Previous studies demonstrated that two accessory proteins, HypA and HypB, play a role in nickel-dependent maturation of both hydrogenase and urease in Helicobacter pylori. Here, the two proteins were purified and characterized. HypA bound two Ni 2؉ ions per dimer with positive cooperativity (Hill coefficient, approximately 2.0). The dissociation constants K 1 and K 2 for Ni 2؉ were 58 and 1.3 M, respectively. Studies on purified site-directed mutant proteins in each of the five histidine residues within HypA, revealed that only one histidine residue (His2) is vital for nickel binding. Nuclear magnetic resonance analysis showed that this purified mutant version (H2A) was similar in structure to that of the wild-type HypA protein. Helicobacter pylori is a spiral, gram negative, microaerophilic bacterium that has been shown to be the etiological agent of gastritis and peptic ulcer disease (3, 4). It expresses two distinct nickel-containing enzymes, both of which are important for its virulence. These are a membrane bound [Ni-Fe] hydrogenuptake hydrogenase, which permits respiratory-based energy production for the bacteria in the mucosa (19,30), and an enzyme critical for early steps in colonization, urease (23,25). Synthesis of metal-containing enzymes often requires the participation of accessory proteins, and the maturation of hydrogenase and urease are no exceptions. Indeed, the complete genome sequence of H. pylori reveals the presence of a full complement of urease (ureIEFGH) and hydrogenase (hypAB CDEF) accessory genes (36). A number of studies exist that address these accessory genes in other bacteria and their role in the Ni-dependent maturation of urease and hydrogenase apoenzymes (7,9,13,14,15,20,22,27,28,34,39). Although the specific role of each of these proteins has not been clarified to date, the existing data indicate that an efficient Ni enzyme maturation process involves a concerted effort of the accessory proteins, likely involving sequential Ni-metabolizing steps.By studying gene-directed mutants in H. pylori, it was found that two of the hydrogenase accessory genes, hypA and hypB, are required for both hydrogenase and urease activities (29).That hypA plays a role in H. pylori urease maturation was confirmed by another research group (F. Wöhl et al., Int. J. Med. Microbiol., vol. 291, suppl. 31, abstr. K-22, 2001), also by use of a gene-directed mutation approach. The lack of urease activity could not be attributed to the lack of hydrogenase activities in these two (hypA and hypB) mutants, since a hydrogenase structural gene mutant, hydB, and other hyp accessory mutants, hypD, hypE, and hypF, showed wild-type levels of urease activity although they were all deficient for hydrogenase activity. Also, the expression levels of the urease apoenzyme in the hypA and hypB mutants were comparable to those of the wild type; however, the nickel content of the urease from the hypA mutant was fourfold less, and that from the hypB mutant was fivefold less, than that of the wild type (29). Therefore, it was pro...

The gastric pathogen Helicobacter pylori is known to be able to use molecular hydrogen as a respiratory substrate when grown in the laboratory. We found that hydrogen is available in the gastric mucosa of mice and that its use greatly increased the stomach colonization by H. pylori. Hydrogenase activity in H. pylori is constitutive but increased fivefold upon incubation with hydrogen. Hydrogen concentrations measured in the stomachs of live mice were found to be 10 to 50 times as high as the H. pylori affinity for hydrogen. A hydrogenase mutant strain is much less efficient in its colonization of mice. Therefore, hydrogen present in animals as a consequence of normal colonic flora is an energy-yielding substrate that can facilitate the maintenance of a pathogenic bacterium.

SummaryThe nickel-containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)-and 200 (HypB:kan)-fold lower than for the wild-type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H 2 -oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease-containing fractions of the mutant strains contained about four (HypA:kan)-and five (HypB:kan)-fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.

SummaryThe gastric pathogen Helicobacter pylori induces a strong inflammatory host response, yet the bacterium maintains long-term persistence in the host. H. pylori combats oxidative stress via a battery of diverse activities, some of which are unique or newly described. In addition to using the well-studied bacterial oxidative stress resistance enzymes superoxide dismutase and catalase, H. pylori depends on a family of peroxiredoxins (alkylhydroperoxide reductase, bacterioferritin co-migratory protein and a thiolperoxidase) that function to detoxify organic peroxides. Newly described antioxidant proteins include a soluble NADPH quinone reductase (MdaB) and an iron sequestering protein (NapA) that has dual roles -host inflammation stimulation and minimizing reactive oxygen species production within H. pylori. An H. pylori arginase attenuates host inflammation, a thioredoxin required as a reductant for many oxidative stress enzymes is also a chaperon, and some novel properties of KatA and AhpC were discovered. To repair oxidative DNA damage, H. pylori uses an endonuclease (Nth), DNA recombination pathways and a newly described type of bacterial MutS2 that specifically recognizes 8-oxoguanine. A methionine sulphoxide reductase (Msr) plays a role in reducing the overall oxidized protein content of the cell, although it specifically targets oxidized Met residues. H. pylori possess few stress regulator proteins, but the key roles of a ferric uptake regulator (Fur) and a post-transcriptional regulator CsrA in antioxidant protein expression are described. The roles of all of these antioxidant systems have been addressed by a targeted mutant analysis approach and almost all are shown to be important in host colonization. The described antioxidant systems in H. pylori are expected to be relevant to many bacterial-associated diseases, as genes for most of the enzymes carrying out the newly described roles are present in a number of pathogenic bacteria.

Based on available annotated gene sequence information, the enteric pathogen salmonella, like other enteric bacteria, contains three putative membrane-associated H 2 -using hydrogenase enzymes. These enzymes split molecular H 2 , releasing low-potential electrons that are used to reduce quinone or heme-containing components of the respiratory chain. Here we show that each of the three distinct membrane-associated hydrogenases of Salmonella enterica serovar Typhimurium is coupled to a respiratory pathway that uses oxygen as the terminal electron acceptor. Cells grown in a blood-based medium expressed four times the amount of hydrogenase (H 2 oxidation) activity that cells grown on Luria Bertani medium did. Cells suspended in phosphatebuffered saline consumed 2 mol of H 2 per mol of O 2 used in the H 2 -O 2 respiratory pathway, and the activity was inhibited by the respiration inhibitor cyanide. Molecular hydrogen levels averaging over 40 M were measured in organs (i.e., livers and spleens) of live mice, and levels within the intestinal tract (the presumed origin of the gas) were four times greater than this. The half-saturation affinity of S. enterica serovar Typhimurium for H 2 is only 2.1 M, so it is expected that H 2 -utilizing hydrogenase enzymes are saturated with the reducing substrate in vivo. All three hydrogenase enzymes contribute to the virulence of the bacterium in a typhoid fever-mouse model, based on results from strains with mutations in each of the three hydrogenase genes. The introduced mutations are nonpolar, and growth of the mutant strains was like that of the parent strain. The combined removal of all three hydrogenases resulted in a strain that is avirulent and (in contrast to the parent strain) one that is unable to invade liver or spleen tissue. The introduction of one of the hydrogenase genes into the triple mutant strain on a low-copy-number plasmid resulted in a strain that was able to both oxidize H 2 and cause morbidity in mice within 11 days of inoculation; therefore, the avirulent phenotype of the triple mutant is not due to an unknown spurious mutation. We conclude that H 2 utilization in a respiratory fashion is required for energy production to permit salmonella growth and subsequent virulence during infection.