs from arabinose positive and negative strains of parahaemolyticus after hybridization with the radiolabelled XbaI-Sali fragment of pVM101. A, v. parahaemolyticus ATCC17802; B, v. parahaemolyticus A127; C, v. parahaemolyticus W424; D, V. parahaemolyticus SN40; E, V. parahaemolyticus SN36; F, V. parahaemolyticus 915/77; G, V. parahaemolyticus A7; H, V. parahaemolyticus D51; I, V. parahaemolyticus 887/77;], V. parahaemolyticus M23/77.

v.

Strain

V. parahaemolyticus 7E 10 V. parahaemolyticus ATCC 17802 V. parahaemolyticus All7 V. parahaemolyticus SN40 V. parahaemolyticus M23/77 V. parahaemolyticus W424 V. parahaemolyticus 915/77 V. parahaemolyticus 887/77 V. parahaemolyticus SN36 V. parahaemolyticus A7 V. parahaemolyticus D51 V. parahaemolyticus S162-71 V. f/uvialis ATCC 33809 V. hollisae V. cholerae ATCC 14035 V.mimicus ATCC 33635 V. alginolyticus ATCC 17749 Aeromonas hydrophila ATCC 15467 a

b

Arabinose fermenting phenotype

DNA probe 1a

2

3

+ + + + + + +

+b + + + + + +

+ + + + + + +

+ + + + + + +

+ +

ND +/+/-

+

ND

1, XbaI fragment; 2, XbaI-Sall fragment; 3, Sail fragment. +, hybridization; -, no hybridization; +/-, weak hybridization; ND, not determined.

Table 3. Hybridization of V. parahaemolyticus ara structural gene probes with DNA from arabinose positive and negative Vibrio strains

Arabinose Fermentation Genes of Vibrio parahaemolyticus 1989). Phenotypic variation involving presence or absence of a trait has been addressed to a lesser extent and often with determinants associated with pathogenicity. In V. parahaemolyticus, the Kanagawa phenomen (KP), i.e. the ability to produce lysis on a special type of blood agar due to elaboration of a thermostable direct hemolysin, is also a variable trait. KP is a common characteristic of strains isolated from clinical cases but is rarely exhibited by strains isolated from seafood or the environment (Kelley et aI., 1991). Nishibuchi et a1. (1985) and Taniguchi et a1. (1985) screened V. parahaemolyticus strains for presence of the thermostable direct haemolysin gene (tdh) by hybridization of their DNAs with tdh gene probes. Most or all of the Ara - strains were missing the gene. Both groups of investigators also found that spontaneously occurring KP negative mutants derived from KP positive strains lacked the tdh gene suggesting that the gene is present on a variable genetic element. The tdh gene has been detected in some V. hollisae strains and on plasmids in some V. cholerae non-01 strains indicating that lateral transfer of the gene has occurred (Nishibuchi et aI., 1985; Honda et aI., 1986). Link and Reiner (1982) found that the genes encoding the catabolism of the pentitol sugars, ribitol and Darabitol, in E. coli C were completely absent from the DNA of E. coli K12 and B strains and that the ribitolarabitol genes of E. coli C were surrounded by 1.4 kb inverted repeats of imperfect homology. The authors suggested these genes may consitute an example of a vestigial transposable element. Their findings support the hypothesis that in the process of evolution, genes serving catabolic functions are subject to lateral transfer (Baumann et aI., 1983). To account for the genetic variability of arabinose fermentation in V. parahaemolyticus, it may be proposed that the arabinose genes were introduced into the V. parahaemolyticus species by lateral transfer of a transposable element which contained the ara genes from another species. The ability to ferment arabinose proved advantageous for the cells in certain environments and the Ara + phenotype achieved widespread distribution. Alternately, V. parahaemolyticus may have evolved as a Ara + species and over the course of time the ara genes were lost in some strains through excision. A favorable mutation occurring in one such strain could have resulted in clonal selection of this strain thus perpetuating the Ara - phenotype. The genomic DNA extracted from strains of the Ara + species, V. f/uvialis and V. hollisae did not hybridize under stringent conditions with two of the V. parahaemolyticus ara gene probes. Some weak hybridization, however, with the XbaI probe was observed. The Xbal probe encompasses the entire araA gene (this paper) and a segment of the araD gene (N. Lee, pers. commun.). It would be interesting to know if the ara genes of V. f/uvialis and V. hollisae and those of V. parahaemolyticus share the same gene order.

Acknowledgements. This work was supported by Public Health Grant R22-AI-14242 under the U.S.-Japan Cooperative Medical Science Program, from the National Institute of Allergy

425

and Infectious Diseases. We are much indebted to Nancy L. Lee for her interest and assistance and for sharing unpublished data with us. We thank Bradley D. Jones and Henry Chenfor competent technical assistance and L. Hefferman for strains.

References

Baumann, P., Baumann, L., Woolkalis, M. J., Bang, S. 5.: Evolutionary relationships in Vibrios and Photobacterium. Ann. Rev. Microbiol. 37, 369-398 (1983 ) Baumann, P., Furniss, A. L., Lee, J. V.: Genus 1. Vibrio, Pacini 1854, pp. 518-538. In: Bergeys Manual of Systematic Bacteriology, Vol. 1 U. G. Holt, and N. R. Krieg, eds.). Baltimore, Williams and Wilkins 1984 Birnboim, H. c., Doly, J.: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1523 (1979) Casabadan, M., Cohen, S. N. : Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. BioI. 138, 179-207 (1980) Fujino, T., Sakazaki, R., Tamura, K.: Designation of the type strain of Vibrio parahaemolyticus and description of 200 strains of the species. Int. J. System. Bact. 24, 447-449 (1974) Greenfield, L., Boone, T. , Wilcox, G.: DNA sequence of araBAD promoter in Escherichia coli B/r. Proc. Nat. Acad. Sci. USA 75, 4724-4728 (1978) Guerry, P., Colwell, R. R.: Isolation of ctyptic plasmid deoxyribonucleic acid from Kanagawa positive strains of Vibrio parahaemolyticus. Infect. Immun. 16,328-334 (1977) Heiberg, 8.: The biochemical reactions of Vibrios. J. Hyg. 36, 114-117 (1936) Honda, T., Nishibuchi, M., Miwatani, T., Kaper, J.: Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-01 strains. Appl. Environ. Microbiol. 52, 1218-1220 (1986) Horwitz, A. H., Hefferman, L., Morandi, c., Lee, J. H., Timko, J., Wilcox, G.: DNA sequence of araBAD-araC controlling region in Salmonella typhimurium LT2. Gene 14, 309-320 (1981) Iuchi, 5., Tanaka, S.: Induction and repression of L-arabinose isomerase in V. parahaemolyticus. Nippon Saikingaku Zasshi 31, 695-704 (1976) Isaki, L. 5., Bever, R. A., Newland, J. W., Voll, M. J.: Isolation and chracterization of small plasmids in strains of Vibrio parahaemolyticus. J. Diar. Dis. Res. 1, 99-106 (1983) Kelley, M. T., Hickmann-Brenner, F. W., Farmer, J. J. III.: Vibrio, pp. 384-395. In: Manual of Clinical Microbiology, 5th ed. (A. Balows, L. J. Hausler, jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy eds.). WashingtonlDC, American Society for Microbiology 1991 Lee, N.: Molecular aspects of ara regulation, pp. 389-409. In: The Operon U. H. Miller and W. S. Reznikoff, eds.). Cold Spring HarbourlNY, Cold Spring Harbour Laboratory 1980 Lei, S. P., Lin, H. c., Heffermann, L., Wilcox, G.: araB gene and nucleotide sequence of the araC gene of Erwinia carotovora. J. Bact. 164, 719-722 (1985) Link, C. D., Reiner, A. M.: Inverted repeats surround the ribitolarabitol genes of E. coli C. Nature 298, 208-218 (1982) Maniatis, T., Fritsch, E. F., Sambrook, J.: Molecular Cloning: A Laboratory Manual. Cold Spring HarbourlNY, Cold Spring Harbour Laboratory 1982 Marmur, J.: A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. BioI. 3, 208-218 (1961)

426

S. Muralidhar and M. J. Voll

McNicol, L. A., De, S. P., Kaper, j. B., West, P. A., Colwell, R. R.: Numerical taxonomy of Vibrio cholerae and related species isolated from areas that are endemic and nonendemic for cholera. J. Clin. Microbiol. 17, 1102-1113 (1983) Nishibuchi, M., Ishibashi, M., Takeda, Y., Kaper,j. B.: Detection of the thermostable direct hemolysin gene and related DNA sequences in Vibrio parahaemolyticus and other Vibrio species by the DNA colony hybridization test. Infect. Immun. 49, 481-486 (1985) Sa-Noguiera, I., de Lencastre, H.: Cloning and characterization of araA, araB and araD, the structural genes for L-arabinose utilization in Bacillus subtilis. J. Bact. 171,4088-4091 (1989) Sa-Noguiera, I., Paveia, H., de Lencastre, H.: Isolation of constitutive mutants for L-arabinose utilization in Bacillus subtilis. J. Bact. 170, 2855-2857 (1988) Schleif, R. F., Wensink, P. c.: Practical Methods in Molecular Biology. New York, Springer-Verlag 1981 Smith, H. L., Goodner, K.: On the classification of vibrios, pp. 4-8. In: Proceedings of the Cholera Research Symposium,

January 1965, Honolulu, Hawaii. Public Health Service Publication No. 1328. Washington, U. S. Government Printing Office 1965 Southern, E.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioI. 98, 503 (1975) Taniguchi, H., Ohta, H., Ogawa, M., Mizuguchi, Y.: Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes. J. Bact. 162, 510-515 (1985) Yanisch-Perron, D., Vieira, j., Messing, j.: Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp18 and pUC19 vectors. Gene 33, 103-119 (1985) Young, j. P. W.: The population genetics of bacteria, pp. 417-438. In: Genetics of Bacterial Diversity (D. A. Hopwood and K. F. Chate, eds.). London, Academic Press 1989 West, P. A., Brayton, P. R., Bryant, T. N., Colwell, R. R.: Numerical taxonomy of Vibrios isolated from aquatic environments. Int. J. System. Bact. 36, 531-543 (1986)

Dr. Mary j. Vall, Dept. of Microbiology, University of Maryland, College Park, MD 20742, USA