Nucleotide sequence of the Vibrio alginolyticus calcium-dependent, detergent-resistant alkaline serine exoprotease A

The nucleotide sequence of the Vibrio alginolyticus alkaline serine exoprotease A (ProA) gene cloned in Escherichia coli was determined. The exoprotease A gene (proA) consisted of 1602 bp which encoded a protein of 534 amino acids (aa) with an Mr of 55,900. The region upstream from the gene was characterized by a putative promoter consensus region (-10 -35), a ribosome-binding site and ATG start codon. The proA gene encodes a typical 21-aa N-terminal signal sequence which, when fused to alkaline phosphatase by means of transposon TnphoA, was able to mediate transport of the alkaline phosphatase to the periplasm in E. coli. Deletions of up to 106 aa from the C terminus of ProA did not result in the loss of extracellular protease activity. Additional V. alginolyticus genes were not involved in the secretion into the medium of the cloned ProA in E. coli. The amino acid sequence of ProA showed low overall homology to a Serratia marcescens serine exoprotease but significant homology was detected with other subtilisin family exoproteases. The fungal proteinase K, another sodium dodecyl sulfate-resistant protease, had 44% aa homology with ProA.     

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.     

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.     

Involvement of LuxR, a quorum sensing regulator in Vibrio harveyi, in the promotion of metabolic genes: argA, purM, lysE and rluA

Quorum sensing, involving signal transduction via the two-component response regulator LuxO to its downstream target LuxR, controls luminescence in the marine bacterium Vibrio harveyi. LuxR is a DNA binding protein that acts as both activator of the lux operon and repressor of its own gene. In order to determine if any other genes are affected by quorum sensing in V. harveyi, an assay for luxR-dependent promotion was devised using a genomic library maintained in a novel luxAB (luciferase) reporter. Screening in Escherichia coli DH-21 (lacI(sq)) entailed the addition of a second plasmid containing luxR under plac control. Four out of 5000 colonies showed luminescence stimulation upon IPTG induction of luxR. The four luxR-dependent promoters were upstream of argA, purM, lysE, and rluA, genes involved in arginine and purine biosyntheses, amino acid efflux, and pseudouridine synthesis, respectively. Based on analysis of luxR-dependent promoters, particularly that of argA, we describe a LuxR binding site, and implicate the coordination of LuxR with ArgR.     

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.