VibD and VibH are required for late steps in vibriobactin biosynthesis in Vibrio cholerae

Vibrio cholerae synthesizes the catechol siderophore vibriobactin. In this report, we present the complete map of a vibriobactin gene region containing two previously unreported vibriobactin biosynthetic genes. vibD encodes a phosphopantetheinyl transferase, and vibH encodes a novel nonribosomal peptide synthase. Both VibD and VibH are required for vibriobactin biosynthesis.     

Identification of additional genes required for O-antigen biosynthesis in Vibrio cholerae O1.

The cloning and expression of the genes encoding the Vibrio cholerae O1 lipopolysaccharide O antigen in a heterologous host have been described previously (P. A. Manning, M. W. Heuzenroeder, J. Yeadon, D. I. Leavesley, P. R. Reeves, and D. Rowley, Infect. Immun. 53:272-277, 1986). It was thus assumed that all the genes required for O-antigen expression were located on a 20-kb SacI restriction fragment. We present evidence for a number of other as yet undescribed genes that are essential for O-antigen biosynthesis in V. cholerae O1 and that these genes are somehow complemented in Escherichia coli K-12. The two genes termed Vibrio cholerae rfbV and rfbU are transcribed in the opposite orientation from the rest of the rfb operon, whereas the galE dehydratase and rfbP (Salmonella enterica) homologs, designated ORF35x7 and rfbW, respectively, are transcribed in the same orientation. The evidence presented here, using chromosomal insertion mutants, clearly shows that the three genes now designated rfbV, rfbU, and rfbW appear to be accessory rfb genes and are essential for O-antigen biosynthesis in V. cholerae but that ORF35x7 is not.     

Identification of the vibriobactin receptor of Vibrio cholerae.

Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.     

The tcp gene cluster of Vibrio cholerae

The toxin co-regulated pilus (TCP) has been identified as a critical colonization factor in both animal models and humans for Vibrio cholerae O1. The major pilin subunit, TcpA (and also TcpB), is similar to type-4 pilins but TCP probably more appropriately belongs to a sub-class which includes the bundle-forming pilus of enteropathogenic Escherichia coli. The genes for TCP biosynthesis and assembly are clustered with the exception of housekeeping functions such as TcpG (=DsbA, a periplasmic disulfide bond epimerase). The nt sequences from El Tor and classical strains show only minor differences corresponding to the major regulatory regions and in TcpA itself. These differences are thought to account for the alternate conditions required for expression of TCP by the two biotypes and the antigenic variation and lack of cross-protection. Aside from the TcpA only a few of the proteins have had their roles in TCP biogenesis defined. Regulation of TCP is controlled by the ToxR regulon via ToxT with a possible involvement of TcpP and the cAMP-CRP system. Experiments using the infant mouse cholera model have now shown that TCP is a colonization factor and protective antigen for both classical and El Tor O1 strains and in the O139 Bengal serotype and that the mannose-sensitive haemagglutinin pilus does not appear to play a comparable role.     

Identification of a new RTX-like gene cluster in Vibrio cholerae

A gene cluster containing two genes in tandem has been identified in Vibrio cholerae ElTor N16961. Each has more than one cadherin domain and is homologous to the RTX toxin family and was common in various V. cholerae strains. Insertional mutagenesis demonstrated that each gene has a role in Hep-2 cell rounding, hemolytic activity towards human and sheep RBCs and biofilm formation. The mutants showed reduced adherence to intestinal epithelial cells as well as reduction of in vivo colonization in suckling mice. These two genes thus code for RTX-like toxins in V. cholerae and are associated with the pathogenecity of this organism.     

Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis.

The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

Cloning and characterization of the gene cluster required for beauvericin biosynthesis in Fusarium proliferatum

Beauvericin, a cyclohexadepsipeptide-possessing natural product with synergistic antifungal, insecticidal, and cytotoxic activities. We isolated and characterized the fpBeas gene cluster, devoted to beauvericin biosynthesis, from the filamentous fungus Fusarium proliferatum LF061. Targeted inactivation of the F. proliferatum genomic copy of fpBeas abolished the production of beauvericin. Comparative sequence analysis of the FpBEAS showed 74% similarity with the BbBEAS that synthesizes the cyclic trimeric ester beauvericin in Beauveria bassiana, which assembles N-methyl-dipeptidol monomer intermediates by the programmed iterative use of the nonribosomal peptide synthetase modules. Differences between the organization of the beauvericin loci in F. proliferaturm and B. bassiana revealed the mechanism for high production of beauvericin in F. proliferatum. Our work provides new insights into beauvericin biosynthesis, and may lead to beauvericin overproduction and creation of new analogs via synthetic biology approaches.     

Induction of melanin biosynthesis in Vibrio cholerae.

Vibrio cholerae synthesized the pigment melanin in response to specific physiological conditions that were stressful to the bacterium. Pigmentation was induced when V. cholerae was subjected to hyperosmotic stress in conjunction with elevated growth temperatures (above 30 degrees C). The salt concentration tolerated by V. cholerae was lowered by additional abiotic factors such as acidic starting pH of the growth medium and limitation of organic nutrients. Although the amount of toxin detected in the culture supernatant decreased significantly in response to stressful culture conditions, no correlation between the physiological conditions that induced melanogenesis and expression of OmpU or cholera toxin was detected. Since conditions that induce melanin production in V. cholerae occur in both the aquatic environment and the human host, it is possible that melanogenesis has a specific function with respect to the survival of the bacterium in these habitats.     

Induction of melanin biosynthesis in Vibrio cholerae.

Vibrio cholerae synthesized the pigment melanin in response to specific physiological conditions that were stressful to the bacterium. Pigmentation was induced when V. cholerae was subjected to hyperosmotic stress in conjunction with elevated growth temperatures (above 30 degrees C). The salt concentration tolerated by V. cholerae was lowered by additional abiotic factors such as acidic starting pH of the growth medium and limitation of organic nutrients. Although the amount of toxin detected in the culture supernatant decreased significantly in response to stressful culture conditions, no correlation between the physiological conditions that induced melanogenesis and expression of OmpU or cholera toxin was detected. Since conditions that induce melanin production in V. cholerae occur in both the aquatic environment and the human host, it is possible that melanogenesis has a specific function with respect to the survival of the bacterium in these habitats.     

Cloning and characterization of mutL and mutS genes of Vibrio cholerae: nucleotide sequence of the mutL gene.

The mutL and mutS genes of Vibrio cholerae have been identified using interspecific complementation of Escherichia coli mutL and mutS mutants with plasmids containing the gene bank of V. cholerae. The recombinant plasmid pJT470, containing a 4.7 kb fragment of V. cholerae DNA codes for a protein of molecular weight 92,000. The product of this gene reduces the spontaneous mutation frequency of the E. coli mutS mutant. The plasmid, designated pJT250, containing a 2.5 kb DNA fragment of V. cholerae and coding for a protein of molecular weight 62,000, complements the mutL gene function of E. coli mutL mutants. These gene products are involved in the repair of mismatches in DNA. The complete nucleotide sequence of mutL gene of V. cholerae has been determined.     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined ?16 to ?25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.     

Elevated zinc induces siderophore biosynthesis genes and a zntA-like gene in Pseudomonas fluorescens

Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.