서강대학교

초록/요약

A facultative anaerobic bacterium, Vibrio vulnificus metabolizes glucose under anaerobic conditions and produces metabolites through mixed-acid fermentation. To examine the anaerobic glucose metabolism in the presence of trimethylamine N-oxide (TMAO) or nitrate/nitrite, the accumulated metabolites in anaerobic cultures of V. vulnificus were analyzed. Compared to the conditions for fermentation or anaerobic respiration with TMAO, the presence of nitrate/nitrite resulted in altered metabolite profile with reduced concentration of formate. The cellular concentration of formate is determined by its production by pyruvate formate lyase (Pfl), its secretion via formate transporter (Foc), and its utilization by formate-dependent nitrite reductase (Nrf). Thus, it was investigated if the expression of these three representative genes were influenced by nitrate/nitrite. Transcription fusion assays showed that only nrfA gene was induced by nitrate/nitrite in a concentration-dependent manner, while the expression of pfl and focA were insensitive to nitrate/nitrite. In Escherichia coli, there is an nrf operon consisting of seven genes, from nrfA to G. In contrast, there are three distinct transcription units in the V. vulnificus genomes, an nrfA gene and two operons, nrfBCDE and nrfGF. Further analyses of each transcript revealed that nrfA and nrfBCDE genes were induced by nitrate/nitrite and the functional Fnr was required for this induction. Screening of other factors involved in anaerobic growth revealed that Fur also activated the transcription of nrfA and nrfBCDE genes. In addition, activation by Fur was dependent upon the presence of iron ion, suggesting the iron-Fur complex activates these gene. This study demonstrates that expression of NrfA~E are induced by both Fnr and Fur in the presence of nitrate/nitrite and iron ion, respectively. These findings suggest that formate produced via fermentation pathways can be utilized as an electron donor in the presence of nitrate/nitrite as electron acceptors or iron ion as the enzyme cofactor, otherwise formate or reactive nitrogen species are detrimental to V. vulnificus.