A pathway leading to a cation-binding pocket determines the selectivity of the NhaP2 antiporter in Vibrio cholerae1.

The Vc-NhaP2 antiporter from Vibrio cholerae exchanges H+ for K+ or Na+ but not for the smaller Li+. The molecular basis of this unusual selectivity remains unknown. Phyre2 and Rosetta software were used to generate a structural model of the Vc-NhaP2. The obtained model suggested that a cluster of residues from different transmembrane segments (TMSs) forms a putative cation-binding pocket in the middle of the membrane: D133 and T132 from TMS V together with D162 and E157 of TMS VI. The model also suggested that L257, G258, and N259 from TMS IX together with T276, D273, Q280, and Y251 from TMS X as well as L289 and L342 from TMS XII form a transmembrane pathway for translocated ions with a built-in filter determining cation selectivity. Alanine-scanning mutagenesis of the identified residues verified the model by showing that structural modifications of the pathway resulted in altered cation selectivity and transport activity. In particular, L257A, G258A, Q280A, and Y251A variants gained Li+/H+ antiport capacity that was absent in the nonmutated antiporter. T276A, D273A, and L289A variants exclusively exchanged K+ for H+, while a L342A variant mediated Na+/H+ exchange only, thus maintaining strict alkali cation selectivity.

Sodium ion cycle in bacterial pathogens: evidence from cross-genome comparisons.

Analysis of the bacterial genome sequences shows that many human and animal pathogens encode primary membrane Na+ pumps, Na+-transporting dicarboxylate decarboxylases or Na+ translocating NADH:ubiquinone oxidoreductase, and a number of Na+ -dependent permeases. This indicates that these bacteria can utilize Na+ as a coupling ion instead of or in addition to the H+ cycle. This capability to use a Na+ cycle might be an important virulence factor for such pathogens as Vibrio cholerae, Neisseria meningitidis, Salmonella enterica serovar Typhi, and Yersinia pestis. In Treponema pallidum, Chlamydia trachomatis, and Chlamydia pneumoniae, the Na+ gradient may well be the only energy source for secondary transport. A survey of preliminary genome sequences of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Treponema denticola indicates that these oral pathogens also rely on the Na+ cycle for at least part of their energy metabolism. The possible roles of the Na+ cycling in the energy metabolism and pathogenicity of these organisms are reviewed. The recent discovery of an effective natural antibiotic, korormicin, targeted against the Na+ -translocating NADH:ubiquinone oxidoreductase, suggests a potential use of Na+ pumps as drug targets and/or vaccine candidates. The antimicrobial potential of other inhibitors of the Na+ cycle, such as monensin, Li+ and Ag+ ions, and amiloride derivatives, is discussed.

Flagellum-independent surface migration of Vibrio cholerae and Escherichia coli.

Surface translocation has been described in a large variety of microorganisms, including some gram-negative enteric bacteria. Here, we describe the novel observation of the flagellum-independent migration of Vibrio cholerae and Escherichia coli on semisolid surfaces with remarkable speeds. Important aspects of this motility are the form of inoculation, the medium composition, and the use of agarose rather than agar. Mutations in several known regulatory or surface structure proteins, such as ToxR, ToxT, TCP, and PilA, did not affect migration, whereas a defect in lipopolysaccharide biosynthesis prevented translocation. We propose that the observed surface migration is an active process, since heat, protease, or chloramphenicol treatments of the cells have strong negative effects on this phenotype. Furthermore, several V. cholerae strains strongly expressing the hemagglutinin/protease but not their isogenic hap-negative mutants, lacked the ability of surface motility, and the treatment of migrating strains with culture supernatants from hap strains but not hap-null strains prevented surface translocation.

Expression and mutagenesis of the NqrC subunit of the NQR respiratory Na(+) pump from Vibrio cholerae with covalently attached FMN.

The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is present in the membranes of a number of marine bacteria and pathogenic bacteria. Two of the six subunits of the Na(+)-NQR, NqrB and NqrC, have been previously shown to contain covalently bound flavin adenine mononucleotide (FMN). In the current work, the cloning of nqrC from Vibrio cholerae is reported. The gene has been expressed in V. cholerae and shown to contain one equivalent of covalently bound FMN. In contrast, no covalent flavin was detected when threonine-225 was replaced by leucine. The data show that the FMN attachment does not require assembly of the enzyme and are consistent with the unusual threonine attachment site.

Role of sodium bioenergetics in Vibrio cholerae.

The ability of the bacterium to use sodium in bioenergetic processes appears to play a key role in both the environmental and pathogenic phases of Vibrio cholerae. Aquatic environments, including fresh, brackish, and coastal waters, are an important factor in the transmission of cholera and an autochthonous source. The organism is considered to be halophilic and has a strict requirement for Na(+) for growth. Furthermore, expression of motility and virulence factors of V. cholerae is intimately linked to sodium bioenergetics and to each other. Several lines of evidence indicated that the activity of the flagellum of V. cholerae might have an impact on virulence gene regulation. As the V. cholerae flagellum is sodium-driven and the Na(+)-NQR enzyme is known to create a sodium motive force across the bacterial membrane, it was recently suggested that the increased toxT expression observed in a nqr-negative strain is mediated by affecting flagella activity. It was suggested that the V. cholerae flagellum might respond to changes in membrane potential and the resulting changes in flagellar rotation might serve as a signal for virulence gene expression. However, we recently demonstrated that although the flagellum of V. cholerae is not required for the effects of ionophores on virulence gene expression, changes in the sodium chemical potential are sensed and thus alternative mechanisms, perhaps involving the TcpP/H proteins, for the detection of these conditions must exist. Analyzing the underlying mechanisms by which bacteria respond to changes in the environment, such as their ability to monitor the level of membrane potential, will probably reveal complex interplays between basic physiological processes and virulence factor expression in a variety of pathogenic species.

Requirements for conversion of the Na(+)-driven flagellar motor of Vibrio cholerae to the H(+)-driven motor of Escherichia coli.

Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H(+) or Na(+) into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions in pomA, pomB, motX, or motY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complemented fliG-null strains of the parent species but not fliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB, motX, and motY became weakly motile when the E. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins of V. cholerae and that the hybrid motors are driven by the proton motive force.

Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae.

The expression of several virulence factors of Vibrio cholerae is coordinately regulated by the ToxT molecule and the membrane proteins TcpP/H and ToxR/S, which are required for toxT transcription. To identify proteins that negatively affect toxT transcription, we screened transposon mutants of V. cholerae carrying a chromosomally integrated toxT::lacZ reporter construct for darker blue colonies on media containing 5-bromo-4-chlor-3-indolyl beta-D galactoside (X-gal). Two mutants had transposon insertions in a region homologous to the nqr gene cluster of Vibrio alginolyticus, encoding a sodium-translocating NADH-ubiquinone oxidoreductase (NQR). In V. alginolyticus, NQR is a respiration-linked Na+ extrusion pump generating a sodium motive force that can be used for solute import, ATP synthesis, and flagella rotation. Inhibition of NQR enzyme function in V. cholerae by the specific inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) resulted in elevated toxT::lacZ activity. Increased toxT::lacZ expression in an nqr mutant strain compared with the parental strain was observed when the TcpP/H molecules alone were strongly expressed, suggesting that the negative effect of the NQR complex on toxT transcription is mediated through TcpP/H. However, the ability of the TcpP/H proteins to activate the toxT::lacZ reporter construct was greatly diminished in the presence of high NaCl concentrations in the growth medium. The flagellar motor of V. cholerae appears to be driven by a sodium motive force, and modulation of flagella rotation by inhibitory drugs, high media viscosity, or specific mutations resulted in increases of toxT::lacZ expression. Thus, the regulation of the main virulence factors of V. cholerae appears to be modulated by endogenous and exogenous sodium levels in a complex way.

TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae.

The production of several virulence factors in Vibrio cholerae O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of V. cholerae generated by Tnbla mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTX phi-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to nqrB from Vibrio alginolyticus, which encodes a subunit of a Na(+)-translocating NADH:ubiquinone oxidoreductase, and tcpI, encoding a chemo-receptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in tcpP, which is in an operon with tcpH, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in V. cholerae, as a tcpP-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from V. cholerae and PsaE from Yersinia pestis. Like ToxR, TcpP activates transcription of the toxT gene, an essential activator of tcp operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for toxT transcription in V. cholerae.

Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: mutants with altered channel gating and pressure sensitivity.

In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic alpha-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning alpha-helix.

Cross-linking studies and membrane localization and assembly of radiolabelled large mechanosensitive ion channel (MscL) of Escherichia coli.

The gene encoding the large conductance mechanosensitive ion channel (MscL) of Escherichia coli and several deletion mutants of mscL were cloned under the control of the T7 RNA polymerase promoter. Transformation of these constructs into an E. coli strain carrying an inducible T7 RNA polymerase gene allowed the specific production and labelling of MscL with [35S]methionine. Preparation of membrane fractions of E. coli cells by sucrose gradient centrifugation indicated that the radiolabelled MscL was present in the inner cytoplasmic membrane in agreement with results of several studies. However, treatment of the labelled cells and cell membrane vesicles with various cross-linkers resulted in the majority of labelled protein migrating as a monomer with a small proportion of molecules (approximately 25%) migrating as dimers and higher order multimers. This result is in contrast with a finding of a study suggesting that the channel exclusively forms hexamers in the cell membrane of E. coli (1) and therefore may have profound implication for the activation and/or "multimerization" of the channel by mechanical stress exerted to the membrane. In addition, from the specific activity of the radiolabelled protein and the amount of protein in the cytoplasmic membrane fraction we estimated the number of MscL ion channels expressed under these conditions to be approximately 50 channels per single bacterium.

Porins of Escherichia coli: unidirectional gating by pressure.

OmpC and PhoE porins of Escherichia coli were examined by the patch-clamp technique following reconstitution in liposomes, and were observed primarily in the open (conducting) state. With application of negative voltage and positive hydrostatic pressure, OmpC exhibited marked gating towards a more closed state whereas PhoE remained largely unaffected by pressure application. Hybrid chimeric OmpC-PhoE proteins showed an increased tendency for pressure-dependent gating as the OmpC proportion in the chimeric molecule increased. In addition, several PhoE mutants with amino acid substitutions and insertions in either the L3 or L4 loop of the monomer exhibited pressure sensitivity comparable with the wild-type OmpC porin. Our data support the structural plasticity model of porins and are consistent with the 'charge-screening-unscreening' hypothesis that describes how these proteins may exist in distinct conformations.

Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli.

The large mechanosensitive ion channel (MscL) of Escherichia coli was expressed on a plasmid encoding MscL as a fusion protein with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene. After purification of the fusion protein using glutathione-coated beads, thrombin cleavage allowed recovery of the MscL protein. The purified protein was reconstituted into artificial liposomes and found to be fully functional when examined with the patch-clamp technique. The reconstituted recombinant MscL protein formed ion channels that exhibited characteristic conductance and pressure sensitivity and were blocked by the mechanosensitive ion channel inhibitor gadolinium. The recombinant MscL protein was also used to raise specific anti-MscL polyclonal antibodies which abolished channel activity when preincubated with the MscL protein.

Genetic characterization of mannose-sensitive hemagglutinin (MSHA)-negative mutants of Vibrio cholerae derived by Tn5 mutagenesis.

El Tor biotype Vibrio cholerae strains express a cell-associated mannose-sensitive hemagglutinin (MSHA) which is a putative attachment factor. Several MSHA-negative mutants from V. cholerae strain JBK70 were previously generated by Tn5 mutagenesis [Finn et al., Infect. Immun. 55 (1987) 942-946]. The chromosomal DNA regions containing the Tn5 insertions were isolated from eight strains for further analysis. Nucleotide sequencing of the insertional junctions and corresponding clones containing the intact chromosomal region from the parental strain revealed the presence of several contiguous ORFs. Only two ORFs of this region had received insertions, and these showed remarkable homology to genes involved in the general secretory pathway found in several Gram- bacterial species. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Marker exchange mutagenesis was used to insert kanamycin-resistance cassettes and TnphoA insertions into different locations of this region in the chromosome of wild-type V. cholerae strains. The phenotypes of these mutants showed that this DNA region is involved in MSHA production, but is not required for general extracellular protein secretion.

Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.

The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins. V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination. The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacterial extracellular zinc-containing metalloproteases.

Extracellular zinc-containing metalloproteases are widely distributed in the bacterial world. The most extensively studied are those which are associated with pathogenic bacteria or bacteria which have industrial significance. They are found practically wherever they are sought in both gram-negative and gram-positive microorganisms, be they aerobic or anaerobic. This ubiquity in itself implies that these enzymes serve important functions for the organisms which produce them. Because of the importance of zinc to enzymatic activity, it is not surprising that there is a pervasive amino acid sequence homology in the primary structure of this family of enzymes regardless of their source. The evidence suggests that both convergent and divergent evolutionary forces are at work. Within the large family of bacterial zinc-containing metalloendopeptidases, smaller family units are observed, such as thermolysin-like, elastase-like, and Serratia protease-like metalloproteases from various bacterial species. While this review was in the process of construction, a new function for zinc-containing metalloproteases was discovered: the neurotoxins of Clostridium tetani and Clostridium botulinum type B have been shown to be zinc metalloproteases with specificity for synaptobrevin, an integral membrane protein of small synaptic vesicles which is involved in neurotransmission. Additional understanding of the mode of action of proteases which contribute to pathogenicity could lead to the development of inhibitors, such as chelators, surrogate substrates, or antibodies, which could prevent or interrupt the disease process. Further studies of this broad family of metalloproteases will provide important additional insights into the pathogenesis and structure-function relationships of enzymes and will lead to the development of products, including "designer proteins," which might be industrially and/or therapeutically useful.

Vibrio cholerae hemagglutinin/protease, colonial variation, virulence, and detachment.

The structural gene, hap, for the secreted hemagglutinin/protease (HA/protease), a putative virulence factor of Vibrio cholerae, has recently been cloned and sequenced (C. C. Häse and R. A. Finkelstein, J. Bacteriol. 173:3311-3317, 1991). The availability of the null mutant, HAP-1, and HAP-1 complemented with pCH2 (which expresses HA/protease), enabled an examination of the role of HA/protease in the virulence of V. cholerae in an animal model. However, the mutants exhibited reversible colonial variation similar but not identical to that which was previously associated with dramatic changes in virulence of parental strain 3083. Regardless of colonial morphology, the mutants were found to be fully virulent in infant rabbits. Thus, the HA/protease is not a primary virulence factor (for infant rabbits). Observations using cultured human intestinal cells indicated, instead, that the HA/protease is responsible for detachment of the vibrios from the cultured cells by digestion of several putative receptors for V. cholerae adhesins.

Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain.

The structural gene hap for the extracellular hemagglutinin/protease (HA/protease) of Vibrio cholerae was cloned and sequenced. The cloned DNA fragment contained a 1,827-bp open reading frame potentially encoding a 609-amino-acid polypeptide. The deduced protein contains a putative signal sequence followed by a large propeptide. The extracellular HA/protease consists of 414 amino acids with a computed molecular weight of 46,700. In the absence of protease inhibitors, this is processed to the 32-kDa form which is usually isolated. The deduced amino acid sequence of the mature HA/protease showed 61.5% identity with the Pseudomonas aeruginosa elastase. The cloned hap gene was inactivated and introduced into the chromosome of V. cholerae by recombination to construct the HA/protease-negative strain HAP-1. The cloned fragment containing the hap gene was then shown to complement the mutant strain.

Comparison of the Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase.

The soluble hemagglutinin/protease (HA/protease) produced by Vibrio cholerae and the elastase of Pseudomonas aeruginosa are both zinc/calcium-dependent proteases. In the present study the two enzymes are compared immunologically and functionally. The N-terminal amino acid sequences of the proteins had 65% identity within the first 20 amino acids. Polyclonal antisera against each purified protein recognized the enzyme of the other species in enzyme-linked immunosorbent assay, checkerboard immunoblot, and Western blot analyses and inhibited the protease activity of both enzymes in milk and elastin agars. Like the HA/protease, the elastase hemagglutinated "responder" but not "nonresponder" chicken erythrocytes, degraded ovomucin, lactoferrin, and fibronectin, and nicked the A subunit of the cholera toxin-related heat-labile enterotoxin from Escherichia coli. Whereas none of the three proteases tested (elastase, HA/protease, or pronase E) had any obvious effect in ileal loop tests in rabbits at doses up to 50 micrograms, all three produced some detectable skin reactions at a dose of 0.1 micrograms and necrosis at a higher dose (i.e., 5 micrograms). We conclude that the V. cholerae HA/protease and the P. aeruginosa elastase are structurally, functionally, and immunologically related.