Recently we described the isolation of spontaneous bacteriophage K139-resistant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galU gene. We inactivated another gal gene, galE, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not the galE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that galU and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found that galU and galE are essential for the formation of a biofilm in a spontaneous phageresistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of galU in V. cholerae virulence, correlated with the observed change in LPS structure, and a role for galU and galE in environmental survival of V. cholerae.The causative agent of the intestinal disease cholera is Vibrio cholerae, a gram-negative motile bacterium. Of the more than 150 known serogroups, only the noncapsulated O1 and the encapsulated O139 serogroup have been found to be associated with epidemic cholera. Epidemic O139 strains are related to and were derived from O1 El Tor strains after genetic alterations of the O-antigen biosynthesis gene cluster (16). The ongoing seventh pandemic, which began in 1961, is caused by O1 El Tor strains (3). V. cholerae is a natural inhabitant of aquatic ecosystems and is known to attach to environmental surfaces such as plants, filamentous green algae, zooplankton, crustaceans, or insects (8). Recently, V. cholerae O1 El Tor was found to form a three-dimensional biofilm on abiotic surfaces (70). Biofilm formation may be important in the life cycle of pathogenic V. cholerae strains, because they reside within natural aquatic habitats during interepidemic periods. O1 El Tor strains are also able to switch to a rugose colony phenotype. This morphology correlates with the constitutively production of an exopolysaccharide allowing biofilm formation on abiotic surfaces (65...

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.

Bacteriophage K139 was recently characterized as a temperate phage of O1 Vibrio cholerae. In this study we have determined the phage adsorption site on the bacterial cell surface. Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor. In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139. Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide. By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection. Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfb region). Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004 into the rfb gene cluster. Treating one wbeW::IS1004 serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance. Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation. This strain had a significant reduction in its ability to colonize the mouse small intestine.Vibrio cholerae strains from serogroups O1 and O139 are the etiologic agents of cholera, a life-threatening acute diarrhea. The O1 serogroup is divided into the main serotypes Inaba and Ogawa, and O1 is subdivided into two distinct biotypes, designated classical and El Tor (22). Lipopolysaccharide (LPS) is the major integral component of the outer membrane and chemically consists of an O antigen, a core oligosaccharide, and lipid A. The O antigen of O1 V. cholerae consists of a homopolymer of approximately 18 (132) linked linear 4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-D-mannose) (23, 36). The LPS also contains the carbohydrate quinovosamine, which at the present time cannot be precisely defined as a component of either the O antigen or the core oligosaccharide (45). The Ogawa and Inaba serotypes differ by the presence of a 2-Omethyl group in the nonreducing terminal carbohydrate in the Ogawa O antigen (19,21). It was shown that Ogawa and Inaba O1 LPS can interconvert and that this serotype variation is due to spontaneous mutations in the wbeT gene (47). Strains of the serogroup O139 contain only a short O antigen but, in contrast to O1 strains, are encapsulated (51). Molecular and epidemiological analyses as well as phage typing revealed that O139 strains are very similar to O1 El Tor strains (2,17,18). One characteristic difference is the replacement of the 22-kb O1 rfb region with a 35-kb DNA fragment encoding the O139 O antigen and capsule (4,5,10,48)...

In this report, we characterize the complete genome sequence of the temperate phage K139, which morphologically belongs to the Myoviridae phage family (P2 and 186). The prophage genome consists of 33,106 bp, and the overall GC content is 48.9%. Forty-four open reading frames were identified. Homology analysis and motif search were used to assign possible functions for the genes, revealing a close relationship to P2-like phages. By Southern blot screening of a Vibrio cholerae strain collection, two highly K139-related phage sequences were detected in non-O1, non-O139 strains. Combinatorial PCR analysis revealed almost identical genome organizations. One region of variable gene content was identified and sequenced. Additionally, the tail fiber genes were analyzed, leading to the identification of putative host-specific sequence variations. Furthermore, a K139-encoded Dam methyltransferase was characterized.At present, 183 different tailed and 10 filamentous Vibrio phages have been described. On the basis of the morphotypes, the tailed phages were grouped into seven basic forms belonging to the families of tailed phages (Myoviridae, Siphoviridae, and Podoviridae) and the filamentous phages were typed to the Inoviridae family (1). Due to the importance of the filamentous phage CTX for the virulence of Vibrio cholerae, sequencing efforts have been focused mainly on this group of phages (CTX [56], fsl [26], and fs-2 [27]). To our knowledge, K139 is the first tailed vibriophage for which information for the entire sequence is available. K139 was originally isolated from the V. cholerae serogroup O139 (48), which emerged for the first time in 1992 as the causative agent of cholera epidemics (1a). Subsequently, we found that the phage can also be recovered very frequently from various V. cholerae strains of serogroup O1 biotype El Tor. The observation that nonlysogenic O139 strains could not be infected with K139 was confirmed by the identification of the O1 antigen as the primary phage receptor (41). Analysis of the lysogeny-lysis switch genes already indicated a relationship to P2-like phages (40), which belong morphologically to the Myoviridae family. Members of this phage group (Escherichia coli phages P2 and 186 [14], Pseudomonas aeruginosa phage CTX [39], and Haemophilus influenzae phages HP1 [15] and HP2 [direct submission, GenBank accession no. NC_003315]) typically contain approximately 31-to 36-kb double-stranded DNA with single-stranded cohesive ends, but several subgroups, defined by the presence of differently derived genes, exist. For example, HP1 and HP2 contain tail genes different from those of the P2/186/CTX group, whereas CTX differs from all P2-like phages in its content of the early and delayed early genes. The evolution of such mosaic-like phage genomes may result from horizontal exchange of whole functional units (modules) (7) or of smaller units (single genes or gene fragments) acquired from a common gene pool shared by all double-stranded DNA tailed bacteriophages (24).Here we completely sequence...