Studies on heterogeneous lipopolysaccharide fractions of Vibrio cholerae 569B

By SDS-PAGE or gel filtration on Sephadex G-25, lipopolysaccharide (LPS) isolated from Vibrio cholerae 569B (Inaba) can be separated into two distinct fractions, one corresponding to smooth LPS and the other to rough LPS. Pulse-labelling of LPS with [14C]glucose showed that the rough form is synthesized first followed by the biosynthesis of the smooth form. A preferential release of the smooth LPS of V. cholerae 569B was also detected during normal growth of cells.     

Cloning and characterisation of a novel ompB operon from Vibrio cholerae 569B

The ompB operon of Vibrio cholerae 569B has been cloned and fully sequenced. The operon encodes two proteins, OmpR and EnvZ, which share sequence identity with the OmpR and EnvZ proteins of a variety of other bacteria. Although the order of the ompR and envZ genes of V. cholerae is similar to that of the ompB operon of E. coli, S. typhimurium and X. nematophilus, the Vibrio operon exhibits a number of novel features. The structural organisation and features of the V. cholerae ompB operon are described.     

Vibrio cholerae O139 Bengal: Combined Physical and Genetic Map and Comparative Analysis with the Genome of V. cholerae O1

A combined physical and genetic map of the genome of strain SG24 of Vibrio cholerae O139 Bengal, a novel non-O1 strain having epidemic potential, has been constructed by using the enzymes NotI, SfiI, and CeuI. The genome of SG24 is circular, and the genome size is about 3.57 Mb. The linkages between 47 NotI and 32 SfiI fragments of V. cholerae SG24 genomic DNA were determined by combining two approaches: (i) identification of fragments produced by enzyme I in fragments produced by enzyme II by the method of fragment excision, redigestion, and end labeling and (ii) use of the linking clone libraries generated from the genome of classical O1 strain 569B. The linkages between nine CeuI fragments were determined primarily by analyses of partial fragments of the CeuI-digested genome. More than 80 cloned homologous and heterologous genes, including several operons, have been positioned on the physical map. The map of the SG24 genome represents the second map of a V. cholerae genome, and a comparison of this map with that of classical O1 strain 569B revealed considerable diversity in DNA restriction sites and allowed identification of hypervariable regions. Several genetic markers, including virulence determinant genes, are in different positions in the SG24 and 569B genomes.Vibrio cholerae, a noninvasive, gram-negative bacterium, is the causative agent of the diarrheal disease cholera. The specificity of the somatic O antigen of V. cholerae resides in the polysaccharide moiety of the lipopolysaccharide present in the outer membrane, which forms the basis of the serological classification of this organism (42). The V. cholerae strains causing epidemic cholera have, until recently, been confined to serogroup O1, which consists of two biotypes, classical and El Tor. The classical biotype was responsible for cholera epidemics till 1961, when the El Tor biotype displaced it. V. cholerae strains other than O1, which are collectively called non-O1 vibrios, can cause only sporadic infections and are believed to lack the potential to cause epidemics (30). One of the two events, the more alarming one, has dominated the global cholera scenario in the present decade; this was the unprecedented emergence in late 1992 in India of a novel strain of V. cholerae which does not agglutinate with O1 polyvalent antiserum but has epidemic and endemic potential, a phenomenon that has never occurred in the recorded history of cholera (1, 13, 36). Strains isolated from different parts of India and Bangladesh during the epidemic were found to be of clonal origin (5, 6) and were classified as new serovar O139, synonym Bengal. The other event was the dramatic and unexpected reappearance of epidemic cholera caused by V. cholerae O1 El Tor in South America in January 1991, after a 100-year absence on that continent (21). These two events have necessitated a renewed look into all aspects of the organism that are related to pathogenesis. The epidemic caused by V. cholerae O139 persisted for about a year (31, 32) and was again displaced by El Tor. Several lines of evidence have, however, suggested that O139 originated from the El Tor biotype (4, 6, 10, 13, 43) by the acquisition of a 35-kb DNA segment which replaced most of the O1 antigen-encoding rfb gene cluster of the recipient strain (8, 14). Thus, serogroup O139 combines the virulent properties of epidemic strains with the outer appearance of nonepidemic strains.By using restriction enzymes which have a single site in either the core region or the direct repeat sequence (RS) of the CTX genetic element (27), it was shown that the genomes of most of the O139 strains have two copies of the CTX genetic element in tandem connected by two RSs (6). The chromosomal location of the CTX genetic element in an O139 strain is the same as that reported for El Tor vibrios. The organization of the virulence gene cassettes in different O139 strains showed genetic heterogeneity in the population. While most of the epidemic O139 strains have two copies of the CTX genetic element, in some strains the number of elements has been amplified and in at least one strain a copy of the element has been deleted (6).The genomes of El Tor strains isolated immediately before and after an O139 outbreak showed extensive restriction fragment length polymorphism (RFLP) among themselves and with the genome of O139 (33, 46). In late 1996, the appearance of a V. cholerae O139 strain having altered antibiotic sensitivity compared to that of the O139 previously seen (29) has complicated the epidemiological scenario of V. cholerae and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in phenotypic traits, which are unexpected in well-characterized clonal strains within such a short period. In view of the fact that the genetic basis of V. cholerae tropism and pathogenesis is still mostly unknown, comparative genome mapping studies to appraise the extent of genome diversity will be of interest, particularly since the emergence of new variants of this organism having epidemic potential with altered genotypes or phenotypes is turning out to be widespread rather than exceptional (20). The physical map of a classical O1 strain has been constructed (12, 25), and there was previously no second map for comparison of the genomes of V. cholerae strains in more detail. It is in this context that the present report describes the construction of a macrorestriction map of the genome of O139 by use of the enzymes NotI, SfiI, and CeuI. About 80 homologous and heterologous genes and operons have been positioned on the physical map. A comparison of the V. cholerae O139 genome with that of classical O1 revealed several gross differences.     

Nucleotide sequence analysis of the CT operon of the Vibrio cholerae classical strain 569B

The complete nucleotide sequence of the Vibrio cholerae classical strain 569B was determined. The results prove the exactness of the amino acid CT B sequence published by Takao et al. (1985, Eur. J. Biochem. 146, 503-508). A comparison is made with already reported CT genes.     

The structure of the O-antigenic side chain of the lipopolysaccharide of Vibrio cholerae 569B (Inaba).

Mineral acid hydrolysis of the lipopolysaccharide from Vibrio cholerae 569B (Inaba) gives an oligosaccharide fraction which was shown, by use of 13C NMR and chemical methods, to be a regular alpha-(1 leads to 2) linked chain of D-perosamine (4-amino-4,6-dideoxy-D-mannose) units. This chain represents the O-antigen of the lipopolysaccharide, in which the amino functions are acylated with 3-hydroxypropionyl groups. The chromatographic properties of some hydroxamic acids are described and used to characterize these acyl groups.     

Cholera toxin is synthesized in precursor form on free polysomes in Vibrio cholerae 569B.

Membrane-bound and free polysomes have been isolated from Vibrio cholerae 569B. Nacent polypeptide chains were completed in a cell-free translation mixture containing Escherichia coli S-300 extracts and [3H]leucine or [35S]methionine. Cholera toxin-related polypeptides synthesized in vitro were immunologically detected after treatment with either anti-subunit A or anti-subunit B serum. Immunoreactive translation products were removed from reaction mixtures with formalinized Cowan's strain of Staphylococcus aureus, electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and visualized by fluorography. Anti-subunit A serum precipitated two major polypeptide species (molecular weights 52,000 and 45,000) from translation mixtures programed with free polysomes, whereas anti-subunit B serum precipitated only the 45,000-molecular-weight polypeptide. No cholera toxin-related polypeptides were detectable in translation mixtures programed with membrane-bound polysomes. Purified subunit A and cholera toxin competed for anti-subunit A binding sites and blocked the immunoprecipitation of the 35S-labeled 52,000- and 45,000-dalton polypeptides from in vitro translation mixtures. The data presented suggest that cholera toxin is synthesized in the cytoplasm in a precursor form on free polysomes and is secreted post-translationally.     

Localization of DnaK and GroEL in Vibrio cholerae

Though the GroEL and DnaK heat shock proteins are well characterized in prokaryotes, only scanty and controversial information exist about their cellular localization. In the present study, the localization of the heat shock proteins DnaK and GroEL in normal and heat shocked cells of Vibrio cholerae, was investigated both by immunogold labeling of ultrathin sections and biochemical methods. Much of the DnaK was found to be localized at the inner membrane in unstressed cells, most probably at the Bayer's adhesion sites. Data suggested that upon heat shock, the DnaK associated with the membrane continued to remain there, but the newly synthesized DnaK appeared mostly in the cytoplasm. GroEL in both stressed and unstressed cells was found mainly in the cytoplasm.     

Genome sequence of the human pathogen Vibrio cholerae Amazonia

Vibrio cholerae O1 Amazonia is a pathogen that was isolated from cholera-like diarrhea cases in at least two countries, Brazil and Ghana. Based on multilocus sequence analysis, this lineage belongs to a distinct profile compared to strains from El Tor and classical biotypes. The genomic analysis revealed that it contains Vibrio pathogenicity island 2 and a set of genes related to pathogenesis and fitness, such as the type VI secretion system, present in choleragenic V. cholerae strains.     

Immunochemical studies of the O-antigens of Vibrio cholerae. The constitution of a lipopolysaccharide from V. cholerae 569B (Inaba)

Binding of Cu(2+) to polynucleotides has been found to occur at 1 Cu/5 polymer P and at similar molar ratios of copper to selected nucleic acid derivatives. Electron spin resonance (ESR) measurements show that the g-values and the hyperfine splitting constants vary substantially among the different complexes. Binding of Cu(2+) to the phosphates as well as to the bases is observed, and in some cases occurs simultaneously.     

Facile identification of protein sequences by mass spectrometry. B subunit of Vibrio cholerae classical biotype Inaba 569B toxin

A mass spectrometric method was applied to the B subunit of Vibrio cholerae classical biotype Inaba 569B toxin to determine its amino acid sequence and to confirm the differences in the amino acid sequences predicted from the nucleotide sequences of the genes of El Tor biotype strains 62746 and 2125 toxins. In this method, the Staphylococcus aureus protease V8 digest of the CNBr-treated B subunit of the classical biotype toxin was examined directly by fast-atom-bombardment mass spectrometry without separation of individual peptides. The values of molecular ion signals observed in the mass spectra were compared with the amino acid sequences of the classical biotype and El Tor biotype toxins. All the observed mass values coincided with those calculated from the published sequences of the B subunit except those of the sequences at positions 12-29 and 69-79. Peptides with these sequences were isolated by high-performance liquid chromatography and analyzed by Edman degradation or by combination of mass spectrometry and enzymatic degradation. The results revealed that the amino acid residues at positions 22 and 70 were Asp instead of Asn in the published sequences of classical biotype toxin. It was also found that Asn at position 44 was partially deaminated to Asp. The amino acid sequence of the classical biotype toxin was found to be different only at positions 18 (His----Tyr), 47 (Thr----Ile) and 54 (Gly----Ser) from that of El Tor biotype toxins.     

Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae non O-1 with plasmid DNA by electroporation

An electroporation procedure for the plasmid-mediated transformation of the genus Vibrio was performed, as part of an effort to develop recombinant DNA techniques for genetic manipulation of the genus Vibrio. Vibrio parahaemolyticus, V. alginolyticus, and V. cholerae non O-1 (9 different strains) were transformed with 3 vector plasmids (pACYC184, pHSG398, and pBR325). The efficiency of transformation was highly dependent on three parameters: the concentration of plasmid DNA; the strength of the electric field; and the combination of plasmid DNA and recipient strain. The drug-resistance genes on the vector plasmid were expressed in the Vibrio strains.     

Scanning the Landscape of Genome Architecture of Non-O1 and Non-O139 Vibrio cholerae by Whole Genome Mapping Reveals Extensive Population Genetic Diversity

Historically, cholera outbreaks have been linked to V. cholerae O1 serogroup strains or its derivatives of the O37 and O139 serogroups. A genomic study on the 2010 Haiti cholera outbreak strains highlighted the putative role of non O1/non-O139 V. cholerae in causing cholera and the lack of genomic sequences of such strains from around the world. Here we address these gaps by scanning a global collection of V. cholerae strains as a first step towards understanding the population genetic diversity and epidemic potential of non O1/non-O139 strains. Whole Genome Mapping (Optical Mapping) based bar coding produces a high resolution, ordered restriction map, depicting a complete view of the unique chromosomal architecture of an organism. To assess the genomic diversity of non-O1/non-O139 V. cholerae, we applied a Whole Genome Mapping strategy on a well-defined and geographically and temporally diverse strain collection, the Sakazaki serogroup type strains. Whole Genome Map data on 91 of the 206 serogroup type strains support the hypothesis that V. cholerae has an unprecedented genetic and genomic structural diversity. Interestingly, we discovered chromosomal fusions in two unusual strains that possess a single chromosome instead of the two chromosomes usually found in V. cholerae. We also found pervasive chromosomal rearrangements such as duplications and indels in many strains. The majority of Vibrio genome sequences currently in public databases are unfinished draft sequences. The Whole Genome Mapping approach presented here enables rapid screening of large strain collections to capture genomic complexities that would not have been otherwise revealed by unfinished draft genome sequencing and thus aids in assembling and finishing draft sequences of complex genomes. Furthermore, Whole Genome Mapping allows for prediction of novel V. cholerae non-O1/non-O139 strains that may have the potential to cause future cholera outbreaks.     

Outer membrane translocation arrest of the TcpA pilin subunit in rfb mutants of Vibrio cholerae O1 strain 569B.

The toxin-coregulated pilus (TCP) of Vibrio cholerae is a type 4-related fimbrial adhesin and a useful model for the study of type 4 pilus biogenesis and related bacterial macromolecular transport pathways. Transposon mutagenesis of the putative perosamine biosynthesis genes in the rfb operon of V. cholerae 569B eliminates lipopolysaccharide (LPS) O-antigen biosynthesis but also leads to a specific defect in TCP export. Localization of TcpA is made difficult by the hydrophobic nature of this bundle-forming pilin, which floats anomalously in sucrose density gradients, but the processed form of TcpA can be found in membrane and periplasmic fractions prepared from these strains. While TcpA cannot be detected by surface immunogold labelling in transmission electron microscope preparations, EDTA pretreatment facilitates immunofluorescent antibody labelling of whole cells, and ultrathin cryosectioning techniques confirm membrane and periplasmic accumulation of TcpA. Salt and detergent extraction, protease accessibility, and chemical cross-linking experiments suggest that although TcpA has not been assembled on the cell surface, subunit interactions are otherwise identical to those within TCP. In addition, TcpA-mediated fucose-resistant hemagglutination of murine erythrocytes is preserved in whole-cell lysates, suggesting that TcpA has obtained its mature conformation. These data localize a stage of type 4 pilin translocation to the outer membrane, at which stage export failure leads to the accumulation of pilin subunits in a configuration similar to that within the mature fiber. Possible candidates for the outer membrane defect are discussed.     

Cloning and characterization of the hemolysin determinants from Vibrio cholerae RV79(Hly+), RV79(Hly-), and 569B

The Hly region from the chromosome of Vibrio cholerae El Tor strain RV79(Hly-) and the nonhemolytic classical strain 569B were cloned into plasmid vector pBR322. Escherichia coli K-12 transformants possessing these recombinant plasmids were nonhemolytic and were detected with a 32P-labeled hly-specific DNA probe. Restriction endonuclease Sau3AI digestions of the cloned hly loci of two independently obtained RV79(Hly+) convertants, when compared with the digests of cloned RV79(Hly-) loci, revealed that an apparent alteration (10 to 15 base pairs) had occurred. In contrast, an apparent 20-base-pair deletion was present in the cloned hly locus of the classical biotype V. cholerae strain 569B. Maxicell analysis and immunoprecipitation of labeled proteins of E. coli which are encoded by the cloned hly loci of RV79(Hly+) and from nuclease BAL 31-deleted plasmids, as well as immunoprecipitation of [35S]methionine-labeled V. cholerae proteins, suggest that the hemolysin is an 84,000-dalton polypeptide.     

Genetic Behavior of R Factors in Vibrio cholerae

The fi⁺ and fi⁻ R factors are unstable in Vibrio cholerae even on selective media containing antibiotics, although a temperature-sensitive kanamycin resistance factor and its recombinants are stably maintained in V. cholerae at low temperatures.     

Genetic markers of epidemic strains of Vibrio cholerae

In this review new data on the key pathogenicity genes of V. cholerae are presented. As shown on the basis of the analysis of the latest information on the structure of the genomes of different V. cholerae strains, structural genes ctxAB coding cholera toxin may not serve as the only marker of epidemically dangerous strains. More complete and reliable information for the evaluation of the epidemic potential of V. cholerae isolated from the environment may be obtained by the simultaneous detection of 4 genetic markers: genes ctxAB, tcpA and hap coding, respectively, cholera toxin, toxin-corregulated adhesion pili and soluble hemagglutinin/protease, as well as regulatory virulence gene toxR.