Role of the Hya hydrogenase in recycling of anaerobically produced H2 in Salmonella enterica serovar Typhimurium.

Due to the anticipated scarcity of fossil fuels, there has been a surge of interest in H2 production for alternative energy means. Numerous studies have attempted to engineer H2-producing organisms, such as photosynthetic bacteria, cyanobacteria, and Escherichia coli, to produce maximal amounts of H2 while minimizing the H2-oxidizing capability of the organism (4, 5, 9, 10, 19). Hydrogenase expression and activity are controlled by multiple regulatory pathways and respond to fluctuations in pH, oxygen levels, and availability of metabolites and metal cofactors (17). In addition, the presence of hydrogen uptake hydrogenases decreases the net H2 yield even under conditions that favor H2 production. It is therefore important to understand the interactions between H2-oxidizing enzymes (i.e., respiratory hydrogenases) and H2-producing enzymes. Gene sequence analysis has revealed that many enteric bacteria contain the genes necessary for hydrogen production and oxidation. The E. coli hydrogenases have been studied extensively, while Salmonella enterica serovar Typhimurium hydrogenases have been studied to a lesser extent. Both E. coli and Salmonella serovar Typhimurium contain the hydrogen-oxidizing hydrogenases Hya and Hyb. Salmonella serovar Typhimurium also contains Hyd, which is another hydrogen-oxidizing hydrogenase (2, 13, 15). Hyc and Hyf are hydrogen-evolving hydrogenases that are present in both E. coli and Salmonella serovar Typhimurium, although it is unknown whether Hyf is functional (1). The Salmonella serovar Typhimurium hydrogenases are important for cellular metabolism. Hyc produces H2 in order to remove excess reductant generated during mixed-acid fermentation. Hyc and formate dehydrogenase constitute the formate hydrogen lyase (FHL) complex (16), which oxidizes formate to produce CO2 and H2 (12). The hyb genes in E. coli and Salmonella serovar Typhimurium are expressed at high levels under anaerobic respiration conditions, and Hyb probably contributes to energy conservation (11, 15, 20). Hyb oxidizes H2 and generates electrons, which are passed through the electron transport chain to terminal acceptors such as fumarate. The protons generated contribute to the proton-motive force. The role of Hya is not as well characterized. Hya may be used to recycle Hyc-produced H2, since the hya operon is expressed at high levels during fermentative growth, or it may play a role in acid stress resistance (6, 14, 20, 21). The hyb genes are expressed at high levels under aerobic conditions in Salmonella serovar Typhimurium, and Hyb may couple H2 oxidation to O2 reduction (20). Redwood et al. recently examined the roles of uptake-type hydrogenases on net hydrogen production in E. coli (10). Cells were pregrown aerobically or anaerobically with formate and then allowed to ferment in anaerobic bottles. H2 gas was collected, and other fermentation products were measured. They found that H2 production increased by 37% in an hya hyb double mutant (compared to that in the wild type) that was grown overnight aerobically with formate. This increase in production was associated with the loss of hyb and not hya. Therefore, in E. coli, Hyb may be responsible for recycling fermentatively produced H2. In this study, we measured the effect of uptake-type hydrogenase mutations on H2 production in Salmonella serovar Typhimurium. We found that the majority of H2-recycling activity in fermenting cells was dependent on the presence of hya, and having only Hya was sufficient to prevent any detectable H2 evolution. These results demonstrate yet another difference between H2 metabolism in E. coli and H2 metabolism in Salmonella serovar Typhimurium.