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.

Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among organisms that are exposed to oxic environments. The single SOD of Helicobacter pylori, encoded by the sodB gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously. We have isolated mutants with interruptions in the sodB gene and have characterized them with respect to their response to oxidative stress and ability to colonize the mouse stomach. The sodB mutants are devoid of SOD activity, based on activity staining in nondenaturing gels and quantitative assays of cell extracts. Though wild-type H. pylori is microaerophilic, the mutants are even more sensitive to O 2 for both growth and viability. While the wild-type strain is routinely grown at 12% O 2 , growth of the mutant strains is severely inhibited at above 5 to 6% O 2 . The effect of O 2 on viability was determined by subjecting nongrowing cells to atmospheric levels of O 2 and plating for survivors at 2-h time intervals. Wild-type cell viability dropped by about 1 order of magnitude after 6 h, while viability of the sodB mutant decreased by more than 6 orders of magnitude at the same time point. The mutants are also more sensitive to H 2 O 2 , and this sensitivity is exacerbated by increased O 2 concentrations. Since oxidative stress has been correlated with DNA damage, the frequency of spontaneous mutation to rifampin resistance was studied. The frequency of mutagenesis of an sodB mutant strain is about 15-fold greater than that of the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became H. pylori positive, while 15 out of 17 mice inoculated with the wild-type strain were shown to harbor the organism. Therefore, SOD is a virulence factor which affects the ability of this organism to colonize the mouse stomach and is important for the growth and survival of H. pylori under conditions of oxidative stress.

Campylobacter jejuni is the leading cause of human food-borne bacterial gastroenteritis. The C. jejuni genome sequence predicts a branched electron transport chain capable of utilizing multiple electron acceptors. Mutants were constructed by disrupting the coding regions of the respiratory enzymes nitrate reductase (napA::Cm), nitrite reductase (nrfA::Cm), dimethyl sulfoxide, and trimethylamine N-oxide reductase (termed Cj0264::Cm) and the two terminal oxidases, a cyanide-insensitive oxidase (cydA::Cm) and cbb3-type oxidase (ccoN::Cm). Each strain was characterized for the loss of the associated enzymatic function in vitro. The strains were then inoculated into 1-week-old chicks, and the cecal contents were assayed for the presence of C. jejuni 2 weeks postinoculation. cydA::Cm and Cj0264c::Cm strains colonized as well as the wild type; napA::Cm and nrfA::Cm strains colonized at levels significantly lower than the wild type. The ccoN::Cm strain was unable to colonize the chicken; no colonies were recovered at the end of the experiment. While there appears to be a role for anaerobic respiration in host colonization, oxygen is the most important respiratory acceptor for C. jejuni in the chicken cecum.