Biotype in Vibrio cholerae

Transcription

1 INFECTION AND IMMUNITY, Dec. 1983, p /83/ $02.00/0 Copyright 1983, American Society for Microbiology Vol. 42, No. 3 Mapping of Chromosomal Genes That Determine the El Tor Biotype in Vibrio cholerae BRUCE A. GREEN,t JOHN W. NEWLAND,f AND RANDALL K. HOLMES* Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Matyland Received 14 July 1983/Accepted 13 September 1983 The El Tor biotype of Vibrio cholerae has several characteristics that differentiate it from the classical biotype of V. cholerae. Among these are production of soluble hemolysin(s), a cell-associated hemagglutinin for chicken erythrocytes, resistance to polymyxin B, and resistance to bacteriophages of Mukerjee group IV. In the present study, we located the determinants for hemolysin (hly), chicken erythrocyte hemagglutinin (cha), and polymyxin B resistance (pmx) on the genetic map of V. cholerae. Transposon-facilitated recombination was used to perform conjugal matings between El Tor donor strains and classical recipient strains of V. cholerae. Recombinants were selected that had inherited specific nutritional markers from the donor strains and streptomycin resistance from the recipient strains. The reconibinants were tested for the presence or absence of the unselected donor markers hly, cha, and pmx. These three El Tor biotype markers were found to be closely linked to each other and were located between the pyra- 201 and his-2 loci on the genetic map of V. cholerae. Both the classical and El Tor biotypes of Vibrio cholerae are capable of causing human disease. Since 1960, epidemics of cholera have most frequently been caused by El Tor strains, whereas previous epidemics were predominantly caused by classical strains (6). The El Tor biotype differs from the classical biotype in several characteristics. El Tor strains usually produce soluble hemolysin(s), have a cell-associated hemagglutinin for chicken erythrocytes, and are resistant to the antibiotic polymyxin B and bacteriophage of group IV (6). Classical strains are usually nonhemolytic, nonhemagglutinating for chicken erythrocytes, and susceptible to polymyxin B and group IV phage. In the present study, we performed conjugal matings between El Tor and classical strains and determined the locations on the genetic map of V. cholerae of the determinants for three El Tor biotype markers: hemolysin (hly), chicken erythrocyte hemagglutinin (cha), and polymyxin B resistance (pmx). Conjugal gene transfer in V. cholerae is mediated by the fertility plasmid P (2). Most early genetic studies of V. cholerae were performed with classical strains (3, 14, 15). The genetic map of the classical V. cholerae strain 162 is circular, and the positions of many genetic markers on the map have been determined (14, 17). Conjugal t Present address: Praxis Biologics, Rochester, NY t Present address: National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Bethesda, MD gene transfer between El Tor and classical biotypes has been demonstrated (17), and preliminary studies on the linkage map of the El Tor biotype have been done (10; J. W. Newland, B. A. Green, and R. K. Holmes, manuscript in preparation). Although the El Tor genetic map closely resembles that of the classical biotype, certain segments of the El Tor map appear to be inverted with respect to the classical map (10; Newland et al., in preparation). The genetic determinants for biotype markers have not previously been located on the genetic map of V. cholerae. Ogg et al. reported that resistance to polymyxin B could be transferred from El Tor donor strains into classical recipient strains by using a recently isolated vibrio-transducing phage, but cotransduction of other markers with polymyxin B resistance was not observed (13). In conjugal matings mediated by the wild-type P plasmid in V. cholerae, recombinants inherit donor alleles at low frequencies from all regions of the genetic map (14, 15). Transposons can be introduced into both the P plasmid and the chromosome in V. cholerae to create new donor strains for transposon-facilitated recombination (Tfr) (10, 17; Newland et al., in preparation). In conjugal matings, Tfr donors transfer the bacterial chromosome to recipient bacteria in an oriented and sequential manner from an origin corresponding to the site of insertion of the transposon in the donor chromosome. Isogenic Tfr donor strains with P plasmids containing the 924

2 VOL. 42, 1983 MAPPING OF BIOTYPE DETERMINANTS IN V. CHOLERAE 925 TABLE 1. Bacterial strains and plasmids Strain Characteristicsa Biotype Source (reference) Bacteria GN6300 prototroph, spc-201 derived from Ul El Tor J. Newlandb GN9006 spc-201, pyra::tns-201 El Tor J. Newlandb GN9003 spc-201, pro::tn5-201 El Tor J. Newlandb GN7007 spc-201, prototroph, Tnl insert between met and trp El Tor J. Newlandb RV88 str-2, leu-1, pro-2, pur-1 Classical W. Romig (15) RV94 str-2, arg-1, ilv-1, his-2, trp-2 Classical W. Romig (15) RV93 str-2, arg-1, ilv-1, his-2, met-2 Classical W. Romig (15) Plasmids P Wild-type fertility plasmid of V. cholerae W. Romig (15) psj5 P::Tnl hybrid; Tnl in (+) orientation W. Romig (10) psj13 P::Tnl hybrid, Tnl in (-) orientation W. Romig (10) pjn2 P::Tnl,Tn5 hybrid; Tn5 in (+) orientation, derived from psj5 J. Newlandb pjn8 P::Tnl,TnS/ hybrid; Tn5 in (-) orientation, derived from psj5 J. Newland' pjn15 P::Tnl,Tn5 hybrid; Tnl in (-) orientation, derived from psj13 J. Newland" a Allele numbers are arbitrary and follow the suggested guidelines of C. Parker (14). Gene symbols are those of Bachmann and Low (1). b Newland et al., manuscript in preparation. appropriate transposon in opposite orientations can, therefore, mobilize chromosomal genes in conjugal matings from a common origin but with opposite polarities (10, 16, 17). In the studies reported here, we used transposon-facilitated recombination mediated by transposon Tnl or transposon TnS for genetic mapping of biotype determinants of V. cholerae. MATERIALS AND METHODS Bacterial strains and plasmids. Bacterial strains and plasmids used in these experiments are listed in Table 1. Media and cultivation conditions. Luria broth (12) and brain heart infusion (Difco Laboratories, Detroit, Mich.) were used for routine liquid cultures. Meat extract agar (15), LB agar (12), syncase agar (7), and minimal medium V (Newland et al., in preparation) were used as solid media for specific experiments as indicated. When required, minimal medium was supplemented with amino acids at 10,ug/ml. Antibiotics were added to solid and liquid media as indicated in the text at the following concentrations: ampicillin (amp) (Sigma Chemical Co., St. Louis, Mo.), 400,ug/ ml; rifampin (rif) (Sigma), 20 jig/ml in synthetic media, 30 p.g/ml in enriched media; kanamycin (kan) (Sigma), 70 jig/ml; polymyxin B sulfate (Sigma), 50,ug/ml; streptomycin (str) (Sigma), 100 pg/ml; spectinomycin (spc) (Sigma), 100,ug/ml. All cultures were incubated at 30 C. Mating procedures. Conjugal matings mediated by P::Tnl,TnS hybrid sex factors (Tfr matings) were performed as described by Newland et al. (in preparation). Characteristics of Tn5 auxotrophic donor strains for Tfr matings are reported elsewhere (Newland et al., in preparation). Overnight cultures of donor and recipient strains were grown in brain heart infusion containing kanamycin and streptomycin, respectively, at 30 C. Samples (100,.1) of each were mixed and spread onto meat extract agar plates; the plates were incubated at 30 C overnight. Bacterial lawns were replica plated, using sterile velveteen squares onto appropriately supplemented minimal media to select for recombinants with desired nutritional markers, and the donor strain was counterselected by streptomycin. Scoring for unselected markers was performed either by transferring inocula from recombinant colonies onto appropriate media or by performing tests for the cell-associated chicken erythrocyte hemagglutinin as described below. Detection of hemolytic activity. Hemolytic activity was determined by stabbing inocula from classical, El Tor, or recombinant colonies into plates with lower layers consisting of syncase agar containing streptomycin and overlays of syncase blood agar. Blood agar overlays were prepared as described by Bramucci and Holmes (4). After inoculation, plates were incubated at 37 C for 24 to 48 h and examined for zones of hemolysis surrounding individual colonies. Chicken erythrocyte hemagglutinin determination. The presence of cell-associated chicken erythrocyte hemagglutinin was determined by a modification of the microtiter plate method developed by Jones et al. (11). Bacteria were grown to stationary phase at 37 C in tryptone broth to minimize production of soluble hemagglutinins. The cultures were allowed to stand overnight at 4 C, and bacteria collected by centrifugation were suspended at 0.1 times the original culture volume in Krebs-Ringer-Tris buffer (9). Chicken erythrocytes were collected in sodium citrate, mixed 1:1 with Alsever's solution (8), and stored at 4 C. After washing 2x in Krebs-Ringer-Tris buffer, a 1% suspension of erythrocytes was made in the same buffer. Control experiments demonstrated that a 1:8 dilution of the bacterial suspensions was optimal for differentiating between the El Tor and classical biotype strains used for our experiments. Samples containing 25,u1 of the diluted bacterial suspensions in Krebs-Ringer-Tris buffer were added to individual wells of 96-well microtiter plates (Dynatech Laboratories, Inc., Alexandria, Va.). Next, 25 Al of the erythrocyte suspension was added to each well, and the plates were incubated at room temperature for 30 min. Hemagglutination was recognized as failure of the erythrocytes to settle into

3 926 GREEN, NEWLAND, AND HOLMES discrete pellets in the presence of the bacteria. When negative reactions were observed, the bacteria were retested at lower dilutions to confirm their nonhemagglutinating phenotype. RESULTS The goal of our experiments was to determine the positions of the El Tor biotype markers hly, pmx, and cha on the genetic map of V. cholerae. All of the El Tor Tfr donor strains used in our studies had these markers, whereas the classical recipient strains did not have these determinants. The results of the conjugal matings that we performed are summarized in Tables 2 to 4. Figure 1 is a segment of the circular genetic map of V. cholerae El Tor and shows the positions of the previously mapped reference loci used for our studies, the sites of insertion of the Tnl or Tn5 transposons in our Tfr donor strains, and our conclusions concerning the locations of the hly, pmx, and cha markers on the genetic map. First, we analyzed the inheritance of hly as an unselected donor marker in matings between Tfr El Tor donor strains and multiply auxotrophic, streptomycin-resistant classical recipient strains of V. cholerae (Table 2). Linkage of the hly marker to the leu, his, and trp loci was clearly demonstrated in matings with GN9003(pJN8) or GN9006(pJN2) as the donor strain. The highest frequency of coinheritance of hly occurred when the selected donor marker was leu+. There was no significant linkage of hly to arg and met when they were mobilized as proximal markers from the donor strains GN9003(pJN2) and GN7007(pJN15), respectively. Both of these strains mobilize the trp-leu segment of the chromosome as distal markers and at low frequency. Although his and trp could be transferred efficiently as proximal markers from donor strains GN9006(pJN2) and GN7007(pJN2), strong linkage of hly to his' or trp+ was observed only with GN9006(pJN8) as the donor strain. Taken together, these results provide strong evidence that hly is located between the his locus and the pyra locus, the site of the chromosomal TnS insertion in strain GN9006, and not between his and the chromosomal Tnl insertion in strain GN7007 (Fig. 1). Similar experiments with pmx as the unselected donor marker demonstrated linkage ofpmx to his' and leu+ with donor strains GN7007(pJN2) and GN9006(pJN2), respectively (Table 2). Control experiments showed no linkage of pmx to met+ and arg+ with donor strains GN7007(pJN15) and GN9006(pJN8), respectively. We concluded that pmx and hly are located in the same region of the V. cholerae chromosome as the his and leu loci. More detailed linkage studies were performed by analyzing the inheritance of multiple genetic GN9003 r GN GN7007 I -arg-1 -pro-202 -pyra-201 _hly-201 _leu-1 _ (pmx-201,cha-201) his-2 -trp-2 met-2 INFECT. IMMUN. FIG. 1. Segment of the genetic map of V. cholerae El Tor strain GN6300 showing the markers used in this study, the sites of chromosomal transposon insertion in the Tfr donor strain (filled triangles adjacent to strain designations), and the locations of the hly, pmx, and cha markers. Distances between genetic markers are approximate. The order of the closely linked pmx and cha loci has not yet been determined. determinants as unselected markers in conjugal matings between El Tor Tfr donor strains and classical recipient strains of V. cholerae. The results of matings between GN7007(pJN2) donor and RV88 or RV94 recipient strains are shown in Table 3. In each of these matings, the origin for transfer of donor markers was the Tnl insertion site between met and trp, and the trp+, his', and leu+ alleles were transferred sequentially to the recipient strains as early markers (Fig. 1). The hly marker was found to be closely linked to leu+ (80% coinheritance) but not to his+ or trp+, and the his and trp loci were closely linked to each other. In contrast, the pmx and cha markers were linked to all three of the selected markers and were rarely separated from each other. Coinheritance of pmx and cha was approximately 94% with leu+, 55% with his+, and 37% with trp+. These data were most consistent

4 VOL. 42, 1983 TABLE 2. MAPPING OF BIOTYPE DETERMINANTS IN V. CHOLERAE 927 Linkage of genetic determinants for hemolysin (hly) and polymyxin B resistance (pmx) to selected nutritional markers in matings between Tfr El Tor donor and classical recipient strains of V. cholerae Selected Avg no. of No. of No. of Donor Recipient donor recombinants hyl/no. pmx/no. markera per plate tested tested GN9003(pJN8) RV88 leu /209 GN9003(pJN2) RV94 arg /102 GN9006(PJN8) RV88 pro /154 GN9006(pJN2) RV88 leu /263 GN9006(pJN2) RV94 his /305 GN9006(pJN2) RV94 trp /300 GN9006(pJN2) RV93 met /104 GN7007(pJN2) RV94 trp /174 GN7007(pJN8) RV94 his /163 GN7007(pJN15) RV93 met /108 GN9006(pJN8) RV94 arg /359 GN9006(pJN2) RV88 leu /370 GN7007(pJN2) RV94 his /170 GN7007(pJN15) RV93 met /237 a In each mating, the selected marker from the recipient was str. with the hypothesis that hly is between the pyra and leu loci, on the opposite site of leu from his and trp, and that the pmx and cha loci are closely linked to the leu locus on the side toward his Ȧdditional mating experiments were performed by using the El Tor donor strain GN9006(pJN2), which transfers the donor alleles leu+, his', and trp+ as early markers from the origin at the pyra locus (Table 4; Fig. 1). Each of the donor markers hly, pmx, and cha was coinherited at high frequency (from 67 to 98%) in recombinants with the selected donor allele leu+, his', or trp+. Two unexpected findings were noted in these experiments and in the experiments shown in Table 3. First, recombinants were isolated that expressed pmx but not cha; however, no reciprocal recombinants with hemagglutinating but polymyxin-susceptible phenotypes were isolated. This was surprising, because the region of the chromosome containing pmx and cha was mobilized in both directions, and selection was imposed for markers located on both sides of pmx and cha (Tables 3 and 4). Second, when trp+ was the selected donor marker in matings involving the GN9006(pJN2) donor parent, the hly, pmx, and cha markers were inherited more frequently than the his' parental marker. Because of these anomalies, it was not possible to assign a precise order for the closely linked pmx and cha loci. DISSCUSSION El Tor biotype strains of V. cholerae possess several characteristics which differentiate them from classical strains. This study was designed to determine the location on the genetic map of V. cholerae of determinants for three of the biotype markers. For mating experiments, we used El Tor Tfr donor strains containing either Tnl or TnS inserted into the bacterial chromosome and auxotrophic classical recipient strains. Preliminary experiments were designed to demonstrate transfer of the El Tor biotype markers to classical recipient strains by conjugation (Table 2). Ogg et al. transduced the polymyxin resistance determinant(s) into classical recipients, using the generalized transducing phage CP-T1, but they were unable to demonstrate linkage of the pmx marker to any other loci (13). Our data showed that pmx and hly were most TABLE 3. Analysis of linkage of El Tor biotype determinants for hemolysin (hly), polymyxin B resistance (pmx), and chicken erythrocyte hemagglutinin (cha) in matings between Tfr El Tor donor strain GN7007(pJN2) and classical recipient strains of V. cholerae Recipient Selected Expression of unselected donor No. donor markers strain strain mre"observed market' hly pmx cha trp+ his' RV88 leu b _b RV94 his RV94 trp a Donors were counterselected by using str in all matings. b +, Expression of the donor marker; -, lack of expression of the donor marker.

5 928 GREEN, NEWLAND, AND HOLMES closely linked to the leu and his loci on the section of the genetic map between the trp and pyra loci (Table 2; Fig. 1). The linkage analysis shown in Tables 3 and 4 showed that the hly, pmx, and cha determinants were all closely linked to leu and provided strong support for the arrangement of these markers shown on the genetic map in Fig. 1. As previously noted, two unexpected findings precluded assignment of a gene order for the pmx and cha loci. These findings were the failure to isolate any polymyxin-susceptible, hemagglutinating recombinants and the unexpectedly high proportion of selected trp+ recombinants that inherited the hly, pmx, and cha markers from the donor strain GN9006(pJN2) (Table 4). These findings raised the possibility that unknown selective factors biased the recovery of specific classes of recombinants. For example, expression of cha without expression of pmx may be lethal, or functions determined by pmx may be necessary for expression of cha. Furthermore, previous studies in our laboratory led to the conclusion that the segment of the El Tor genetic map including the loci pro-201-pyra-201-leu- 1-ura-201 was inverted with respect to the map of the classical V. cholerae strain 162 (Newland et al., in preparation). Since the hly, pmx, and cha markers are closely linked to leu, the possible presence of an inversion in this region of the genetic map might have unpredictable results on inheritance of hly, pmx, or cha in some matings between El Tor donor and classical recipient strains of V. cholerae. The linkage relationships of hly, pmx, and cha were most difficult to interpret in the matings shown in Table 4, in which the origin for transfer of the donor chromosome was located within a segment of the V. cholerae chromosome presumed to be inverted between El Tor and classical strains. If mutations affecting hly, pmx, and cha can be isolated in El Tor donor strains of V. cholerae, it will be possible in future studies to analyze the locations of these genes in matings between homologous donor and recipient strains. This approach should eliminate potential complications caused by chromosomal inversions between the donor and recipient strains. Additional studies will also be required to identify the gene products encoded by the hly, pmx, and cha determinants and to characterize their functions. The classical and El Tor biotypes of V. cholerae are closely related bacteria that show extensive DNA homology (5) and undergo genetic exchange mediated by conjugation or transduction (2, 13, 15). Nevertheless, they also have distinctive phenotypic characteristics, and most clinical isolates of V. cholerae can be identified as belonging to either the classical or the El Tor biotype. The results reported here demonstrate INFECT. IMMUN. TABLE 4. Analysis of linkage of El Tor biotype determinants for hemolysin (hly), polymyxin B resistance (pmx), and chicken erythrocyte hemagglutinin (cha) in matings between Tfr El Tor donor strain GN9006(pJN2) and classical recipient strains of V. cholerae Selected Expression of unselected Recipient donor No. donor markers strain dnr observed marker' hly pmx cha trp his RV88 leu b _b RV94 his RV94 trp _ a Donors were counterselected by using str. b +, Expression of the donor marker; -, lack of expression of the donor marker. that three of the El Tor biotype determinants, hly, pmx, and cha, are closely linked on the genetic map of V. cholerae. It remains to be established whether determinants for other biotype-specific characteristics are also located in this region of the chromosome of V. cholerae. The close genetic linkage of biotype markers probably contributes to the phenotypic stability of the classical and El Tor biotypes, but barriers to free genetic exchange between classical and El Tor strains of V. cholerae in natural environments may also play a role in biotype stability (3). ACKNOWLEDGMENTS This study was supported by Uniformed Services University of the Health Sciences protocol number R We thank F. Finkelman for providing the chicken erythrocytes used in our studies and I. Dinger for secretarial assistance. LITERATURE CITED 1. Bachmann, B. J., and K. B. Low Linkage map of Escherichia coli K-12, edition 6. Microbiol. Rev. 44: Bhaskaran, K Recombination of characteristics between mutant stocks of Vibrio cholerae, strain 162. J. Gen. Microbiol. 23: Bhaskaran, K Vibrio genetics, p In D. Barua and W. Burrows (ed.), Cholera. The W. B. Saunders Co., Philadelphia, Pa.

6 VOL. 42, 1983 MAPPING OF BIOTYPE DETERMINANTS IN V. CHOLERAE Bramucci, M. G., and R. K. Holmes Radial passive immune hemolysis assay for detection of heat-labile enterotoxin produced by individual colonies of Escherichia coli or Vibrio cholerae. J. Clin. Microbiol. 8: Citarella, R. V., and R. R. Colwell Polyphasic taxonomy of the genus Vibrio: polynucleotide sequence relationships among selected Vibrio species. J. Bacteriol. 104: Finkelstein, R. A Cholera. Crit. Rev. Microbiol. 2: Finkelstein, R. A., P. Atthasampunna, M. Chulaspamaya, and P. Charumathee Pathogenesis of experimental cholera: biological activities of purified procholeragen A. J. Immunol. 96: Garvey, J. S., N. E. Cremer, and D. H. Sussdorf (ed) Methods in immunology, 3rd ed., p Benjamin Cummings Publishing Co., Reading, Mass. 9. Holmgren, J., A.-M. Svennerholm, and M. Lindblad Receptor-like glycocompounds in human milk that inhibit classical and El Tor Vibrio cholerae cell adherence (hemagglutination). Infect. Immun. 39: Johnson, S. R., and W. R. Romig Transposonfacilitated recombination in Vibrio cholerae. Mol. Gen. Genet. 170: Jones, G. W., G. D. Abrams, and R. Freter Adhesive properties of Vibrio cholerae: adhesion to isolated rabbit brush border membranes and hemagglutinating activity. Infect. Immun. 14: Miller, J. H Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p Ogg, J. E., T. L. Timme, and M. M. Alemohammad General transduction in Vibrio cholerae. Infect. Immun. 31: Parker, C Expanded linkage map of Vibrio cholerae. Genetics 91: Parker, C., and W. R. Romig Self-transfer and genetic recombination mediated by P, the sex factor of Vibrio cholerae. J. Bacteriol. 112: Saunders, D. W., K. J. Schanbacher, and M. G. Bramucci Mapping of a gene in Vibrio cholerae that determines the antigenic structure of cholera toxin. Infect. Immun. 38: Sublett, R. D., and W. R. Romig Transposonfacilitated recombination in classical biotypes of Vibrio cholerae. Infect. Immun. 32: