Occurrence of P-flavin binding protein in Vibrio fischeri and properties of the protein.
(25/1761)
In previous studies involving Photobacterium species we proposed that (i) P-flavin is the product of luciferase, (ii) the physiological function of the lux operon is not to produce light but to produce FP(390) (luxF protein), including its prosthetic group, P-flavin, and (iii) FP(390) reactivates oxidatively inactivated cobalamin-dependent methionine synthase similar to flavodoxin but at relatively high ionic strength. It seems difficult to extend this idea to all luminous bacteria because the luxF gene is not present in the lux operon in Vibrio or Xenorhabdus. But we predicted that a luciferase fragment which binds P-flavin should function like FP(390) in these species. In this study, we isolated P-flavin binding protein from Vibrio fischeri ATCC 7744. The obtained protein was a modified luciferase as expected, in which the beta-subunit was intact but about 25 amino acid residues at the C-terminus of the alpha-subunit were deleted and the prosthetic group was the fully reduced P-flavin. These results strongly support that the physiological function of the lux operon is as described above even in luminous bacteria other than Photobacterium species. We propose that chromophore B reported by Tu and Hastings [Tu, S.-C. and Hastings, J.W. (1975) Biochemistry 14, 1975-1980] is the reduced P-flavin. (+info)
Induction of protease activity in Vibrio anguillarum by gastrointestinal mucus.
(26/1761)
The effect of gastrointestinal mucus on protease activity in Vibrio anguillarum was investigated. Protease activity was measured by using an azocasein hydrolysis assay. Cells grown to stationary phase in mucus (200 microg of mucus protein/ml) exhibited ninefold-greater protease activity than cells grown in Luria-Bertani broth plus 2% NaCl (LB20). Protease induction was examined with cells grown in LB20 and resuspended in mucus, LB20, nine-salts solution (NSS [a carbon-, nitrogen-, and phosphorus-free salt solution]), or marine minimal medium (3M) ( approximately 10(9) CFU/ml). Induction of protease activity occurred 60 to 90 min after addition of mucus and was >/=70-fold greater than protease activity measured in cells incubated in either LB20 or 3M. Mucus was fractionated into aqueous and chloroform-methanol-soluble fractions. The aqueous fraction supported growth of V. anguillarum cells, but did not induce protease activity. The chloroform-methanol-soluble fraction did not support growth, nor did it induce protease activity. When the two fractions were mixed, protease activity was induced. The chloroform-methanol-soluble fraction did not induce protease activity in cells growing in LB20. EDTA (50 mM) inhibited the protease induced by mucus. Upon addition of divalent cations, Mg(2+) (100 mM) was more effective than equimolar amounts of either Ca(2+) or Zn(2+) in restoring activity, suggesting that the mucus-inducible protease was a magnesium-dependent metalloprotease. An empA mutant strain of V. anguillarum did not exhibit protease activity after exposure to mucus, but did grow in mucus. Southern analysis and PCR amplification confirmed that V. anguillarum M93 contained empA. These data demonstrate that the empA metalloprotease of V. anguillarum is specifically induced by gastrointestinal mucus. (+info)
Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity.
(27/1761)
TraR, a member of the LuxR family of quorum-sensing transcription factors, is responsible for the population density-dependent regulation of Ti plasmid conjugal transfer. The protein requires as coinducer an acyl-homoserine lactone signal molecule called AAI (Agrobacterium autoinducer) that is produced by the bacteria themselves. TraR only activates its target genes, making it difficult to determine whether interaction with AAI is required for binding DNA or for initiating transcription. To assess this, we converted TraR into a repressor by placing a copy of the tra box, an 18-bp inverted repeat believed to be the recognition site for this protein, over the -10 region of a promoter driving expression of lacZ. Repression of this promoter by TraR depended on AAI or, at higher concentrations, VAI, the closely related signal of Vibrio fischeri. C-terminal deletions as short as 2 aa and N-terminal deletions as short as 4 aa in TraR abolished both repressor and activator functions. The C-terminal mutants were strongly dominant over TraR, suggesting that they can form heteromultimers with the wild-type activator. Mutants of TraR with substitutions at Asp-10 and Gly-123 failed to activate a positively controlled reporter but continued to repress the chimeric promoter in an AAI-dependent manner. We conclude that TraR recognizes the tra box as its binding site, that binding of TraR to this site depends on AAI, and that the N-terminal half of the protein contains one or more domains that are required for activation but not for multimerization, for interaction with the acyl-homoserine lactone, or for DNA binding. (+info)
Functional interaction between PomA and PomB, the Na(+)-driven flagellar motor components of Vibrio alginolyticus.
(28/1761)
Four proteins, PomA, PomB, MotX, and MotY, appear to be involved in force generation of the sodium-driven polar flagella of Vibrio alginolyticus. Among these, PomA and PomB seem to be associated and to form a sodium channel. By using antipeptide antibodies against PomA or PomB, we carried out immunoprecipitation to verify whether these proteins form a complex and examined the in vivo stabilities of PomA and PomB. As a result, we could demonstrate that PomA and PomB functionally interact with each other. (+info)
Random mutagenesis of the pomA gene encoding a putative channel component of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus.
(29/1761)
PomA and PomB are integral membrane proteins and are essential for the rotation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. On the basis of their similarity to MotA and MotB, which are the proton-conducting components of the H(+)-driven motor, they are thought to form the Na(+)-channel complex and to be essential for mechanochemical coupling in the motor. To investigate PomA function, random mutagenesis of the pomA gene by using hydroxylamine was carried out. We isolated 37 non-motile mutants (26 independent mutations) and most of the mutations were dominant; these mutant alleles are able to inhibit the motility of wild-type cells when greatly overexpressed. The mutant PomA proteins could be detected by immunoblotting, except for those with deletions or truncations. Many of the dominant mutations were mapped to the putative third or fourth transmembrane segments, which are the most conserved regions. Some mutations that showed strong dominance were in highly conserved residues. T1861 is the mutation of a polar residue located in a transmembrane segment that might be involved in ion translocation. P199L occurred in a residue that is thought to mediate conformational changes essential for torque generation in MotA. These results suggest that PomA and MotA have very similar structures and roles, and the basic mechanism for torque generation will be similar in the proton and sodium motors. (+info)
Perkinsus marinus extracellular protease modulates survival of Vibrio vulnificus in Eastern oyster (Crassostrea virginica) hemocytes.
(30/1761)
The in vitro effects of the Perkinsus marinus serine protease on the intracellular survival of Vibrio vulnificus in oyster hemocytes were examined by using a time-course gentamicin internalization assay. Results showed that protease-treated hemocytes were initially slower to internalize V. vulnificus than untreated hemocytes. After 1 h, the elimination of V. vulnificus by treated hemocytes was significantly suppressed compared with hemocytes infected with invasive and noninvasive controls. Our data suggest that the serine protease produced by P. marinus suppresses the vibriocidal activity of oyster hemocytes to effectively eliminate V. vulnificus, potentially leading to conditions favoring higher numbers of vibrios in oyster tissues. (+info)
Cross-reaction between a strain of Vibrio mimicus and V. cholerae O139 Bengal.
(31/1761)
Of 200 isolates of Vibrio mimicus screened, one from water (N-57) agglutinated with V. cholerae O139 polyclonal antiserum (absorbed with a rough strain of V. cholerae only) and not with O139 polyclonal diagnostic antiserum (absorbed with the rough strain and V. cholerae O22 and O155). The antigenic relationship between V. cholerae 0139 and N-57 is of a,b-a,c type, where a is the common antigenic epitope and b and c are unique epitopes. Strain N-57 was assigned to a new serogroup of V. cholerae O194. It gave negative results in a monoclonal antibody-based rapid test and a PCR test specific for V. cholerae O139. It did not possess the ctx gene or produce cholera toxin. Antiserum to strain N-57 cross-protected infant mice against cholera on challenge with V. cholerae O139. Structural studies of the surface polysaccharides and studies of the rfb genes will shed more light on the extent of relatedness between V. mimicus N-57 and V. cholerae O139. (+info)
Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein.
(32/1761)
Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V. fischeri gene, ainS, directs the synthesis of N-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N-(3-hydroxybutyryl)-L-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N-octanoylhomoserine lactone from S-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes. (+info)