Sugars Inhibit Expression of the Rugose Phenotype
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微生物临床杂志 2005年第3期
Department of Epidemiology and Preventive Medicine
Department of Pathology, School of Medicine, University of Maryland Baltimore
Veterans Affairs Maryland Health Care System, Baltimore, Maryland
ABSTRACT
Vibrio cholerae can shift to a rugose colony phenotype, reflecting expression of an exopolysaccharide that provides protection against a variety of environmental stresses. Our data indicate that expression of the rugose phenotype is inhibited by a variety of sugars, including sucrose, dextrose, arabinose, fructose, and maltose. Inhibition by sucrose may be one factor in explaining the failure of rugose strains to grow on thiosulfate citrate bile salts sucrose agar, the primary selective medium for V. cholerae.
TEXT
Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments. In countries where cholera is endemic, such as Bangladesh and India, the disease has seasonal peaks with epidemics followed by an interepidemic period during which only sporadic or no cases of cholera can be detected (2, 3). Although epidemic strains of V. cholerae can persist for years in natural waters, the underlying basis for the survival of V. cholerae, particularly during interepidemic periods, is not well understood.
As originally described by White in 1938 (11), V. cholerae can assume a "rugose" phenotype, producing "wrinkled" rather than typical smooth colonies on nonselective media such as L agar. Rugose variants secrete copious amounts of exopolysaccharide, which confers resistance to chlorine, acid pH, serum killing, and osmotic and oxidative stresses (5, 8, 10, 13). Recently, we have observed that certain strains of V. cholerae undergo high-frequency (as high as 80%) rugose exopolysaccharide production after growing smooth cells for 2 to 3 days in modified alkaline peptone water (1). Furthermore, high-frequency rugose exopolysaccharide production was found more often in epidemic strains than in nonepidemic and environmental strains. Because of the rugose phenotype's characteristics, others and we hypothesize that it plays a critical role in the survival and persistence of V. cholerae in the environment, particularly under stressful growth conditions (1, 8, 13).
Despite its superior ability to grow under stressful growth conditions, the significance of the rugose phenotype to the environmental persistence and long-term survival of V. cholerae is poorly understood because the rugose phenotype is not expressed on thiosulfate citrate bile salts sucrose (TCBS) agar (4), the primary selective medium used to isolate V. cholerae. Subculturing of smooth forms of a rugose variant from TCBS agar (Difco, Detroit, Mich.) onto L agar resulted in rugose colonies (1), suggesting that a component(s) of TCBS agar inhibits expression of the rugose phenotype. In contrast to L agar, which allows expression of the rugose phenotype, TCBS agar contains sucrose among other ingredients as a major, readily available carbon source. Sucrose has been found to increase production of exopolysaccharide in other bacteria, such as Escherichia coli and Pseudomonas aeruginosa (9). In this study, in contrast, we found that sucrose, dextrose, fructose, maltose, and arabinose inhibited expression of the rugose phenotype when rugose cultures were grown on L agar supplemented with any of the sugars.
L agar supplemented with various sugars inhibits expression of the rugose phenotype of V. cholerae Chemicals and sugars used in this study were purchased from Sigma-Aldrich (St. Louis, Mo.). The optical activity of each carbohydrate used in this study is as follows: D-(+)-sucrose, D-dextrose, D-(–)-fructose, D-(+)-maltose, D-(–)-arabinose, D-(+)-melibiose, D-mannitol, D-arbutin, D-(–)-salicin, L-(+)-rhamnose, and D-(+)-raffinose. In initial studies, we examined the effect of sucrose in L agar (1% tryptone, 1% NaCl, 0.5% yeast extract, 1.5% agar) supplemented (after autoclaving) with various concentrations of filter-sterilized sucrose. L agar was chosen as the basal medium for our experiments because it equally supports the growth of the rugose and smooth colonial variants of V. cholerae. According to our hypothesis, if sucrose inhibits expression of the rugose phenotype, (i) rugose colonies grown on L agar should shift to a smooth colonial phenotype on L-sucrose agar, (ii) smooth colonies of rugose variants should continue displaying a smooth phenotype until the supply of the sugar is exhausted, and (iii) after the sugar is consumed by the growing cultures, the cells will use the relatively complex carbon sources (possibly tryptone and/or yeast extract's components) in L agar, thereby shifting the smooth colonies to a rugose phenotype. In order to examine our hypothesis, single colonies of a rugose variant (N16961R derived from its parent N16961S as previously described [1]) and a smooth variant (N16961S) of V. cholerae El Tor strain, grown overnight at 37°C on L agar, were appropriately diluted in saline (0.85% NaCl) and spread (in triplicate) on L-sucrose agar plates. The plates were incubated at 37°C for 24 h, then incubated at room temperature for various times, and then examined for the presence of smooth and rugose colonies with a stereoscope (Nikon, Tokyo, Japan). The data presented in Fig. 1 support our hypothesis that sucrose inhibits the expression of the rugose phenotype of V. cholerae. The smooth variant (N16961S) remained smooth on L-sucrose agar throughout the entire experimental period; however, the rugose variant (N16961R) exhibited variable results on L-sucrose agar plates, depending on the sucrose concentration and the incubation time. Five percent (wt/vol) sucrose-L agar inhibited expression of the rugose phenotype during the experimental period. In contrast, 2, 1, and 0.5% (wt/vol, final concentration) sucrose-LB agar exhibited variable results. At 24 h, all of the sucrose-L agar media inhibited rugose expression (i.e., rugose cells reverted to smooth cells); however, the smooth colonies reverted to the rugose colonial phenotype after growth for 48, 96, and 120 h on 0.5, 1, and 2% sucrose L agar, respectively (Fig. 1).
Similar results were also noted for 5, 2, 1, and 0.5% (wt/vol, final concentration) dextrose-, fructose-, arabinose-, and maltose-L agar (Fig. 1), which suggests that these sugars also prevent expression of the rugose phenotype. As expected, a nonmetabolizable carbon source such as melibiose (a disaccharide composed of galactose and glucose) did not inhibit expression of the rugose phenotype (data not shown). Glycerol (not metabolized by strain N16961) and mannitol (a metabolizable carbon source) did not inhibit the rugose phenotype (data not shown), which suggests that polyhydric alcohols, unlike sugars, do not affect the rugose phenotype. Arabinose, a hexose sugar not metabolized by V. cholerae, inhibited expression of the rugose phenotype (Fig. 1). However, the colonies grown on arabinose agar plates were very small (data not shown), which suggests that arabinose may inhibit expression of the rugose phenotype by limiting bacterial growth by an as-yet-unknown mechanism(s). Since pentoses, hexoses, and disaccharides (Fig. 1) inhibited the expression of the rugose phenotype, we were interested in determining whether other sugars, including trisaccharides (e.g., raffinose), methyl pentoses (e.g., rhamnose), and glycosides (e.g., salicin, arbutin, and esculin), inhibit its expression. Therefore, appropriate dilutions of an L broth culture of rugose strain N16961 were plated (in triplicate) on individual L agar plates supplemented with the above-mentioned sugars at 0.5, 1, 2, and 5% (wt/vol, final concentration) and the plates were incubated (37°C for 1 day and then at room temperature for 7 days). Unlike sucrose, dextrose, arabinose, fructose, and maltose (Fig. 1), the lowest concentrations (0.5, 1, and 2%) of rhamnose and raffinose did not inhibit expression of the rugose phenotype. On the other hand, the highest concentration (5%) of the sugars initially inhibited the rugose phenotype's expression, resulting in a rugose-to-smooth conversion by 1 day postincubation at 37°C. However, all of the smooth colonies grown on L agar containing 5% rhamnose and raffinose reverted to the rugose phenotype after subsequent incubation for 3 days at room temperature. These two observations suggest that the highest concentration of those sugars transiently masked the rugose phenotype by an as-yet-unknown mechanism(s). Despite repeated attempts, we were unable to grow rugose strain N16961 on L agar containing 2 or 5% esculin; therefore, we examined the effect of arbutin and salicin (two other glycosides) on the rugose phenotype's expression. Low concentrations (0.5 and 1%) of those two glycosides did not inhibit the phenotype's expression; however, higher concentrations (2 and 5%) inhibited its expression. The smooth colonies formed by the rugose strain grown on 2 and 5% arbutin and salicin for 1 day at 37°C did not revert to the rugose phenotype after incubation at room temperature for another 7 days. Thus, our observations suggest that a high concentration (5%) of rhamnose and raffinose differs from that of arbutin and salicin in altering expression of the rugose phenotype. We observed that metabolizable sugar (mannitol) did not affect the expression of the rugose phenotype; in contrast, nonmetabolizable sugars (salicin, arbutin, rhamnose, and raffinose) partially affected the rugose phenotype's expression. On the basis of these observations, we conclude that inhibition of expression of the rugose phenotype on L agar supplemented with various sugars (used in this study) is not exclusively related to the ability of V. cholerae to metabolize those sugars. Our present study did not focus on elucidating the genetic mechanisms that promote inhibition of the rugose phenotype's expression by various sugars. However, other investigators have reported that mutations in V. cholerae genes galU and galE, encoding UDP-glucose pyrophosphorylase (which catalyzes the production of UDP-glucose) and UDP-galactose epimerase, respectively, are required for expression of the rugose phenotype (6, 7, 12). Therefore, our future studies will focus on determining the mechanism by which certain sugars inhibit the rugose phenotype's expression.
We speculated that the acid pH (in and around the growing colony) resulting from the metabolism of the sugars might have contributed to the observed effect. In order to further assess our hypothesis, a single colony of a rugose variant was added to triplicate samples of (i) L broth (Miller; pH 7.2), (ii) L broth (Miller; pH 7.2) supplemented with 2 and 5% sucrose (a readily available carbon source), and (iii) a minimal medium composed of minimal salts, 5x (Difco, pH 6.8), containing 2% sucrose. The cultures were grown overnight at 37°C with shaking, and appropriate dilutions of each culture were plated onto L agar to determine the rugose or smooth colonial phenotype. As expected, the rugose variant grown in L broth yielded 100% rugose colonies. Interestingly, rugose variant grown in L-sucrose broth and sucrose minimal medium also resulted in 100% rugose colonies. The pHs of the L broth and L-sucrose broth cultures were found to be 8.4, 8.2 (L-sucrose broth with 2% sucrose), and 5.5 (L-sucrose broth with 5% sucrose), respectively (data not shown). Furthermore, the pH of the sucrose minimal broth was 5.77. These observations suggest that (i) acid pH does not promote rugose-to-smooth conversion of V. cholerae, and (ii) explicit conversion of rugose colonies to smooth colonies on TCBS agar and L agar supplemented with various sugars may be attributed to as-yet-identified factors and processes.
To examine whether inhibition of rugose phenotype expression on L-sucrose agar was due to the effect of osmolarity, expression of the phenotype on 2% L-sucrose agar was compared with that on L agar supplemented with another osmolant, NaCl (0.2, 0.4, or 0.6 M). V. cholerae N16961R exhibited rugose colonies on L-agar supplemented with NaCl (0.2 to 0.6 M), but it reverted back to a smooth variant on L-sucrose agar after 18 h of incubation at 37°C. This observation indicates that the osmolarity of the medium did not influence expression of the rugose phenotype.
Since sugars other than dextrose inhibit expression of the rugose phenotype, their effect appears not to be caused by catabolite repression. Our findings contrast with that reported (9) for exopolysaccharide production by E. coli, in which sucrose induces increased synthesis of polysaccharide without significantly changing the growth rate of the bacterium.
TCBS agar contains factors inhibiting rugose phenotype expression in V. cholerae We hypothesized that the results we obtained with 2% sucrose-L agar (Fig. 1) simulate the results obtained with V. cholerae growing on TCBS agar containing 2% sucrose as a readily available carbon source. To examine our hypothesis, the rugose variant (N16961R) was spread on TCBS agar and incubated as described above. However, the smooth form of the rugose variant did not revert to the rugose variant by 120 h of incubation, as expected (Fig. 2). This observation suggests that, in addition to sucrose, TCBS agar contains an additional factor(s) capable of inhibiting expression of the rugose phenotype. To evaluate this idea, an appropriately diluted culture of the rugose variant was spread on TCBS agar lacking sucrose and incubated as described above. TCBS agar without sucrose exhibited a partial rugose phenotype at 36 to 48 h (Fig. 2). Comparable results were also noted (Fig. 2) when the rugose and smooth variants were grown on L agar supplemented with the ingredients in TCBS agar without sucrose (i.e., sodium thiosulfate, sodium citrate, ferric citrate, and Bacto Oxgall, used at final concentrations of 1, 1, 0.1, and 0.8% [wt/vol], respectively).
TCBS agar contains multiple ingredients, including 2% sucrose, which inhibits expression of the rugose phenotype. To identify other specific constituents that might inhibit rugose colony formation, L agar was supplemented with the individual ingredients of TCBS agar (including sodium thiosulfate, sodium citrate, ferric citrate, and Bacto Oxgall at final concentrations of 1, 1, 0.1, and 0.8% [wt/vol], respectively) and inoculated with the rugose variant. None of the ingredients alone inhibited the expression of the rugose phenotype, suggesting that inhibition was related either to a combination of ingredients or to other metabolites or by-products formed specifically in the production of TCBS agar.
The data obtained during our studies suggest that sucrose, dextrose, fructose, and maltose inhibit expression of the rugose phenotype when these sugars are added to an agar. Thus, the commonly used TCBS agar (which contains sucrose as a key ingredient) or any agar containing these sugars should not be used to isolate rugose variants of V. cholerae. The possible significance of the rugose phenotype for the survival of V . cholerae in aquatic environments has recently received increased attention from the scientific community. However, using TCBS agar to determine the incidence of the rugose variant in clinical and environmental samples may mislead researchers.
ACKNOWLEDGMENTS
We thank Sean C. Daugherty for technical assistance, Arnold Kreger for copy editing assistance, and James Kaper for comments on the manuscript. We also thank the anonymous reviewers for suggestion to improve the manuscript.
This work was supported by a Department of Veterans Affairs grant to Judith A. Johnson and an NIH grant (RO1 GM60791) to J. G. Morris, Jr.
REFERENCES
Ali, A., M. H. Rashid, and D. K. R. Karaolis. 2002. High-frequency rugose exopolysaccharide production by Vibrio cholerae. Appl. Environ. Microbiol. 68:5773-5778.
Barua, D. 1992. History of cholera, p. 1-36. In D. Barua and W. B. Greenough III (ed.), Cholera. Plenum, New York, N.Y.
Kaper, J. B., J. G. Morris, Jr., and M. M. Levine. 1995. Cholera. Clin. Microbiol. Rev. 8:48-86.
Kobayashi, T., S. Enomoto, R. Sakazaki, and S. Kuwahara. 1963. A new selective isolation medium for the vibrio group; on a modified Na-kanishi's medium (TCBS agar medium). Jpn. J. Bacteriol. 18:387-392.
Morris, J. G., Jr., M. B. Sztein, E. W. Rice, J. P. Nataro, G. A. Losonsky, P. Panigrahi, C. O. Tacket, and J. A. Johnson. 1996. Vibrio cholerae O1 can assume a chlorine-resistant rugose survival form that is virulent for humans. J. Infect. Dis. 174:1364-1368.
Nesper, J., C. M. Lauriano, K. E. Klose, D. Kapfhammer, A. Kraiss, and J. Reidl. 2001. Characterization of Vibrio cholerae O1 El Tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation. Infect. Immun. 69:435-445.
Rashid, M. H., C. Rajanna, A. Ali, and D. K. R. Karaolis. 2003. Identification of genes involved in the switch between the smooth and rugose phenotypes of Vibrio cholerae. FEMS Microbiol. Lett. 227:113-119.
Rice, E. W., C. H. Johnson, R. M. Clark, K. R. Fox, D. J. Reasoner, M. E. Dunnigan, P. Panigrah, J. A. Johnson, and J. G. Morris, Jr. 1993. Chlorine and survival of "rugose" Vibrio cholerae. Int. J. Environ. Health Res. 3:89-98.
Samrakandi, M. M., C. Rouques, and G. Michel. 1997. Influence of trophic conditions on exopolysaccharide production: bacterial biofilm susceptibility to chlorine and monochloramine. Can. J. Microbiol. 43:751-758.
Wai, S. N., Y. Mizunoe, A. Takade, S. I. Kawabata, and S. I. Yoshida. 1998. Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation. Appl. Environ. Microbiol. 64:3648-3655.
White, P. B. 1938. The rugose variant of vibrios. J. Pathol. Bacteriol. 46:1-6.
Yildiz, F. H., X. S. Liu, A. Heydorn, and G. K. Schoolnik. 2004. Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant. Mol. Microbiol. 53:497-515.
Yildiz, F. H., and G. K. Schoolnik. 1999. Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proc. Natl. Acad. Sci. USA 96:4028-4033.(Afsar Ali, J. Glenn Morri)
Department of Pathology, School of Medicine, University of Maryland Baltimore
Veterans Affairs Maryland Health Care System, Baltimore, Maryland
ABSTRACT
Vibrio cholerae can shift to a rugose colony phenotype, reflecting expression of an exopolysaccharide that provides protection against a variety of environmental stresses. Our data indicate that expression of the rugose phenotype is inhibited by a variety of sugars, including sucrose, dextrose, arabinose, fructose, and maltose. Inhibition by sucrose may be one factor in explaining the failure of rugose strains to grow on thiosulfate citrate bile salts sucrose agar, the primary selective medium for V. cholerae.
TEXT
Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments. In countries where cholera is endemic, such as Bangladesh and India, the disease has seasonal peaks with epidemics followed by an interepidemic period during which only sporadic or no cases of cholera can be detected (2, 3). Although epidemic strains of V. cholerae can persist for years in natural waters, the underlying basis for the survival of V. cholerae, particularly during interepidemic periods, is not well understood.
As originally described by White in 1938 (11), V. cholerae can assume a "rugose" phenotype, producing "wrinkled" rather than typical smooth colonies on nonselective media such as L agar. Rugose variants secrete copious amounts of exopolysaccharide, which confers resistance to chlorine, acid pH, serum killing, and osmotic and oxidative stresses (5, 8, 10, 13). Recently, we have observed that certain strains of V. cholerae undergo high-frequency (as high as 80%) rugose exopolysaccharide production after growing smooth cells for 2 to 3 days in modified alkaline peptone water (1). Furthermore, high-frequency rugose exopolysaccharide production was found more often in epidemic strains than in nonepidemic and environmental strains. Because of the rugose phenotype's characteristics, others and we hypothesize that it plays a critical role in the survival and persistence of V. cholerae in the environment, particularly under stressful growth conditions (1, 8, 13).
Despite its superior ability to grow under stressful growth conditions, the significance of the rugose phenotype to the environmental persistence and long-term survival of V. cholerae is poorly understood because the rugose phenotype is not expressed on thiosulfate citrate bile salts sucrose (TCBS) agar (4), the primary selective medium used to isolate V. cholerae. Subculturing of smooth forms of a rugose variant from TCBS agar (Difco, Detroit, Mich.) onto L agar resulted in rugose colonies (1), suggesting that a component(s) of TCBS agar inhibits expression of the rugose phenotype. In contrast to L agar, which allows expression of the rugose phenotype, TCBS agar contains sucrose among other ingredients as a major, readily available carbon source. Sucrose has been found to increase production of exopolysaccharide in other bacteria, such as Escherichia coli and Pseudomonas aeruginosa (9). In this study, in contrast, we found that sucrose, dextrose, fructose, maltose, and arabinose inhibited expression of the rugose phenotype when rugose cultures were grown on L agar supplemented with any of the sugars.
L agar supplemented with various sugars inhibits expression of the rugose phenotype of V. cholerae Chemicals and sugars used in this study were purchased from Sigma-Aldrich (St. Louis, Mo.). The optical activity of each carbohydrate used in this study is as follows: D-(+)-sucrose, D-dextrose, D-(–)-fructose, D-(+)-maltose, D-(–)-arabinose, D-(+)-melibiose, D-mannitol, D-arbutin, D-(–)-salicin, L-(+)-rhamnose, and D-(+)-raffinose. In initial studies, we examined the effect of sucrose in L agar (1% tryptone, 1% NaCl, 0.5% yeast extract, 1.5% agar) supplemented (after autoclaving) with various concentrations of filter-sterilized sucrose. L agar was chosen as the basal medium for our experiments because it equally supports the growth of the rugose and smooth colonial variants of V. cholerae. According to our hypothesis, if sucrose inhibits expression of the rugose phenotype, (i) rugose colonies grown on L agar should shift to a smooth colonial phenotype on L-sucrose agar, (ii) smooth colonies of rugose variants should continue displaying a smooth phenotype until the supply of the sugar is exhausted, and (iii) after the sugar is consumed by the growing cultures, the cells will use the relatively complex carbon sources (possibly tryptone and/or yeast extract's components) in L agar, thereby shifting the smooth colonies to a rugose phenotype. In order to examine our hypothesis, single colonies of a rugose variant (N16961R derived from its parent N16961S as previously described [1]) and a smooth variant (N16961S) of V. cholerae El Tor strain, grown overnight at 37°C on L agar, were appropriately diluted in saline (0.85% NaCl) and spread (in triplicate) on L-sucrose agar plates. The plates were incubated at 37°C for 24 h, then incubated at room temperature for various times, and then examined for the presence of smooth and rugose colonies with a stereoscope (Nikon, Tokyo, Japan). The data presented in Fig. 1 support our hypothesis that sucrose inhibits the expression of the rugose phenotype of V. cholerae. The smooth variant (N16961S) remained smooth on L-sucrose agar throughout the entire experimental period; however, the rugose variant (N16961R) exhibited variable results on L-sucrose agar plates, depending on the sucrose concentration and the incubation time. Five percent (wt/vol) sucrose-L agar inhibited expression of the rugose phenotype during the experimental period. In contrast, 2, 1, and 0.5% (wt/vol, final concentration) sucrose-LB agar exhibited variable results. At 24 h, all of the sucrose-L agar media inhibited rugose expression (i.e., rugose cells reverted to smooth cells); however, the smooth colonies reverted to the rugose colonial phenotype after growth for 48, 96, and 120 h on 0.5, 1, and 2% sucrose L agar, respectively (Fig. 1).
Similar results were also noted for 5, 2, 1, and 0.5% (wt/vol, final concentration) dextrose-, fructose-, arabinose-, and maltose-L agar (Fig. 1), which suggests that these sugars also prevent expression of the rugose phenotype. As expected, a nonmetabolizable carbon source such as melibiose (a disaccharide composed of galactose and glucose) did not inhibit expression of the rugose phenotype (data not shown). Glycerol (not metabolized by strain N16961) and mannitol (a metabolizable carbon source) did not inhibit the rugose phenotype (data not shown), which suggests that polyhydric alcohols, unlike sugars, do not affect the rugose phenotype. Arabinose, a hexose sugar not metabolized by V. cholerae, inhibited expression of the rugose phenotype (Fig. 1). However, the colonies grown on arabinose agar plates were very small (data not shown), which suggests that arabinose may inhibit expression of the rugose phenotype by limiting bacterial growth by an as-yet-unknown mechanism(s). Since pentoses, hexoses, and disaccharides (Fig. 1) inhibited the expression of the rugose phenotype, we were interested in determining whether other sugars, including trisaccharides (e.g., raffinose), methyl pentoses (e.g., rhamnose), and glycosides (e.g., salicin, arbutin, and esculin), inhibit its expression. Therefore, appropriate dilutions of an L broth culture of rugose strain N16961 were plated (in triplicate) on individual L agar plates supplemented with the above-mentioned sugars at 0.5, 1, 2, and 5% (wt/vol, final concentration) and the plates were incubated (37°C for 1 day and then at room temperature for 7 days). Unlike sucrose, dextrose, arabinose, fructose, and maltose (Fig. 1), the lowest concentrations (0.5, 1, and 2%) of rhamnose and raffinose did not inhibit expression of the rugose phenotype. On the other hand, the highest concentration (5%) of the sugars initially inhibited the rugose phenotype's expression, resulting in a rugose-to-smooth conversion by 1 day postincubation at 37°C. However, all of the smooth colonies grown on L agar containing 5% rhamnose and raffinose reverted to the rugose phenotype after subsequent incubation for 3 days at room temperature. These two observations suggest that the highest concentration of those sugars transiently masked the rugose phenotype by an as-yet-unknown mechanism(s). Despite repeated attempts, we were unable to grow rugose strain N16961 on L agar containing 2 or 5% esculin; therefore, we examined the effect of arbutin and salicin (two other glycosides) on the rugose phenotype's expression. Low concentrations (0.5 and 1%) of those two glycosides did not inhibit the phenotype's expression; however, higher concentrations (2 and 5%) inhibited its expression. The smooth colonies formed by the rugose strain grown on 2 and 5% arbutin and salicin for 1 day at 37°C did not revert to the rugose phenotype after incubation at room temperature for another 7 days. Thus, our observations suggest that a high concentration (5%) of rhamnose and raffinose differs from that of arbutin and salicin in altering expression of the rugose phenotype. We observed that metabolizable sugar (mannitol) did not affect the expression of the rugose phenotype; in contrast, nonmetabolizable sugars (salicin, arbutin, rhamnose, and raffinose) partially affected the rugose phenotype's expression. On the basis of these observations, we conclude that inhibition of expression of the rugose phenotype on L agar supplemented with various sugars (used in this study) is not exclusively related to the ability of V. cholerae to metabolize those sugars. Our present study did not focus on elucidating the genetic mechanisms that promote inhibition of the rugose phenotype's expression by various sugars. However, other investigators have reported that mutations in V. cholerae genes galU and galE, encoding UDP-glucose pyrophosphorylase (which catalyzes the production of UDP-glucose) and UDP-galactose epimerase, respectively, are required for expression of the rugose phenotype (6, 7, 12). Therefore, our future studies will focus on determining the mechanism by which certain sugars inhibit the rugose phenotype's expression.
We speculated that the acid pH (in and around the growing colony) resulting from the metabolism of the sugars might have contributed to the observed effect. In order to further assess our hypothesis, a single colony of a rugose variant was added to triplicate samples of (i) L broth (Miller; pH 7.2), (ii) L broth (Miller; pH 7.2) supplemented with 2 and 5% sucrose (a readily available carbon source), and (iii) a minimal medium composed of minimal salts, 5x (Difco, pH 6.8), containing 2% sucrose. The cultures were grown overnight at 37°C with shaking, and appropriate dilutions of each culture were plated onto L agar to determine the rugose or smooth colonial phenotype. As expected, the rugose variant grown in L broth yielded 100% rugose colonies. Interestingly, rugose variant grown in L-sucrose broth and sucrose minimal medium also resulted in 100% rugose colonies. The pHs of the L broth and L-sucrose broth cultures were found to be 8.4, 8.2 (L-sucrose broth with 2% sucrose), and 5.5 (L-sucrose broth with 5% sucrose), respectively (data not shown). Furthermore, the pH of the sucrose minimal broth was 5.77. These observations suggest that (i) acid pH does not promote rugose-to-smooth conversion of V. cholerae, and (ii) explicit conversion of rugose colonies to smooth colonies on TCBS agar and L agar supplemented with various sugars may be attributed to as-yet-identified factors and processes.
To examine whether inhibition of rugose phenotype expression on L-sucrose agar was due to the effect of osmolarity, expression of the phenotype on 2% L-sucrose agar was compared with that on L agar supplemented with another osmolant, NaCl (0.2, 0.4, or 0.6 M). V. cholerae N16961R exhibited rugose colonies on L-agar supplemented with NaCl (0.2 to 0.6 M), but it reverted back to a smooth variant on L-sucrose agar after 18 h of incubation at 37°C. This observation indicates that the osmolarity of the medium did not influence expression of the rugose phenotype.
Since sugars other than dextrose inhibit expression of the rugose phenotype, their effect appears not to be caused by catabolite repression. Our findings contrast with that reported (9) for exopolysaccharide production by E. coli, in which sucrose induces increased synthesis of polysaccharide without significantly changing the growth rate of the bacterium.
TCBS agar contains factors inhibiting rugose phenotype expression in V. cholerae We hypothesized that the results we obtained with 2% sucrose-L agar (Fig. 1) simulate the results obtained with V. cholerae growing on TCBS agar containing 2% sucrose as a readily available carbon source. To examine our hypothesis, the rugose variant (N16961R) was spread on TCBS agar and incubated as described above. However, the smooth form of the rugose variant did not revert to the rugose variant by 120 h of incubation, as expected (Fig. 2). This observation suggests that, in addition to sucrose, TCBS agar contains an additional factor(s) capable of inhibiting expression of the rugose phenotype. To evaluate this idea, an appropriately diluted culture of the rugose variant was spread on TCBS agar lacking sucrose and incubated as described above. TCBS agar without sucrose exhibited a partial rugose phenotype at 36 to 48 h (Fig. 2). Comparable results were also noted (Fig. 2) when the rugose and smooth variants were grown on L agar supplemented with the ingredients in TCBS agar without sucrose (i.e., sodium thiosulfate, sodium citrate, ferric citrate, and Bacto Oxgall, used at final concentrations of 1, 1, 0.1, and 0.8% [wt/vol], respectively).
TCBS agar contains multiple ingredients, including 2% sucrose, which inhibits expression of the rugose phenotype. To identify other specific constituents that might inhibit rugose colony formation, L agar was supplemented with the individual ingredients of TCBS agar (including sodium thiosulfate, sodium citrate, ferric citrate, and Bacto Oxgall at final concentrations of 1, 1, 0.1, and 0.8% [wt/vol], respectively) and inoculated with the rugose variant. None of the ingredients alone inhibited the expression of the rugose phenotype, suggesting that inhibition was related either to a combination of ingredients or to other metabolites or by-products formed specifically in the production of TCBS agar.
The data obtained during our studies suggest that sucrose, dextrose, fructose, and maltose inhibit expression of the rugose phenotype when these sugars are added to an agar. Thus, the commonly used TCBS agar (which contains sucrose as a key ingredient) or any agar containing these sugars should not be used to isolate rugose variants of V. cholerae. The possible significance of the rugose phenotype for the survival of V . cholerae in aquatic environments has recently received increased attention from the scientific community. However, using TCBS agar to determine the incidence of the rugose variant in clinical and environmental samples may mislead researchers.
ACKNOWLEDGMENTS
We thank Sean C. Daugherty for technical assistance, Arnold Kreger for copy editing assistance, and James Kaper for comments on the manuscript. We also thank the anonymous reviewers for suggestion to improve the manuscript.
This work was supported by a Department of Veterans Affairs grant to Judith A. Johnson and an NIH grant (RO1 GM60791) to J. G. Morris, Jr.
REFERENCES
Ali, A., M. H. Rashid, and D. K. R. Karaolis. 2002. High-frequency rugose exopolysaccharide production by Vibrio cholerae. Appl. Environ. Microbiol. 68:5773-5778.
Barua, D. 1992. History of cholera, p. 1-36. In D. Barua and W. B. Greenough III (ed.), Cholera. Plenum, New York, N.Y.
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