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

Double and triple uptake-type hydrogenase mutants were used to determine which hydrogenase recycles fermentatively produced hydrogen. The Deltahyb Deltahya and Deltahyd Deltahya double mutants evolved H(2) at rates similar to that of the triple mutant strain, so Hya alone oxidizes the bulk of H(2) produced during fermentation. When only Hya was present, no hydrogen production was observed in nutrient-limited medium. H(2) uptake assays showed that Hya can oxidize both exogenously added H(2) and formate hydrogen lyase-evolved H(2) anaerobically. Even after anaerobic growth, all three uptake-type hydrogenases could function in the presence of oxygen, including using O(2) as a terminal acceptor.

Salmonella enterica serovar Typhimurium NiFe uptake-type hydrogenases are differentially expressed in vivo.

Salmonella enterica serovar Typhimurium, a common enteric pathogen, possesses three NiFe uptake-type hydrogenases. The results from mouse infection studies suggest that the H(2) oxidation capacity provided by these hydrogenases is important for virulence. Since the three enzymes are similar in structure and function, it may be expected that they are utilized under different locations and times during an infection. A recombination-based method to examine promoter activity in vivo (RIVET) was used to determine hydrogenase gene expression in macrophages, polymorphonuclear leukocyte (PMN)-like cells, and a mouse model of salmonellosis. The hyd and hya promoters showed increased expression in both murine macrophages and human PMN-like cells compared to that in the medium-only controls. Quantitative reverse transcription-PCR results suggested that hyb is also expressed in phagocytes. A nonpolar hya mutant was compromised for survival in macrophages compared to the wild type. This may be due to lower tolerance to acid stress, since the hya mutant was much more acid sensitive than the wild type. In addition, hya mutant cells were internalized by macrophages the same as wild-type cells. Mouse studies (RIVET) indicate that hyd is highly expressed in the liver and spleen early during infection but is expressed poorly in the ileum in infected animals. Late in the infection, the hyd genes were expressed at high levels in the ileum as well as in the liver and spleen. The hya genes were expressed at low levels in all locations tested. These results suggest that the hydrogenases are used to oxidize hydrogen in different stages of an infection.

Nickel enzyme maturation in Helicobacter hepaticus: roles of accessory proteins in hydrogenase and urease activities.

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, possesses a hydrogenase and a urease, both of which are nickel-containing enzymes. Analysis of the genome sequence of H. hepaticus revealed a full set of accessory genes which are required for the nickel maturation of each enzyme in other micro-organisms. Erythromycin-resistant mutants were constructed in four of these genes, hypA, hypB, ureE and ureG. Controls for polar effect were provided for hypA or hypB mutants by disrupting each gene located immediately downstream, i.e. hp0809 or hypC, respectively. Urease and hydrogenase activities were determined for each strain with or without supplemented nickel in the medium. As expected, the ureE and the ureG mutants had negligible urease activity, but they retained normal levels of hydrogenase activity. Urease levels could not be increased by the addition of nickel to the medium. The H. hepaticus hypA and hypB strains were deficient in both urease and hydrogenase activities, suggesting that both gene products act in a similar fashion as their counterparts in H. pylori. However, in contrast with the analogous mutants of H. pylori, the addition of nickel into the growth medium failed to restore either urease or hydrogenase enzyme levels in the H. hepaticus hypA or hypB mutants, indicating a probably unique role for these genes in the mouse liver pathogen.

Differential expression of NiFe uptake-type hydrogenase genes in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium possesses three similar NiFe hydrogenases important to its virulence. Here we show that the three hydrogenase operons hyb, hya and hyd are expressed under different environmental conditions and are subject to control by different regulatory proteins. Hydrogenase promoter-lacZ fusion plasmids were transferred into the wild-type strain or into arcA, fnr, iscR, narL and narP deletion mutants, or into a fnr/arcA double mutant. The hyb promoter had highest beta-galactosidase activity under growth conditions promoting anaerobic respiration (glycerol plus fumarate) and may be subject to glucose repression, since cells grown with glucose had about half the transcriptional activity of cells grown with mannose. Based on the phenotype of regulatory mutant strains, IscR represses hyb aerobically, and ArcA plays a role in both hyb and hyd regulation. The hyd promoter had about five times more activity in cells grown under aerobic conditions compared to anaerobic levels, and its activity tripled in an arcA mutant grown anaerobically. The hya promoter had the highest activity when cells were grown anaerobically with glucose, and the growth yield of the hya mutant was about 25 % lower than for wild-type cells grown fermentatively, suggesting that Hya may be utilized during fermentation. The hya promoter is repressed by nitrate and this repression was abolished when the NarL-binding site was mutated, or in a narL mutant background. FNR is involved in hyb and hya regulation, since beta-galactosidase activity decreased significantly in a fnr mutant. These findings suggest that the three hydrogenases are used under different conditions, likely enhancing the pathogen's capacity to survive in a variety of environments.