LuxO controls extracellular protease, haemolytic activities and siderophore production in fish pathogen Vibrio alginolyticus

Aims: To characterize the luxO gene in fish pathogen Vibrio alginolyticus MVP01 and investigate its roles in regulation of extracellular products (ECP) and siderophore production. Methods and Results: The luxO gene was cloned from V. alginolyticus MVP01. Genetic analysis revealed that it encoded a protein with high similarity to other LuxO homologues. The luxO in-frame deletion mutant and rpoN null mutant were constructed with suicide plasmids. We demonstrated that sole deletion in LuxO increased the secretion of extracellular protease and haemolytic products, but decreased siderophore production for V. alginolyticus MVP01. Mutants with null rpoN displayed significantly enhanced protease level and siderophore production while notable reduction in haemolytic activities of ECP. Conclusions: Vibrio alginolyticus harbours functional luxO gene that regulates the secretion of extracellular protease and haemolytic materials as well as siderophore production in either σ⁵⁴ dependent or independent manners. Significance and Impact of the Study: The current study demonstrated that V. alginolyticus MVP01 produces extracellular protease and haemolytic activity material as well as siderophore, which may be characteristics of the virulence of the strain. Revelations that secretion of these products is under the regulation of LuxO and σ⁵⁴ as well as the potential quorum sensing systems in V. alginolyticus MVP01 will expedite the understanding of vibriosis pathogenesis.     

Characterization of extracellular alkaline proteases and collagenase induction in Vibrio alginolyticus

The number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28 000, 22 500 and 19 500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15 500 and 14 500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 degrees C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45 000, 38 500, 33 500 and 31 000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.     

Growth, copper-tolerant cells, and extracellular protein production in copper-stressed chemostat cultures of Vibrio alginolyticus.

The influence of elevated copper concentrations on cell numbers and extracellular protein production was investigated in chemostat cultures of Vibrio alginolyticus. High (20 microM) copper in the medium reservoir resulted in a dramatic drop in cell numbers which was overcome with time. The copper-stressed cultures established a new equilibrium cell concentration slightly (ca. 20%) lower than control cultures. Copper-stressed chemostat populations contained an increased number of copper-resistant cells, but these averaged only 26% of the copper-adapted population. Previously copper-stressed populations exhibited resistance to a second challenge with copper. Proteins with properties identical to those of copper-induced, copper-binding proteins (CuBPs) observed in batch cultures of V. alginolyticus were observed in the supernatants of copper-stressed chemostat cultures and not in controls. CuBPs from batch and chemostat cultures were identical in terms of their induction by copper, molecular weight, and retention volumes on both immobilized copper ion-affinity chromatography and reverse-phase high-performance liquid chromatography columns. The concentration of CuBP in the chemostat was dependent on copper concentration in the medium reservoir. Either one or two forms of CuBP were observed in various analyses from both batch and chemostat cultures. Gel-to-gel variability was implicated as a factor determining whether one or two forms were resolved in a given analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Bacillus amyloliquefaciens amylase and Vibrio alginolyticus protease A fusion genes.

Previously we reported [Deane, S. M., Maharaj, R., Robb, F. T. & Woods, D. R. (1987) Journal of General Microbiology 133, 2295-2302] that the production of a Vibrio alginolyticus SDS-resistant alkaline serine protease (Pro A) cloned in Escherichia coli was characterized by a 12 h delay between the synthesis of an inactive precursor and secretion of active Pro A. Replacement of the V. alginolyticus promoter region by the alpha-amylase promoter region from Bacillus amyloliquefaciens resulted in the simultaneous synthesis and secretion of Pro A in E. coli. The V. alginolyticus pro A gene cloned on a shuttle vector did not produce active Pro A in Bacillus subtilis. Although Pro A has a typical Gram-positive signal sequence, it was not functional in B. subtilis. Replacement of the Pro A signal sequence with the alpha-amylase signal sequence resulted in the production of active Pro A in B. subtilis.     

Chemotactic responses of Vibrio alginolyticus to algal extracellular products.

A capillary assay was used to evaluate the chemotactic responses of Vibrio alginolyticus to three common algal extracellular products. Acrylate and glycolate attracted the motile marine bacterium. The peak response occurred with 10(-2) M of each chemical. Acrylic and glycolic acid also attracted V. alginolyticus, with the peak response occurring at 5 x 10(-4) M of each chemical. Higher concentrations of the organic acids resulted in a decreased response. The bacteria also displayed positive chemotaxis to dimethyl sulfide.     

Xylanase production in Aspergillus nidulans: induction and carbon catabolite repression

Abstract The induction of the synthesis of extracellular xylanases was investigated in the fungus Aspergillus nidulans using a number of compounds, including xylans of different origin, monosaccharides, xylooligosaccharides and xylose derivatives. Certain xylans (wheat arabinoxylan, oat spelt xylan, birchwood xylan and 4-O-methyl-D-glucurono-D-xylan) were found to be the most powerful inducers. Also, xylooligosaccharides such as xylobiose, xylotriose and xylotetraose served as inducers, their efficiency being directly related to their chain length. Xylose, on the contrary, was not a true inducer. Of the three endo-β-(1,4)-xylanases secreted by A. nidulans , that of 24 kDa was not under carbon catabolite repression, whereas the other two, of 22 and 34 kDa, were under glucose repression mediated by the creA gene product.     

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus

The production of an extracellular collagenase and an alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37°C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5°C Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30°C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.     

Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus.

Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes.     

Effects of extracellular products of Vibrio alginolyticus on penaeid prawn plasma components

The effects of both crude extracellular products (ECP) and a partially purified protease of Vibrio alginolyticus on the plasma components of kuruma prawn ( Penaeus japonicus ) and tiger prawn ( P. monodon ) were studied using crossed immunoelectrophoresis (CIE). A component of the plasma, tentatively identified as coagulogen, apparently disappeared after incubation with the ECP, while the amount of a component tentatively identified as haemocyanin decreased. The coagulogen and an unknown component (component 1) in the penaeid plasma showed an increased migration rate after incubation with a partially purified 33 kDa protease of the bacterium. In contrast, incubation with protease had no detectable effect on the amount of haemocyanin. These events may significantly contribute to the pathogenicity of Vibrio alginolyticus in penaeids.     

Induction and carbon catabolite repression of isoamylase production in Rhizopus oryzae PR7

The growth and the extracellular isoamylase production by Rhizopus oryzae PR7 MTCC 9642 were studied in a stationary culture at 28°C, with maximum isoamylase production obtained after 72 hours. Glycogen was found to be the best inducer for isoamylase synthesis, followed by maltose and dextrin. The enzyme was found to be repressed by glucose and this repression was not overcome by the addition of cGMP. The abrupt reduction in enzyme synthesis after the addition of exogenous glucose in a glycogen-induced culture medium confirmed the repressive action of glucose. An almost similar rate of repression was found to be exerted by α- and β-cyclodextrins. The inhibition of enzyme production after the addition of cycloheximide, a translation blocker, indicated the existence of de novo synthesis of the enzyme.     

Glucose-induced production of recombinant proteins in Hansenula polymorpha mutants deficient in catabolite repression

The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system.     

Regulation of extracellular alkaline protease activity by histidine in a collagenolytic Vibrio alginolyticus strain

Vibrio alginolyticus synthesized an inducible extracellular collagenase in a peptone medium during the stationary growth phase. These cultures also possessed extracellular alkaline serine protease activity. The alkaline protease activity did not require a specific inducer and it was produced in tryptone or minimal media. The collagenase was not produced in either the tryptone or minimal media. The alkaline protease activity was sensitive to catabolite repression by a number of carbon sources, including glucose, and by amino acids and ammonium ions. Cyclic AMP, dibutyryl cyclic AMP and cyclic GMP did not relieve catabolite repression. Histidine and urocanic acid stimulated the production of alkaline protease activity in tryptone and minimal media. Other compounds associated with the histidine utilization (hut) pathway did not increase alkaline protease activity. Histidine reversed the repression of alkaline protease activity by glucose of (NH4)2SO4 in minimal medium. Histidine and the compounds associated with the hut pathway inhibited collagenase production.     

Chitin colonization, chitin degradation and chitin-induced natural competence of Vibrio cholerae are subject to catabolite repression

Although Vibrio cholerae is a human pathogen its primary habitat are aquatic environments. In this environment, V.cholerae takes advantage of the abundance of zooplankton, whose chitinous exoskeletons provide a nutritious surface. Chitin also induces the developmental programme of natural competence in several species of the genus Vibrio. Because the chitin surface can serve as the sole carbon source for V.cholerae, the link between carbon catabolite repression and chitin-induced natural competence for transformation was investigated in this study. Provision of competing phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS)-dependent carbon sources in addition to chitin significantly lowered natural transformability. These sugars are known to interfere with the accumulation of 3',5'-cyclic AMP (cAMP); therefore, the contributions of the cAMP-producing enzyme, adenylate cyclase and the cAMP receptor protein (CRP) to chitin surface colonization, chitin degradation and natural transformation were also analysed. The results provided here indicate that cAMP and CRP are important in at least three interlinked areas of the chitin-induced natural competence programme. First, cAMP and CRP are required for the efficient colonization of the chitin surface; second both contribute to chitin degradation and utilization, and third, cAMP plus CRP play a role in increasing competence gene expression. These findings highlight the complex regulatory circuit of chitin-induced natural competence in V.cholerae.