The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE. We previously demonstrated that vvpE expression is differentially directed by two different promoters in a growth phase-dependent manner. The activity of the stationaryphase promoter (promoter S (PS)) is dependent on RpoS and is also under the positive control of cyclic AMP receptor protein (CRP). In this study, primer extension analyses revealed that SmcR, the Vibrio harveyi LuxR homolog, is also involved in the regulation of vvpE transcription by activating PS. Although the influence of CRP on PS is mediated by SmcR, the level of PS activity observed when CRP and SmcR function together was found to be greater than the sum of the PS activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of RpoS, CRP, and SmcR were not significantly affected by one other, indicating that CRP and SmcR function cooperatively to activate PS rather than sequentially in a regulatory cascade. The binding sites for CRP and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro DNase I protection assays. The binding sites for CRP and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of PS, respectively. Accordingly, these results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoSdependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase.The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases, including life-threatening septicemia and possibly gastroenteritis, in individuals with underlying predisposed conditions such as liver damage, excess levels of iron, and immunocompromised conditions. Wound infections also result from exposure to seawater or from the handling of shellfish contaminated with V. vulnificus. The mortality from septicemia is very high (Ͼ50%), and death can occur within 1-2 days after the first signs of illness. Several potential virulence factors, including an endotoxin, polysaccharide capsule, iron-sequestering systems, cytolytic hemolysin, elastase, phospholipase A 2 , and other exotoxins, have been identified in V. vulnificus (for recent reviews, see Refs. 1 and 2).The elastase activity is from a neutral metalloprotease and represents the major proteolytic activity of V. vulnificus (3, 4). Our previous study revealed the existence of at least two proteases that are produced by V. vulnificus (5). Therefore, vvpE was designed to differentiate the elastase gene from other genes encoding other potential proteases of V. vulnificus (5). As such, a gene encoding the V. vulnificus elastase was recently cloned and sequenced by us (5) and by others (6). The deduced gene product was predicted to be a 609-amino acid polypeptide, and the mature elastase is a 45-kDa protein consisting of 413 amino acids generated by the deletion of the N-termi...

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Vibrio vulnificus is an opportunistic gram-negative pathogen that commonly contaminates oysters. Predisposed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people are susceptible to serious wound infection after contact with contaminated seafood or seawater. Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of V. vulnificus infections. Among the putative virulence factors is an elastolytic metalloprotease. We cloned and sequenced the vvpE gene encoding an elastase of V. vulnificus ATCC 29307. The functions of the elastase were assessed by constructing vvpE insertional knockout mutants and evaluating phenotypic changes in vitro and in mice. Although other types of protease activity were still observed in vvpE mutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinant vvpE gene. In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of the V. vulnificus vvpE gene did not affect the ability of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the liver, in both iron-treated and normal mice. Furthermore, a vvpE mutant was not affected with regard to cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro. Therefore, it appears that elastase is less important in the pathogenesis of V. vulnificus than would have been predicted by examining the effects of administering purified proteins to animals. However, V. vulnificus utilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors.The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases such as life-threatening septicemia and possibly gastroenteritis in individuals with underlying predisposing conditions such as liver damage, excess levels of iron, and immunocompromised conditions (2, 14). Wound infections result from exposure to seawater or from the handling of shellfish contaminated with V. vulnificus. Mortality from septicemia is very high (Ͼ50%), and death may occur within 1 to 2 days after the first signs of illness (14,47). Several potential virulence factors including an endotoxin, a polysaccharide capsule (46,55,57), iron-sequestering systems (19, 54), a cytolytic hemolysin (43, 53), an elastase (16, 24, 36), a phospholipase A 2 (48), and other exotoxins have been identified for V. vulnificus. However, to date, only the capsule (55) and ironsequestering systems (19) have been confirmed as virulence factors by using the molecular version of Koch's postulates (6, 11), in which mutations are constructed in genes encoding putative virulence factors, followed by complementation of any observed attenuating phenotypes. It is interesting that a m...