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.     

Role of lectin (hlyA) in the hemolytic and hemagglutinating activity of Vibrio cholerae

Data on the nature of the substance which determines the structural gene hlyA in V. cholerae are presented. Computer analysis and experimental data on hemolysin preparations and V. cholerae strains testify that gene hlyA determines the synthesis of ricin-like galactose-specific lectin. Its lectin domain takes part in the lysis of sheep (but not rabbit!) red blood cells, as well as in the hemagglutinating capacity of non-toxigenic and toxigenic V. cholerae 569 B.     

Identification, characterization, and expression of a unique secretory lipase from the human pathogen Leishmania donovani

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a ~33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.     

Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non-haemolytic classical strain 569B

The EI T or haemolysin, product of hlyA, is exported from Vibrio cholerae as a Mr 80,000 protein which can be subsequently cleaved to give two proteins of Mr 65,000 and 15,000. Nucleotide sequence analysis has demonstrated that hlyA encodes a protein of Mr 82,250 with a potential 18-amino-acid signal sequence. The non-haemolytic classical strain 569B has been shown to have a structural gene defect rather than a defect in secretion. By non-reciprocal recombination it was possible to transfer this defect onto a plasmid and show that a truncated hlyA product of Mr 27,000 is made in Escherichia coli K-12 minicells. Nucleotide sequence analysis demonstrates an 11-base-pair deletion which would result in a Mr 26,940 protein probably loosely associated with the membrane.     

DNA binding and ToxR responsiveness by the wing domain of TcpP,an activator of virulence gene expression in Vibrio cholerae

Virulence in Vibrio cholerae requires activation of toxT by two membrane-localized activators, TcpP and ToxR. We isolated 12 tcpP activation mutants that fell into two classes: class I mutants were inactive irrespective of the presence of ToxR, and class II mutants exhibited near wild-type activity when coexpressed with ToxR. Most class I mutants had lesions in the wing domain predicted by homology with the winged helix-turn-helix family of activators. Class I mutants bound promoter DNA poorly and were largely unable to interact with ToxR in a crosslinking assay, whereas class II mutants retained physical interaction with ToxR. One mutant constructed in vitro bound DNA poorly but nevertheless responded to ToxR by activating toxT and also maintained ToxR interaction. We propose that ToxR interaction, but not DNA binding, is essential for TcpP function and that the wing domain of TcpP enables contact with ToxR required for productive TcpP-RNA polymerase association.     

Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae

Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.     

Identification and characterization of a Vibrio cholerae gene,mbaA, involved in maintenance of biofilm architecture

The formation of biofilms is thought to play a key role in the environmental survival of the marine bacterium Vibrio cholerae. Although the factors involved in V. cholerae attachment to abiotic surfaces have been extensively studied, relatively little is known about the mechanisms involved in the subsequent maturation of the biofilms. Here we report the identification of a novel gene, which we have named mbaA (for maintenance of biofilm architecture), that plays a role in the formation and maintenance of the highly organized three-dimensional architecture of V. cholerae El Tor biofilms. We demonstrate that although the absence of mbaA does not significantly affect the initial attachment of cells onto the surface, it leads to the formation of biofilms that lack the typical structure, including the pillars of cells separated by fluid-filled channels that are evident in mature wild-type biofilms. Microscopic analysis indicates that the absence of mbaA leads to an increase in the amount of extracellular matrix material in the biofilms. The predicted mbaA product is a member of a family of regulatory proteins, containing GGDEF and EAL domains, suggesting that MbaA regulates the synthesis of some component of the biofilm matrix.     

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.     

Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH

Vibriobactin [N(1)-(2,3-dihydroxybenzoyl)-N(5),N(9)-bis[2-(2, 3-dihydroxyphenyl)-5-methyloxazolinyl-4-carboxamido]norspermidine] , is an iron chelator from the cholera-causing bacterium Vibrio cholerae. The six-domain, 270 kDa nonribosomal peptide synthetase (NRPS) VibF, a component of vibriobactin synthetase, has been heterologously expressed in Escherichia coli and purified. VibF has an unusual NRPS domain organization: cyclization-cyclization-adenylation-condensation-peptidyl carrier protein-condensation (Cy(1)-Cy(2)-A-C(1)-PCP-C(2)). VibF activates and covalently loads its PCP with L-threonine, and together with vibriobactin synthetase proteins VibE (adenylation) and VibB (aryl carrier protein) condenses and heterocyclizes 2, 3-dihydroxybenzoyl-VibB with L-Thr to 2-dihydroxyphenyl-5-methyloxazolinyl-4-carboxy-VibF in the first demonstration of oxazoline formation by an NRPS cyclization domain. This enzyme-bound aryl oxazoline can be transferred by VibF to various amine acceptors but most efficiently to N(1)-(2, 3-dihydroxybenzoyl)norspermidine (k(cat) = 122 min(-1), K(m) = 1.7 microM), the product of 2,3-dihydroxybenzoyl-VibB, norspermidine, and VibH. This diacylated product undergoes a second aryl oxazoline acylation on its remaining secondary amine, also catalyzed by VibF, to yield vibriobactin. Vibriobactin biosynthesis in vitro has thus been accomplished from four proteins, VibE, VibB, VibF, and VibH, with the substrates 2,3-dihydroxybenzoic acid, L-Thr, norspermidine, and ATP. Vibriobactin synthetase is an unusual NRPS in that all intermediates are not covalently tethered as PCP thioesters and in that it represents an NRPS pathway with two branch points.     

Cloning, sequence analysis and heterologous expression of the DNA adenine-(N(6)) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans

We cloned and sequenced the DNA adenine-N(6) methyltransferase gene of the human pathogen Actinobacillus actinomycetemcomitans (M.AacDAM). Restriction digestion shows that the enzyme methylates adenine in the sequence GATC. Expression of the enzyme in a DAM(-) background shows in vivo activity. A PSI-BLAST search revealed that M.AacDAM is most related to M.HindIV, M.EcoDAM, M.StyDAM, and M.SmaII. The ClustalW alignment shows highly conserved regions in the enzyme characteristic for type a MTases. Phylogenetic tree analysis shows a cluster of enzymes recognizing the sequence GATC, within a branch of orphan MTases harboring M.AacDAM. The cloning and sequencing of this first methyltransferase gene described for A. actinomycetemcomitans open the path for studies on the potential regulatory impact of DNA methylation on gene regulation and virulence in this organism.     

Covalent binding of flavins to RnfG and RnfD in the Rnf complex from Vibrio cholerae

Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na+-NQR complexes.     

The transcriptional activator HIyU of Vibrio cholerae: nucleotide sequence and role in virulence gene expression

HlyU upregulates expression of the haemolysin, HlyA, of Vibrio cholerae. DNA sequence analysis indicates that HlyU is an 11.9 kDa protein containing a putative helix-turn-helix motif and belonging to a family of small regulatory proteins, including NolR (Rhizobium meliloti), SmtB (Synechococcus PCC 7942) and ArsR (piasmids R773, Escherichia coli; p1258, Staphylococcus aureus; and pSX267, Staphylococcus xylosus). An hlyU mutant was constructed by insertional inactivation, and found to be deficient in the production of both the haemolysin and a 28kDa secreted protein. The mutant was assessed for virulence in the infant mouse cholera model, revealing a 100-fold increase in the LD_50.. This suggests that HlyU promotes expression of virulence determinant(s) in vivo.