Differences in the Propensity of Different Antimicrobial Resistance Determinants to Be Disseminated via Transformation in Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are leading zoonotic foodborne pathogens, and the drugs of choice for human campylobacteriosis are macrolides (e.g., erythromycin) and fluoroquinolones. C. jejuni and C. coli are naturally competent for transformation via naked DNA uptake, but potential differences in transformation frequency (TF) for different antimicrobial resistance (AMR) markers remain poorly understood. We determined TFs for resistance to different antibiotics using as recipient a derivative of C. jejuni NCTC 11168 (strain SN:CM) with donor DNA from multidrug-resistant C. jejuni or C. coli. TF for nalidixic acid resistance ranked significantly highest (~1.4 × 10-3), followed by resistance to streptomycin and gentamicin. Tetracycline resistance via chromosomal tet(O) was less commonly transferred (~7.6 × 10-7), while transformation to erythromycin resistance was rare (≤4.7 × 10-8). We also determined TFs with the contemporary poultry-derived strains C. jejuni FSIS 11810577 and C. coli FSIS 1710488 as recipients. TFs to nalidixic acid and streptomycin resistance remained the highest (~7 × 10-4). However, TF for gentamicin resistance was remarkably low in certain recipient-donor combinations, while average TF for erythromycin resistance was noticeably higher (~3 × 10-6) than with SN:CM. Findings from this experimental model provide insights into factors that may impact transformation-mediated transfer of AMR leading to AMR dissemination in the agricultural ecosystem.

Interaction of Copper Toxicity and Oxidative Stress in Campylobacter jejuni.

Copper is both a required micronutrient and a source of toxicity in most organisms, including Campylobacter jejuni Two proteins expressed in C. jejuni (termed CopA and CueO) have been shown to be a copper transporter and multicopper oxidase, respectively. We have isolated strains with mutations in these genes, and here we report that they were more susceptible to both the addition of copper in the growth media and to induced oxidative stress. Both mutant strains were defective in colonization of an avian host, and copper in the feed exacerbated the colonization deficiency. Overexpression of a cytoplasmic peptide derived from the normally periplasmic copper-binding region of CueO also caused copper intolerance compared to nonexpressing strains or strains expressing the non-copper-binding versions of the peptide. Taken together, the results indicate that copper toxicity in C. jejuni is due to a failure to effectively sequester cytoplasmic copper, resulting in an increase in copper-mediated oxidative damage.IMPORTANCE Copper is a required micronutrient for most aerobic organisms, but it is universally toxic at elevated levels. These organisms use homeostatic mechanisms that allow for cells to acquire enough of the element to sustain metabolic requirements while ensuring that lethal levels cannot build up in the cell. Campylobacter jejuni is an important foodborne pathogen that typically makes its way into the food chain through contaminated poultry. C. jejuni has a metabolic requirement for copper and encodes a copper detoxification system. In the course of studying this system, we have learned that it is important for avian colonization. We have also gained insight into how copper exerts its toxic effects in C. jejuni by promoting oxidative stress.

Complete Genome Sequence of Campylobacter jejuni subsp. jejuni ATCC 35925.

Here, we report the complete genome sequence of Campylobacter jejuni ATCC 35925, an avian isolate from Sweden. The genome gives insight into the ATCC 35925 strain's remarkable ability to tolerate copper and its permissiveness to plasmid transformation.

Identification of a Campylobacter coli methyltransferase targeting adenines at GATC sites.

Campylobacter coli can infect humans and colonize multiple other animals, but its host-associated genes or adaptations are poorly understood. Adenine methylation at GATC sites, resulting in MboI resistance of genomic DNA, was earlier frequently detected among C. coli from swine but not among turkey-derived isolates. The underlying genetic basis has remained unknown. Comparative genome sequence analyses of C. coli 6461, a swine-derived strain with MboI-resistant DNA, revealed two chromosomal ORFs, 0059 and 0060, encoding a putative DNA methyltransferase and a conserved hypothetical protein, respectively, which were lacking from the genome of the turkey-derived C. coli strain 11601, which had MboI-susceptible DNA. To determine whether ORF0059 mediated MboI resistance and hence encoded a putative N6-adenine DNA methyltransferase, the gene was cloned immediately upstream of a chloramphenicol resistance cassette (cat) and a PCR fragment harboring ORF0059-cat was transformed into C. coli 11601. The transformants had MboI-resistant DNA, suggesting a direct role of this gene in methylation of adenines at GATC sites. In silico analyses suggested that the ORF0059-ORF0060 cassette was more frequent among C. coli from swine than certain other sources (e.g. cattle, humans). Potential impacts of ORF0059-mediated methylation on C. coli host preference and other adaptations remain to be elucidated.

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6.

The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound.

The impairment of methylmenaquinol:fumarate reductase affects hydrogen peroxide susceptibility and accumulation in Campylobacter jejuni.

The methylmenaquinol:fumarate reductase (Mfr) of Campylobacter jejuni is a periplasmic respiratory (redox) protein that contributes to the metabolism of fumarate and displays homology to succinate dehydrogenase (Sdh). Since chemically oxidized redox-enzymes, including fumarate reductase and Sdh, contribute to the generation of oxidative stress in Escherichia coli, we assessed the role of Mfr in C. jejuni after exposure to hydrogen peroxide (H2 O2 ). Our results show that a Mfr mutant (∆mfrA) strain was less susceptible to H2 O2 as compared to the wildtype (WT). Furthermore, the H2 O2 concentration in the ∆mfrA cultures was significantly higher than that of WT after exposure to the oxidant. In the presence of H2 O2 , catalase (KatA) activity and katA expression were significantly lower in the ∆mfrA strain as compared to the WT. Exposure to H2 O2 resulted in a significant decrease in total intracellular iron in the ∆mfrA strain as compared to WT, while the addition of iron to the growth medium mitigated H2 O2 susceptibility and accumulation in the mutant. The ∆mfrA strain was significantly more persistent in RAW macrophages as compared to the WT. Scanning electron microscopy showed that infection with the ∆mfrA strain caused prolonged changes to the macrophages' morphology, mainly resulting in spherical-shaped cells replete with budding structures and craters. Collectively, our results suggest a role for Mfr in maintaining iron homeostasis in H2 O2 stressed C. jejuni, probably via affecting the concentrations of intracellular iron.

Respiratory proteins contribute differentially to Campylobacter jejuni's survival and in vitro interaction with hosts' intestinal cells.

BACKGROUND: The genetic features that facilitate Campylobacter jejuni's adaptation to a wide range of environments are not completely defined. However, whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejuni's adaptation. Therefore, our objectives were to characterize the contributions of selected RPs to C. jejuni's i- key survival phenotypes under different temperature (37°C vs. 42°C) and oxygen (microaerobic, ambient, and oxygen-limited/anaerobic) conditions and ii- its interactions with intestinal epithelial cells from disparate hosts (human vs. chickens). RESULTS: C. jejuni mutant strains with individual deletions that targeted five RPs; nitrate reductase (ΔnapA), nitrite reductase (ΔnrfA), formate dehydrogenase (ΔfdhA), hydrogenase (ΔhydB), and methylmenaquinol:fumarate reductase (ΔmfrA) were used in this study. We show that only the ΔfdhA exhibited a decrease in motility; however, incubation at 42°C significantly reduced the deficiency in the ΔfdhA's motility as compared to 37°C. Under all tested conditions, the ΔmfrA showed a decreased susceptibility to hydrogen peroxide (H(2)O(2)), while the ΔnapA and the ΔfdhA showed significantly increased susceptibility to the oxidant as compared to the wildtype. Further, the susceptibility of the ΔnapA to H(2)O(2) was significantly more pronounced at 37°C. The biofilm formation capability of individual RP mutants varied as compared to the wildtype. However, the impact of the deletion of certain RPs affected biofilm formation in a manner that was dependent on temperature and/or oxygen concentration. For example, the ΔmfrA displayed significantly deficient and increased biofilm formation under microaerobic conditions at 37°C and 42°C, respectively. However, under anaerobic conditions, the ΔmfrA was only significantly impaired in biofilm formation at 42°C. Additionally, the RPs mutants showed differential ability for infecting and surviving in human intestinal cell lines (INT-407) and primary chicken intestinal epithelial cells, respectively. Notably, the ΔfdhA and the ΔhydB were deficient in interacting with both cell types, while the ΔmfrA displayed impairments only in adherence to and invasion of INT-407. Scanning electron microscopy showed that the ΔhydB and the ΔfdhA exhibited filamentous and bulging (almost spherical) cell shapes, respectively, which might be indicative of defects in cell division. CONCLUSIONS: We conclude that the RPs contribute to C. jejuni's motility, H(2)O(2) resistance, biofilm formation, and in vitro interactions with hosts' intestinal cells. Further, the impact of certain RPs varied in response to incubation temperature and/or oxygen concentration. Therefore, RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogen's remarkable potential for adaptation.

Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies.

This research focused on optimizing the upstream process time for production of polyhydroxybutyrate (PHB) from sucrose by two-stage batch and fed-batch fermentation with Alcaligenes latus ATCC 29714. The study included selection of strain, two-stage batch fermentations with different time points for switching to nitrogen limited media (14, 16 or 18 h) and fed-batch fermentations with varied time points (similar to two stage) for introducing nitrogen limited media. The optimal strain to produce PHB using sucrose as carbon source was A. latus ATCC 29714 with maximum-specific growth rate of 0.38 ± 0.01 h(-1) and doubling time of 1.80 ± 0.05 h. Inducing nitrogen limitation at 16 h and ending second stage at 26 h gave optimal performance for PHB production, resulting in a PHB content of 46.7 ± 12.2 % (g PHB per g dry cell weight) at the end of fermentation. This was significantly higher (P ≤ 0.05) (approximately 7 %) than the corresponding fed batch run in which nitrogen limitation was initiated at 16 h.

Barriers to Horizontal Gene Transfer in Campylobacter jejuni.

Campylobacter jejuni is among the most frequent agent of foodborne gastroenteritis in the world, but its physiology and pathogenesis is less well understood than other bacterial enteric pathogens. This is due in part to the incompatibility of the molecular tools that have enabled advances in the characterization of other bacterial species. Most notably, the dearth of plasmid-based complementation, reporter assays, and plasmid-based unmarked mutagenesis procedures in many of the type strains has hindered research progress. The techniques themselves are not inadequate in Campylobacter species, but rather the barrier to genetic transfer of these genetic constructs from non-Campylobacter cloning stains such as Escherichia coli. Here, we review the modes of genetic transfer in C. jejuni and review the current state of research into the mechanism of each. Also reviewed are two systems (CRISPR-Cas and restriction modification) that are common to many strains of C. jejuni and are at least partly responsible for these barriers.

Molybdenum and tungsten in Campylobacter jejuni: their physiological role and identification of separate transporters regulated by a single ModE-like protein.

Campylobacter jejuni is an important human pathogen that causes millions of cases of food-borne enteritis each year. The C. jejuni respiratory chain is highly branched and contains at least four enzymes predicted to contain a metal binding pterin (MPT), with the metal being either molybdenum or tungsten. Also predicted are two separate transport systems, one for molybdenum encoded by modABC and a second for tungsten encoded by tupABC. Both transport systems were mutated and the activities of the four predicted MPT-containing enzymes were assayed in the presence of molybdenum and tungsten in wild-type and mod and tup backgrounds. Results indicate that mod is primarily a molybdenum transporter that can also transport tungsten, while tup is a tungsten-specific transporter. The MPT containing enzymes nitrate reductase, sulphite oxidase, and SN oxide reductase are strict molybdoenzymes while formate dehydrogenase prefers tungsten. A ModE-like protein regulates both transporters, repressing mod in the presence of both molybdenum and tungsten and tup only in the presence of tungsten. Like other ModE proteins, the C. jejuni ModE binds DNA through a helix-turn-helix DNA binding domain, but unlike other members of the ModE family it does not have a metal binding domain.

The dual-functioning fumarate reductase is the sole succinate:quinone reductase in Campylobacter jejuni and is required for full host colonization.

Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA::cat(+) strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA::cat(+) strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA::cat(+) strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA::cat(+) strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA::cat(+) strain colonized chickens at significantly lower levels than the wild type, while the sdhA::cat(+) strain colonized chickens at wild-type levels.

The role of respiratory donor enzymes in Campylobacter jejuni host colonization and physiology.

The human pathogen Campylobacter jejuni utilizes oxidative phosphorylation to meet all of its energy demands. The genome sequence of this bacterium encodes a number of respiratory enzymes in a branched electron transport chain that predicts the utilization of a number of electron transport chain donor and acceptor molecules. Three of these electron donor enzymes: hydrogenase, formate dehydrogenase, and 2-oxoglutarate:acceptor oxidoreductase (OOR), oxidize hydrogen, formate and alpha-ketoglutarate as electron donors, respectively. Mutations were created in these donor enzymes to isolate mutants in hydrogenase (HydB::CM), formate dehydrogenase (Fdh::CM), and OOR (OorB::CM), as well as a strain with insertions in both hydrogenase and formate dehydrogenase (Hyd::Fdh). These mutants are deficient in their respective enzyme activities and do not reduce the components of the electron transport chain when provided with their respective substrates. The presence of either hydrogen or formate in the media stimulated the growth of wild type (WT) C. jejuni (but not the associated mutant strains) and at least one of these alternative substrates is required for growth of the OOR mutant strain OorB::CM. Finally, the importance of hydrogenase, formate dehydrogenase and OOR as well as the complex I of C. jejuni are elucidated by chicken colonization assays, where the double mutant Hyd::Fdh, OorB::CM and nuo mutants are severely impaired in host colonization.

Role of Campylobacter jejuni respiratory oxidases and reductases in host colonization.

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.

The Campylobacter jejuni NADH:ubiquinone oxidoreductase (complex I) utilizes flavodoxin rather than NADH.

Campylobacter jejuni encodes 12 of the 14 subunits that make up the respiratory enzyme NADH:ubiquinone oxidoreductase (also called complex I). The two nuo genes not present in C. jejuni encode the NADH dehydrogenase, and in their place in the operon are the novel genes designated Cj1575c and Cj1574c. A series of mutants was generated in which each of the 12 nuo genes (homologues to known complex I subunits) was disrupted or deleted. Each of the nuo mutants will not grow in amino acid-based medium unless supplemented with an alternative respiratory substrate such as formate. Unlike the nuo genes, Cj1574c is an essential gene and could not be disrupted unless an intact copy of the gene was provided at an unrelated site on the chromosome. A nuo deletion mutant can efficiently respire formate but is deficient in alpha-ketoglutarate respiratory activity compared to the wild type. In C. jejuni, alpha-ketoglutarate respiration is mediated by the enzyme 2-oxoglutarate:acceptor oxidoreductase; mutagenesis of this enzyme abolishes alpha-ketoglutarate-dependent O2 uptake and fails to reduce the electron transport chain. The electron acceptor for 2-oxoglutarate:acceptor oxidoreductase was determined to be flavodoxin, which was also determined to be an essential protein in C. jejuni. A model is presented in which CJ1574 mediates electron flow into the respiratory transport chain from reduced flavodoxin and through complex I.

Role of a bacterial organic hydroperoxide detoxification system in preventing catalase inactivation.

In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H. pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase. H. pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H2O2. However, it inactivated is by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation.

Helicobacter pylori FlgR is an enhancer-independent activator of sigma54-RNA polymerase holoenzyme.

Helicobacter pylori FlgR activates transcription with sigma54-RNA polymerase holoenzyme (sigma54-holoenzyme) from at least five flagellar operons. Activators of sigma54-holoenzyme generally bind enhancer sequences located >70 bp upstream of the promoter and contact sigma54-holoenzyme bound at the promoter through DNA looping to activate transcription. H. pylori FlgR lacks the carboxy-terminal DNA-binding domain present in most sigma54-dependent activators. As little as 42 bp of DNA upstream of the flaB promoter and 26 bp of DNA sequence downstream of the transcriptional start site were sufficient for efficient FlgR-mediated expression from a flaB'-'xylE reporter gene in H. pylori, indicating that FlgR does not use an enhancer to activate transcription. Other examples of sigma54-dependent activators that lack a DNA-binding domain include Chlamydia trachomatis CtcC and activators from the other Chlamydia spp. whose genomes have been sequenced. FlgR from Helicobacter hepaticus and Campylobacter jejuni, which are closely related to H. pylori, appear to have carboxy-terminal DNA-binding domains, suggesting that the loss of the DNA-binding domain from H. pylori FlgR occurred after the divergence of these bacterial species. Removal of the amino-terminal regulatory domain of FlgR resulted in a constitutively active form of the protein that activated transcription from sigma54-dependent genes in Escherichia coli. The truncated FlgR protein also activated transcription with E. coli sigma54-holoenzyme in an in vitro transcription assay.

Characterization of Helicobacter pylori nickel metabolism accessory proteins needed for maturation of both urease and hydrogenase.

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 microM, 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. A chromosomal site-directed mutant of hypA (in the codon for His2) lacked hydrogenase activity and possessed only 2% of the wild-type urease activity. Purified HypB had a GTPase activity of 5 nmol of GTP hydrolyzed per nmol of HypB per min. Site-directed mutagenesis within the lysine residue in the conserved GTP-binding motif of HypB (Lys59) nearly abolished the GTPase activity of the mutant protein (K59A). In native solution, both HypA and HypB exist as homodimers with molecular masses of 25.8 and 52.4 kDa, respectively. However, a 1:1 molar mixture of HypA plus HypB gave rise to a 43.6-kDa species composed of both proteins. A 43-kDa heterodimeric HypA-HypB complex was also detected by cross-linking. The cross-linked adduct was still observed in the presence of 0.5 mM GTP or 1 microM nickel or when the mutant version of HypA (altered in His2) and HypB (altered in Lys59) were tested. Individually, HypA and HypB formed homodimeric cross-linked adducts. An interaction between HypA and the Hp0868 protein (encoded by the gene downstream of hypA) could not be detected via cross-linking, although such an interaction was predicted by yeast two-hybrid studies. In addition, the phenotype of an insertional mutation within the Hp0868 gene indicated that its presence is not critical for either the urease or the hydrogenase activity.

Association of Helicobacter pylori antioxidant activities with host colonization proficiency.

To assess the importance of two separate antioxidant activities in Helicobacter pylori, we tested the abilities of strains with mutations in either tpx (encoding thiolperoxidase) or ahpC (encoding alkyl hydroperoxide reductase [AhpC]) to colonize the stomachs of mice. The tpx strain was clearly more sensitive than the parent strain to both oxygen and cumene hydroperoxide. The strain colonized only 5% of the inoculated mice. Two different classes of oxygen-sensitive ahpC mutants in the type strain (ATCC 43504) were recently described (A. A. Olczak, J. W. Olson, and R. J. Maier, J. Bacteriol. 184:3186-3193, 2002). The same two classes of mutants were recovered upon ahpC mutagenesis of the mouse-adapted strain, SS1. Neither of these mutants was able to colonize mouse stomachs, whereas 78% of the mice inoculated with the parent strain became H. pylori positive.

Molecular hydrogen as an energy source for Helicobacter pylori.

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.

Oxidative-stress resistance mutants of Helicobacter pylori.

Within a large family of peroxidases, one member that catalyzes the reduction of organic peroxides to alcohols is known as alkyl hydroperoxide reductase, or AhpC. Gene disruption mutations in the gene encoding AhpC of Helicobacter pylori (ahpC) were generated by screening transformants under low-oxygen conditions. Two classes of mutants were obtained. Both types lack AhpC protein, but the major class (type I) isolated was found to synthesize increased levels (five times more than the wild type) of another proposed antioxidant protein, an iron-binding, neutrophil-activating protein (NapA). The other class of mutants, the minor class (type II), produced wild-type levels of NapA. The two types of AhpC mutants differed in their frequencies of spontaneous mutation to rifampin resistance and in their sensitivities to oxidative-stress chemicals, with the type I mutants exhibiting less sensitivity to organic hydroperoxides as well as having a lower mutation frequency. The napA promoter regions of the two types of AhpC mutants were identical, and primer extension analysis revealed their transcription start site to be the same as for the wild type. Gene disruption mutations were obtained in napA alone, and a double mutant strain (ahpC napA) was also created. All four of the oxidative-stress resistance mutants could be distinguished from the wild type in oxygen sensitivity or in some other oxidative-stress resistance phenotype (i.e., in sensitivity to stress-related chemicals and spontaneous mutation frequency). For example, growth of the NapA mutant was more sensitive to oxygen than that of the wild-type strain and both of the AhpC-type mutants were highly sensitive to paraquat and to cumene hydroperoxide. Of the four types of mutants, the double mutant was the most sensitive to growth inhibition by oxygen and by organic peroxides and it had the highest spontaneous mutation frequency. Notably, two-dimensional gel electrophoresis combined with protein sequence analysis identified another possible oxidative-stress resistance protein (HP0630) that was up-regulated in the double mutant. However, the transcription start site of the HP0630 gene was the same for the double mutant as for the wild type. It appears that H. pylori can readily modulate the expression of other resistance factors as a compensatory response to loss of a major oxidative-stress resistance component.