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.     

Hemolysin from Vibrio cholerae eltor: cloning and expression of genes in Escherichia coli]

A 6.56-kb V. cholerae eltor DNA fragment encoding hemolysin synthesis was cloned in pUC18. The resultant recombinant plasmid pES4H (9.25 kb) was mapped by restriction analysis and shown to express in different E. coli strains as well as in nonhemolytic V. cholerae strains. Application of the cloned fragment as a molecular probe revealed homologous sequences in all V. cholerae strains tested independently on their biotypes, hemolytic activity and presence of vct-genes in their genomes while none of other Vibrio species and related microorganisms contained such sequences. A recombinant E. coli strain, a V. cholerae eltor hemolysin producer, was constructed. The simultaneous expression of hemolytic and toxinogenic properties by the same V. cholerae strains is discussed.     

Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1

Vibrio cholerae serogroup O139 Bengal is the first documented serogroup other than O1 to cause epidemic cholera. The O139 Bengal strains are very similar to V. cholerae serogroup O1 biotype El Tor strains. The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule. We previously described three Tn phoA mutants of O139 strain AI1837 which abolish both O antigen and capsule production. These Tn phoA insertions were mapped to a 21.5 kb Eco RI fragment of the O139 chromosome. We describe here the cloning and mapping of this 21.5 kb Eco RI fragment and it was shown to complement each of the mutants in trans to produce O antigen and capsule. The Eco RI fragment contains 13 kb of DNA that is specific to O139 and 8.5 kb of DNA that is common to O1 and O139. Sequence analysis of the 13 kb of O139-specific DNA revealed that it contains 11 open reading frames all of which are transcribed in the same direction. Eight of the 11 open reading frames are homologous to sugar biosynthesis genes from other organisms. Using extended polymerase chain reactions, we show that the extent of the DNA region in O139 that is not present in O1 is approximately 35kb. The site of insertion of this O139-specific DNA in the O1 chromosome was mapped to the rfb _O1 region. We also demonstrate that O139 Bengal strain AI1837 contains a deletion of 22 kb that in serogroup O1 strains contains the rfb region. Therefore, O139 Bengal probably arose from an O1 strain that had undergone genetic rearrangements including deletion of the O1 rfb region and acquisition of a 35 kb region of DNA which encodes O139 surface polysaccharide.     

Replication and integration of a Vibrio cholerae cryptic plasmid linked to the CTX prophage

We identified a 4.7 kb cryptic plasmid in all ctxAB ⁺ Vibrio cholerae strains we tested. An isolate of the V. cholerae classical biotype strain O395 that harbours the cryptic plasmid at high copy number was found. Hybridization analysis demonstrated that sequences highly related or identical to this plasmid exist in all toxigenic strains of V. cholerae but were notably absent in all non-toxigenic environmental isolates that lacked the genes for toxin-co-regulated pili and the filamentous CTX prophage. Accordingly, we have named the cryptic plasmid pTLC for toxin-linked cryptic. The complete nucleotide sequence of pTLC from the high-copy-number isolate was determined. The largest open reading frame in the plasmid is predicted to encode a protein similar to the replication initiation protein (pII) of Escherichia coli F-specific filamentous phages. The nucleotide sequence of pTLC also facilitated the structural characterization of the DNA homologous to pTLC in other strains of V. cholerae . pTLC-related DNA exists in these strains as both low-copy-number, covalently closed circular DNA and tandemly duplicated, chromosomally integrated DNA. Remarkably, the chromosomally integrated form of pTLC is adjacent to the CTX prophage. The strain distribution, chromosomal location and DNA sequence of pTLC suggests that it may be a genetic element that plays some role in the biology of CTXφ, perhaps facilitating either its acquisition or its replication.     

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.     

Molecular analysis of the rstR and orfU genes of the CTX prophages integrated in the small chromosomes of environmental Vibrio cholerae non-O1, non-O139 strains

The ctxAB genes encoding cholera toxin, reside in the genome of a filamentous bacteriophage CTXphi. The presence of CTX prophage in non-epidemic environmental Vibrio cholerae strains is rare. The CTX prophage, the lysogenic form of CTXphi in V. cholerae, is comprised of the 'RS2' and the 'Core'. Analysis of the rstR gene present in the RS2 region of the CTX prophage revealed the presence of new alleles of the prophages in four environmental non-O1, non-O139 strains VCE22 (O36), VCE228 (O27), VCE232 (O4) and VCE233 (O27), and the CTX prophages are located in the small chromosomes. Phylogenetic analysis based on the nucleotide sequences of the rstR and orfU (present in the core) genes of these prophages placed them in a single unique cluster, which is distally located compared with that of epidemic V. cholerae O1 strains. Further analysis indicated that the genome of the prophage present in the strain VCE22 is devoid of the ctxAB genes, called pre-CTX prophage and the strain also possess the toxin-coregulated pilus protein coding gene tcpA of classical type, another important pathogenicity determining locus of the epidemic V. cholerae strains. Comparative analysis of the nucleotide sequences of the rstR and orfU genes indicated that the pre-CTX prophage of VCE22 might be the progenitor of new alleles of the CTX prophages present in these environmental strains.     

Physical and genetic map of the genome of Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species

We constructed a physical map of the genomic DNA (5.1 Mb) for Vibrio parahaemolyticus strain AQ4673 by combining 17 adjacent NotI fragments. This map shows two circular replicons of 3.2 and 1.9 Mb. Pulsed-field gel electrophoresis (PFGE) of undigested genomic DNA revealed two bands of corresponding sizes. Analysis both by NotI digestion and by Southern blot of the two isolated bands confirmed the existence of two replicons. The presence of genes for 16S rRNA on both the replicons indicates that the replicons are chromosomes rather than megaplasmids. The two bands were also seen after PFGE of undigested genomic DNA of V. parahaemolyticus strains other than AQ4673, and of strains belonging to other Vibrio species, such as V. vulnificus, V. fluvialis and various serovars and biovars of V. cholerae. It is noteworthy that V. cholerae O1 strain 569B, a classical biovar, was also shown to have two replicons of 2.9 and 1.2 Mb, which does not agree with a physical map proposed in a previous study. Our results suggest that a two-replicon structure is common throughout Vibrio species.     

Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.     

Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli

A cosmid gene bank of Vibrio cholerae 395, classical Ogawa, was screened in Escherichia coli HB101 for expression of the vibrio neuraminidase (NANase) gene nanH (N-acylneuraminate glycohydrolase). Positive clones were identified by their ability to cleave the fluorogenic NANase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Seven NANase-positive clones were detected after screening 683 cosmid isolates with a rapid, qualitative plate assay method. The nanH gene was subcloned from one of the cosmids and was located within a 4.8-kilobase-pair BglII restriction endonuclease fragment. Evidence that nanH was the NANase structural gene was obtained by transposon mutagenesis and by purification and comparison of the cloned gene product with the secreted NANase purified from the parent V. cholerae strain. The sequence of the first 20 amino-terminal amino acids of the secreted NANase purified from V. cholerae was determined by automated Edman degradation and matched perfectly with the amino acid sequence predicted from nucleotide sequencing of nanH. The sequence data also revealed the existence of a potential signal peptide that was apparently processed from NANase in both V. cholerae and E. coli. In contrast to V. cholerae, E. coli nanH+ clones did not secrete NANase into the growth medium, retaining most of the enzyme in the periplasmic compartment. Kinetic studies in V. cholerae showed that nanH expression and NANase secretion were temporally correlated as cells in batch culture entered late-exponential-phase growth. Similar kinetics were observed in at least one of the E. coli nanH+ clones, suggesting that nanH expression in E. coli might be controlled by some of the same signals as in the parent V. cholerae strain.     

Comparative genomic analyses of the vibrio pathogenicity island and cholera toxin prophage regions in nonepidemic serogroup strains of Vibrio cholerae

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTXphi), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPIphi. In order to determine the prevalence of horizontal transfer of VPI and CTXphi among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI(+) strains have also acquired the CTXphi. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTXphi prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTXphi prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTXphi independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.     

A study of the prevalence of regulatory genes controlling virulence gene expression among Vibrio choleraeeltor biovariant strains varying in their pandemic potential

The evolution of the genome of the pathogenic agent of the seventh cholera pandemia Vibrio cholerae eltor biovariant was thought to occur by acquiring not only structural genes of virulence but also regulatory systems as a result of horizontal transfer events. The polymerase chain reaction revealed the presence of the following regulatory genes that control the virulence gene expression in the chromosome of pre-pandemic and pandemic strains of cholera vibrios eltor: toxR, toxT, tcpP, tcpH, luxS, luxO, crp, vicH, pepA. The avirulent V. cholerae strain ATCC14033 isolated in 1910 (hypothetical predecessor of the cholera eltor agent) was shown to be lacking the regulatory genes toxT, tcpP, tcpHlocalized in the pathogenicity island VPI-1, and to be capable of realizing positive control over the expression of the virulence genes involved in the ToxR regulon. The virulent strains isolated from cholera patients during the local cholera outbreak in Indonesia in 1937 did not differ from the strains that caused cholera eltor pandemic in 1961. The strains had identical content of the regulatory genes tested. Only one strain of the four isolates studied contained no tcpPgene. Two key regulatory genes, toxR and toxT, were sequenced in all the isolates. The toxR nucleotide sequence of three pre-pandemic strains was shown to be indistinguishable from that of the pandemic isolates. On the other hand, the clinical strain MAK757 isolated prior to the emergence of the epidemic demonstrated an altered nucleotide sequence in its toxR gene. Experiments with the intra-intestinal challenge of suckling rabbits were indicative of similar virulence levels for the pre-pandemic and pandemic clinical strains. These results may serve as the evidence of the in vivo activity of the pre-pandemic strains of the toxT, tcpH, and tcpP positive regulatory genes that acquired in V. cholerae during the evolutionary process.     

A 195-kb cosmid walk encompassing the human Xq28 color vision pigment genes

By using cosmid walking, we have cloned a 195-kb region from chromosome band Xq28 that encompasses the red and green color pigment genes and 85 kb of flanking sequences. This has allowed us to confirm that the color pigment genes are within very homologous units arranged in tandem array. Each unit contains two BssHII sites and one NruI site that are frequently methylated in male leukocyte DNA. A NotI and an EagI site are present 6 kb upstream from the red pigment gene promoter; the NotI site was shown to be unmethylated in the active X chromosome in leukocytes and may represent a CpG island for the whole cluster. We have identified another CpG island, 61 kb 3' from the last green pigment gene, that is unmethylated in leukocytes on the active X chromosome, but methylated on the inactive X. This island is flanked by sequences conserved in evolution and may thus correspond to an expressed gene. We also describe an informative three-allele restriction fragment length polymorphism within the pigment gene cluster.     

我国霍乱弧菌的脂肪酸分型研究

目的 对脂肪酸分型方法在霍乱弧菌菌株鉴定、菌株相似性分析等方面的应用价值进行评价。方法 选取了分离自我国的两个主要致病血清群的194株霍乱弧菌菌株（1961年以来的El Tor型和1992年以来的O139群）,提取脂肪酸,应用MIDI公司的脂肪酸分型系统,进行数据分析。结果 检测的所有菌株都含有的脂肪酸成分有13种。霍乱弧菌的判断符合率为88.6%。二维聚类分析没有成明显可区分的群, O139群霍乱弧菌的脂肪酸组成与O1群的相似,产毒与非产毒霍乱弧菌的脂肪酸成分没有显著差异。结论 脂肪酸分型对弧菌种的快速鉴定有应用价值,对霍乱弧菌的现场分离鉴定有辅助意义,在小样本暴发资料的研究中能够反映菌株之间的亲缘关系,但其对霍乱弧菌种属内的各种特征性菌群不具有鉴别能力。     

Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains.

Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (FP1). The nontoxigenic V. cholerae O1 also yielded four fragments but constituted a different FP group (FP2). A total of 15 different patterns were observed among the V. cholerae non-O1 strains. Two patterns were observed most frequently for V. cholerae non-01 strains, 25% of which have FP3, with five fragments, and 16.7% of which have FP4, with two fragments. Three fragments, 1.75, 0.79, and 0.5 kb, were found to be common to both toxigenic and nontoxigenic V. cholerae O1 strains as well as to group FP3, containing V. cholerae non-O1 strains. Two fragments of group FP3, 1.3 and 1.0 kb, were present in FP1 and FP2 respectively. The 0.5-kb fragment was common to all strains and serogroups of V. cholerae analyzed. It is concluded from the results of this study, based on DNA FPs of environmental isolates, that it is possible to detect an emerging virulent strain in a cholera-endemic region. ERIC-PCR constitutes a powerful tool for determination of the virulence potential of V. cholerae O1 strains isolated in surveillance programs and for molecular epidemiological investigations.     

Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin

Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1.3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1.3 kb Vibrio DNA fragment, two proteins were made in E. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains.

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.     

霍乱弧菌甘露醇PTS操纵子中mtlR为转录抑制基因

产毒和非产毒的El Tor生物型霍乱弧菌对甘露醇发酵利用的速率有明显差别,在霍乱致病株的快速判断中有重要的参考价值。通过比较快发酵菌株(非产毒株)和慢发酵菌株(产毒株)mtlR缺失突变株与野生株在含0.2%甘露醇的M9培养液及甘露醇发酵液中生长、产酸等的变化,定性地证明了mtlR基因的抑制作用;另外通过定量RT-PCR进一步验证了MtlR蛋白在mtlCBA转录水平发挥负调控作用。但是mtlR还不是引起快慢发酵菌株对甘露醇发酵差异的直接原因。本研究也为我们研究霍乱弧菌甘露醇快慢发酵差异机制提供了必要的参考依据。     

Distribution of three genes involved in the PJM1 iron-sequestering system in various Vibrio anguillarum serogroups

Gene sequences with high homologies to the two genes fatA and fatD, associated with the pJM1 virulence plasmid-encoded iron-sequestering system (anguibactin) in Vibrio anguillarum serogroup O1 strains, were found in a range of serogroup O2a strains and one NT 4 strain that either contained no plasmids or only small-sized plasmids. None of these strains contained the angR gene responsible for regulation of the anguibactin synthesis. DNA sequences of presumed fatA and fatD fragments from one O2a strain and one NT4 strain were identical to each other. The fatA sequence of these strains included 35 nucleotide substitutions and 7 amino acid substitutions out of 555 nucleotides when compared with the fatA sequence of the O1 strain 76775. The fatD sequence included 10 nucleotide substitutions and 1 amino acid substitution out of 543 nucleotides when compared with 76775. The fact that the majority of nucleotide substitutes in the fatA and fatD fragments did not result in amino acid substitutes suggest that these sequences may to some degree be preserved by selective pressure. The functional role of these genes in the O2a serogroup is most likely different from their role in the pJM1 system.