Abstract
Abstract Vibrio vulnificus, a gram-negative marine bacterium, is an opportunistic pathogen with a high invasive capability that causes high mortality in infected immunocompromised patients. In order to solve the problem of the bacterial infection, it is essential to understand the pathogenesis of the pathogen, particularly the virulence-associated genes and their roles in the infection process. In this study we have established a kanamycin resistance gene-based transposon mutagenesis system for use in V. vulnificus. A mutant WY005 which exhibited a high hemolytic ability was isolated. The transposon insertion site was cloned by seletion on kanamycin containing agar and the identity of the disrupted gene was determined by nucleotide sequencing. Sequence comparison in the GenBank database has demonstrated that the gene is pilT that is located on V. vulnificus plasmid pYJ016. PilT is a putative nucleotide-binding protein involved in function of type IV pili including movement and attachment to the host. In Neisseria, a PilT mutant has been reported to participate in release of heme from red blood cells. We also attempt to investigate the expression of response regulator gene in V.vulnificus. A total of 38 response regulators of V. vulnificus that contain a putative DNA binding motif were cloned and expressed. Among them 28 has successfully yielded a protein of expected sizes. Finally, using the RNA dot blotting, expression levels of 7 response regulator genes under different growth conditions were analyzed. The expression level of PhoB and gltR genes were found to be activated significantly under ferric limitation and minimum medium culture. The results suggest that the two response regulators play a role in counter nutrient limitation conditions in V.vulnificus.