INTRODUCTION

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A new era in genome science started with the publication of the complete genome of Haemophilus influenzae (82). Eleven other prokaryotic genomes, including two strains of the gastric pathogen Helicobacter pylori (3, 280), and the eukaryotic genome of Saccharomyces cerevisiae (102) have been sequenced, allowing for genome comparisons. Microbial genomics is providing increasing and useful information on fundamental and applied microbiology, taxonomy, molecular biology, microbial ecology, and medical, veterinary, and agricultural microbiology.

Complete genome sequence information has extended our knowledge of bacterial genomic systems, showing that the organization of orthologous genes on the chromosome is not conserved during evolution and that the genome itself is quite complex and flexible. The advent of these sequences has afforded novel insights into the molecular mechanisms of genetic change and adaptive mutations characteristic of bacteria. The construction of putative minimal gene sets sufficient for sustaining cellular life has become a more realistic possibility through comparison of bacterial genomes.

Genome sequence data also are utilized to obtain a physiological integration of phenotypic characteristics of an organism based on functional identification of genes and network reconstruction of pathways and functional units. A current and widely used method for functional analysis is based on searching for similarities in databases (34). Mapping out metabolic and regulatory networks, signal transduction, etc., follows the functional identification of genes. In practice, these are iterative processes: when a pathway is reconstructed incompletely from the predicted genes, the functional identification of genes has to be reviewed, and either genes are reassigned to the missing steps or a new set of genes encoding alternative proteins that can fill the missing functions is found. In bacterial genomes in which the operon structure is relatively well conserved, the clustering of genes can act as a good indicator for reconstruction of functional units, e.g. the aerobic respiratory chain. Through physiological integration, the results of functional identification of genes and network reconstruction are assembled into coherent wholes describing overall phenotypic features of the organism, e.g., aspects of its physiology, adaptation to an ecological niche, and pathogenicity. Physiological integration also serves to explain characteristics of the phenotype not yet fully understood and predicts properties which had not been observed experimentally.

The problems encountered in functional identification, network reconstruction, and physiological integration of organisms are being solved as a result of the growing number of complete genomes sequenced and the efforts to develop new databases and computational algorithms and techniques. Nevertheless, at present genome data on their own are not sufficient even for complete functional identification of genes. Thus, knowledge of biochemical pathways, mechanisms of gene expression, genetics, etc., must be used to interpret the results of sequence similarity searches performed on complete genomes and to place all this information into a coherent set that provides insight into the physiology of a microorganism. It is not surprising that the best-annotated genome is that of Escherichia coli, which is in fact the best-studied organism.

H. pylori is a gram-negative bacterium that is associated with gastric inflammation (290) and peptic ulcer disease (179) and is a risk factor for gastric cancer (83) and non-Hodgkin's lymphomas of the stomach (230). Almost half of the world's population harbors an H. pylori infection, with the nature and severity of the disease depending on both host characteristics and environmental factors. There is a considerable body of evidence indicating that the bacterial genotype is also an important factor (201, 203) in the nature of the disease. Sequencing of the genome of H. pylori 26695 has provided insights into its biology, and the recently published comparison of this genome to that of strain J99 has served to deepen our knowledge of the diversity of this bacterium.

Unless explicitly mentioned, we discuss the published genome of strain 26695, whose genes are numbered and preceded by "HP." After a brief description of the general features of the genome, the links between experimental data and the information provided by sequence annotations are reviewed within the framework of functional identification, network reconstruction, and physiological integration. The review is divided into five areas: metabolism; replication, transcription, and translation; regulation of gene expression; diversity; and colonization factors.

The genomes of H. pylori 26695 and J99 were sequenced from circular chromosomes containing 1,667,867 and 1,643,831 bp, respectively. These sizes are similar to that of Haemophilus influenzae and about one-third that of E. coli (30, 82).

The average G+C content is 39%, but five regions in the genome of strain 26695 (nine in strain J99) have a distinct G+C composition (3, 280). Region 2 (35% G+C) of strain 26695 is the cag pathogenicity island associated with production of the CagA antigen and upregulation of interleukin 8 (42). The other four regions have not been yet characterized experimentally. Regions 1 and 3 (33% G+C) contain copies of the insertion sequence IS605, 5S rRNA genes, and a 521-bp repeat. In addition, region 1 contains the virB4 gene, which encodes the protein involved in the transfer of the T-DNA in Agrobacterium tumefaciens and in the secretion of the Bordetella pertussis toxin (294). Region 4 (43% G+C) contains fused rpoB and rpoC genes encoding the  and ' subunits of the RNA polymerase. The fusA gene, which codes for the translation elongation factor EF-G, is also associated with this region. Finally, region 5 (33% G+C) contains two restriction/modification systems.

Eight repeat families, with a sequence identity greater than 97% have been found in the H. pylori DNA. They are localized either in intergenic regions (one family) or within coding sequences, where they may represent gene duplications.

Tomb et al. (280) identified 1,590 open reading frames (ORFs), which represent 91% of the H. pylori chromosome of strain 26695, and Alm et al. (3) identified 1,495 ORFs, which represent 90.8% of the chromosome of strain J99. No genome-wide strand bias was observed. The noncoding regions of strain 26695 (9%) are divided into three classes. Intergenic sequences represent 6% of the noncoding regions, while noncoding repeats account for 2.3% and stable RNA accounts for 0.7%. Among the 1,590 ORFs, 1,091 were found to have counterparts in other organisms, allowing putative biological roles to be assigned to them, although not all have orthologues of known function. The 499 ORFs which exhibited no database matches may be considered at this time to be specific to H. pylori. In the reannotation of this genome carried out by Alm et al. (3), the number of ORFs in strain 26695 is 1,552, of which 1,185 have orthologues found in other species, 367 are H. pylori specific, and 69 are specific to strain 26695. The proportion of orphan genes in H. pylori is similar to that found in other bacteria sequenced to date. However, it ought to be kept in mind that orthologous genes of some of these ORFs will probably be identified when further whole genomes are completely sequenced.

A better understanding of the biochemistry of H. pylori is of fundamental interest to microbiology and also could help in developing new anti-H. pylori therapies. In this section, the principal metabolic pathways of the bacterium are reviewed by comparing the experimental data with those derived from the whole genome sequence (280).

Soon after its discovery, H. pylori was classified as a Campylobacter species and, like other members of this genus, was reported to be unable to catabolize carbohydrates. Several studies of the physiology of the bacterium (39, 43, 181, 183, 185, 187, 188) have provided evidence that it can metabolize glucose by both oxidative and fermentative pathways, although it is an obligate microaerophile. Moreover, glucose appears to be the only carbohydrate utilized by the bacterium (188). More recently, the whole-genome analysis of H. pylori has supported these findings (280).

Glucose is imported into the cells by a permease which is specific for D-glucose and galactose. This transporter is sodium dependent and is unaffected by inhibitors known to affect other bacterial glucose permeases (39, 181). Analysis of the genome established the presence of the GluP glucose/galactose transporter, and no other saccharide permease has been identified (280). As suggested by Mendz et al. (188), intracellular phosphorylation of glucose is performed by a glucokinase rather than a hexokinase, and there is no evidence for the glucose phosphotransferase system involving the phosphorylation of an E-III enzyme. In agreement with these data, the analysis of the H. pylori genome shows its capacity to code for a glucokinase. The HP1103 gene has 59.5% similarity to the glk gene of E. coli, and genes encoding either a phosphotransferase system or a less specific hexokinase were not found by Tomb et al. (280). This feature could explain the limited range of carbohydrates used by H. pylori. Glucose utilization shows biphasic characteristics, with a slow initial period followed by faster catabolism. The rates of decline of glucose levels in both phases depend on the growth conditions of the bacteria, suggesting that this metabolite is not a preferred energy substrate but can be used when other sources of energy have been exhausted (185, 188). It would be of interest to attempt to relate the characteristics of glucose uptake and catabolism to the properties of the specific proteins encoded in the chromosome.

Mendz and Hazell (183) demonstrated the presence of enzymatic activities which are part of the oxidative and nonoxidative steps of the pentose-phosphate pathway in H. pylori. This pathway is an efficient mechanism to provide NADPH and NADH for reductive biosynthesis and C₅ phosphorylated carbohydrates essential for nucleotide synthesis (Fig. 1). The genes HP1386, HP1102, HP1101, HP1495, HP1088, and HP0354, orthologous to rpe, devB, g6pD, tal, tktA, and tktB, respectively, encoding enzymes of the pentose phosphate pathway, were identified in the genome, but no sequence similarity to the 6-phosphogluconate dehydrogenase gene, a key enzyme of the pathway, was found in the H. pylori DNA. This suggests the existence of a protein with a similar function to and different characteristics from other 6-phosphogluconate dehydrogenases.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Glycolysis, gluconeogenesis, pentose phosphate, and Entner-Doudoroff pathways. Glycolysis: glk, glucokinase; pgi, phosphoglucose isomerase; pfk, phosphofructokinase; fda, fructose-1,6-bisphosphate aldolase; tpi, triose-phosphate isomerase; gap, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphoglycerate kinase; pgm, phosphoglycerate mutase; eno, enolase; pyk, pyruvate kinase. Gluconeogenesis: the same enzymes as in glycolysis but with unidirectional steps, i.e., ppsA, phosphoenol pyruvate synthase; fbp, fructose-1,6 bisphosphatase; and g6p, glucose-6 phosphatase. Pentose phosphate: g6pD (devB), glucose-6-phosphate dehydrogenase; lactonase; gnd, 6-phosphogluconate dehydrogenase; rpe, D-ribulose-5-phosphate 3 epimerase; tal, transaldolase; tkt, transketolase. Entner-Doudoroff: edd, 6-phosphosgluconate dehydratase; eda, 2-keto-3-deoxy-6-phosphogluconate aldolase. Asterisks denote enzymes for which no gene was identified in the H. pylori sequence. The circle denotes an enzyme whose enzymatic activity has not been observed but whose corresponding gene was identified.

Alternatively, glucose-6-phosphate can be utilized by the Entner-Doudoroff pathway, regarded as an alternative to glycolysis (43, 187). The specific steps of this pathway consist of two reactions: a dehydratase-catalyzed formation of 2-keto-3-deoxygluconate-6-phosphate from gluconate-6-phosphate and an aldolase-catalyzed production of pyruvate and glyceraldehyde-3-phosphate (Fig. 1). These activities were found in H. pylori lysates (187), and ORFs HP1099 and HP1100, with homologies to the 2-keto-3-deoxy-6-phosphogluconate aldolase (eda) gene and the 6-phosphogluconate dehydratase (edd) gene from E. coli, respectively, were identified by Tomb et al. (280). The Entner-Doudoroff pathway is inducible in E. coli and is rarely employed by wild-type strains (85); however, it appears to be constitutive in H. pylori. Although its potential energy yield is less than that of glycolysis, it offers the possibility of metabolizing aldonic acids.

In 1996, Hoffman et al. (121) reported glycolytic activity and gluconeogenesis in H. pylori. The two pathways have seven common reversible reactions and are distinguished by three opposed irreversible steps (Fig. 1). There has been controversy about the existence of glycolysis since Mendz et al. (187) and Chalk et al. (43) were not able to detect some of the enzyme activities of this pathway. In particular, phosphoglycerate mutase activity has not been observed, although a gene (HP0974) coding for a protein with 44.6% similarity to the pgm gene product has been identified. Analysis of the H. pylori genome suggests that other genes coding for the enzymes of the glycolytic pathway are present, with the important exceptions of phosphofructokinase, which affects the irreversible phosphorylation of fructose-6-phosphate to fructose-1,6-biphosphate in glycolysis and of pyruvate kinase. Tomb et al. (280) identified a gene (HP1385) coding for a putative fructose-1,6-bisphosphatase which catalyzes the opposed irreversible reaction in gluconeogenesis and also found a gene (HP0121) encoding phosphoenolpyruvate synthase, an enzyme complex responsible for another of the irreversible reactions in gluconeogenesis. However, a gene coding for glucose-6-phosphatase, the enzyme involved in the third irreversible reaction of gluconeogenesis, has not been identified, and only a gene (HP1103) coding for glucokinase, which catalyzes the opposed reaction of glycolysis, was identified. Thus, in the case of glycolysis and gluconeogenesis, deductions from the H. pylori genome analysis have been useful in confirming some experimental data and at the same time have shown the need for further verification of the presence of these pathways.

Pyruvate is an end product of both the glycolytic and Entner-Doudoroff pathways. The metabolic fate of pyruvate in H. pylori was investigated by several groups. In one study, pyruvate was metabolized to lactate, ethanol, and acetate under anaerobic conditions whereas the major product of pyruvate under aerobic conditions was acetate (43). In another study (190), cells incubated with pyruvate under microaerobic conditions yielded lactate, acetate, formate, succinate, and alanine. The formation of succinate suggests the incorporation of pyruvate into the Krebs cycle, and the presence of alanine supports the view that pyruvate could play an important role in biosynthetic processes. The formation of lactate, ethanol, and acetate suggests the use of pyruvate in fermentative metabolism. In agreement with this observation, Tomb et al. (280) found ORFs HP1222 and HP0357 with similarities to the H. influenzae genes encoding D-lactate dehydrogenase, necessary to convert pyruvate to lactate, and short-chain alcohol dehydrogenase, respectively. The corresponding enzymes catalyze reactions occurring in conjunction with the oxidation of NADH. Formation of acetate from pyruvate requires three steps: the oxidative decarboxylation of pyruvate to produce acetyl coenzyme A (acetyl-CoA) and formate, the formation of acetyl-phosphate, and its dephosphorylation to acetate. H. pylori ORFs corresponding to genes coding for enzymes responsible for the last two steps in E. coli were identified by using sequence similarities: HP0904 to the phosphate acetyltransferase pta gene, and HP0903 to the acetate kinase ackA gene.

To enter the Krebs cycle, as well as to form acetate, pyruvate must be converted to acetyl-CoA. Hughes et al. (126) demonstrated that oxidative decarboxylation of pyruvate is carried out in H. pylori by a pyruvate:acceptor oxidoreductase (POR), instead of the aerobic pyruvate dehydrogenase (AceEF) or the strictly anaerobic pyruvate-formate lyase (Pfl) associated with mixed-acid fermentation. Flavodoxin is believed to be the in vivo electron acceptor for POR. This type of oxidoreductase is found commonly in obligate anaerobes, such as Clostridium spp. The pyruvate-flavodoxin oxidoreductase of H. pylori is composed of four subunits and is related to pyruvate-ferrodoxin oxidoreductases previously detected only in hyperthermophilic organisms (126). In the H. pylori genome sequence, genes HP1108 to HP1111 have similarities to the POR genes of the hyperthermophilic archaeon Pyrococcus furiosus and those of the hyperthermophilic bacterium Thermotoga maritima, depending on the subunit considered. Neither aceEF nor pfl genes were identified (280). The genome analysis of H. pylori supports the experimental data, particularly its utilization of pyruvate in fermentative metabolism. The biochemical characterization of the pyruvate-flavodoxin oxidoreductase and the data provided by the analysis of the genome data provide insight into the microaerophily of the bacterium.

Elements of the Krebs cycle are present in H. pylori (121, 188, 237, 267). The absence of the -ketoglutarate dehydrogenase complex was reported by Hoffman et al. (121) and confirmed by other groups (52, 237), and Pitson et al. (237) did not find succinyl-CoA synthetase in the bacterium. Accordingly, genes encoding the two subunits of the -ketoglutarate dehydrogenase complex (sucA and sucB) or the subunits of the succinyl-CoA synthetase (sucC and sucD) were not identified (280).

Hoffman et al. (121) and Pitson et al. (237) reported the existence of -ketoglutarate:acceptor oxidoreductase (OOR) which catalyzes the conversion of -ketoglutarate to succinate. Hughes et al. (126) purified the OOR enzyme and showed that it was composed of four heterogeneous subunits, with significant sequence similarity to archaeal enzymes. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. The analysis performed by Tomb et al. (280) yielded the ORFs HP0589, HP0590, and HP0591, which had similarities to the ferrodoxin oxidoreductase genes korA, korB, and korG from Methanococcus jannaschii and which presumably could encode three of the subunits of the H. pylori OOR enzyme. However, the amino acid sequences of H. pylori OorA and OorB determined by Hughes et al. (126) are more closely related to those of the two-subunit POR of the aerobic halophile Halobacterium halobium. The last subunit, OorD, appears to be a 10-kDa integral ferredoxin-like protein. Like OOR, POR is oxygen labile, and both are likely to be major factors in the requirement of H. pylori for low oxygen tensions.

In H. pylori, the Krebs cycle appears to be a branched noncyclic pathway, in which the dicarboxylic acid arm proceeds reductively from oxaloacetate through malate and fumarate to succinate and the tricarboxylic acid arm operates oxidatively from oxaloacetate through citrate and isocitrate to -ketoglutarate (114, 188, 237) (Fig. 2). The discoveries of genes encoding citrate synthase (gltA, HP0026), aconitase (acnB, HP0779), isocitrate dehydrogenase (icd, HP0027), fumarase (fumC, HP1325), and fumarate reductase (frdABC, HP0191 to HP0193) and the absence of ORFs with similarity to the genes encoding -ketoglutarate dehydrogenase support this hypothesis (280). Malate dehydrogenase and malate synthase activities have been measured in H. pylori (237), but no genes encoding these enzymes have been identified, suggesting that they are likely to have an amino acid sequence quite different from the others so far determined. These examples illustrate the caution that must be exercised when predicting metabolic and functional characteristics of organisms based solely on genomic analysis.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Dicarboxylic and tricarboxylic acid branches of the noncyclic Krebs cycle. gltA, citrate synthase; acnB, aconitase; icd, isocitrate dehydrogenase; sucAB, -ketoglutarate dehydrogenase; frdABC, fumarate reductase; fumC, fumarase; mdh, malate dehydrogenase; aceB, malate synthase. Asterisks denote enzymes for which no genes were identified in the H. pylori sequence; crosses denote enzymes whose enzymatic activities were observed but the corresponding genes were not identified. Reprinted from reference 237 with permission of the publisher.

An interesting feature of the reductive branch is that fumarate, via fumarate reductase, may act as the terminal electron acceptor in anaerobic respiration. Mendz et al. (189) investigated the properties of the H. pylori enzyme activity in vivo and established that it is a potential therapeutic target against the bacterium. In E. coli, fumarate reductase is made up of four subunits, i.e., a flavoprotein, an iron-sulfur protein, and two membrane anchor proteins (12); and in Wolinella succinogenes, fumarate reductase consists of three subunits (151). Ge et al. (92) cloned and characterized the H. pylori fumarate reductase operon, which is composed of three structural genes coding for subunits highly similar to those of W. succinogenes.

The putative anaerobic respiration with fumarate as the acceptor and the presence of pyruvate-flavodoxin and -ketoglutarate:acceptor oxidoreductases support the existence of anaerobic metabolism in H. pylori, even though oxygen is required for growth, and contribute to the characterization of the microaerophilic phenotype of the bacterium.

Concerning the Krebs cycle, some features deduced from the genome are in agreement with experimental data, but other deductions need further experimental verification. Conversely, the metabolic data suggest the need for more genomic analyses of the bacterium.

Amino acids are potential sources of carbon, nitrogen, and energy. The development of a defined medium for growing H. pylori and the subsequent determination of the amino acid requirements of the bacterium were important steps in understanding its metabolism (215, 246). All the strains tested required arginine, histidine, isoleucine, leucine, methionine, phenylalanine, and valine. Some of them also required alanine (8 of 10 strains), and serine was also needed for 5 of them. Analyses of genome molecular data support these experimental results.

The requirement for arginine and histidine in the growth medium may be explained by the fact that no protein involved in the arginine or histidine biosynthetic pathways is encoded by the H. pylori chromosome, except glutamate dehydrogenase (GdhA), which catalyzes the synthesis of glutamate from -ketoglutarate (98, 244, 295). ORF HP0380 has 72.7% similarity to E. coli gdhA.

Aspartate synthesis is a key step in the syntheses of several other amino acids. Aspartate is formed from oxaloacetate by transamination with glutamate as the amino donor (244), and the syntheses of methionine, threonine, and isoleucine are linked to that of aspartate (107, 231). The genes HP1189, HP1229, HP0822, HP1050, and HP0098 in the H. pylori DNA sequence are similar to asd, lysC, metL, thrB, and thrC, respectively, which encode enzymes involved in the threonine synthesis pathway. However, the genes encoding threonine deaminase and acetohydroxy acid synthase, which are necessary to form isoleucine, are lacking; thus, this amino acid is required for growth. Furthermore, a requirement for valine would follow from the absence of the gene which encodes acetohydroxy acid synthase, the first enzyme of the valine biosynthesis pathway (284). In the specific pathway leading to methionine synthesis, only the metB gene (HP0106), encoding cystathionine -synthase, seems to be present, which would explain the need for this amino acid. The absence of the leuA gene, encoding isopropyl malate dehydrogenase, is consistent with the need for leucine, which cannot be synthesized from -ketoglutarate (284).

Chorismate, a precursor of aromatic amino acids, is formed from phosphoenol pyruvate and erythrose 4-phosphate by means of seven enzymes whose genes are present in H. pylori DNA (238). The synthesis of tyrosine and phenylalanine from chorismate requires three enzymes, one of which (tyrosine aminotransferase) is common to both pathways. The tyrB gene, encoding tyrosine aminotransferase, was not identified from the nucleotide sequence, but its function can be supplemented by the branched-amino-acid aminotransferase enzyme, whose gene, ilvE (HP1468), is found in the chromosomal DNA of H. pylori. Since the tyrA gene (HP1380), encoding chorismate mutase-prephenate dehydrogenase, is also present in the genome, the bacterium can synthesize tyrosine. However, H. pylori is not able to synthesize phenylalanine, because the pheA gene, encoding chorismate mutase-prephenate dehydratase, is absent from the chromosome. The five enzymes specific for the biosynthesis of tryptophan have been identified in the genome of H. pylori, and the six sequential genes HP1277 to HP1282 encoding them have a similar organization to the corresponding genes of E. coli, which form the trpEDCBA operon.

The genome sequence of H. pylori suggests that other amino acids are synthesized via their conventional pathways (149, 156, 231, 238, 244, 269, 284).

Although Reynolds and Penn (246) observed that alanine enhances bacterial growth in the presence of glucose, Mendz and Hazell (182) demonstrated that H. pylori grows well in a glucose-free medium supplemented with arginine, aspartate, asparagine, glutamate, glutamine, and serine as sole substrates. The principal metabolic products observed from amino acid catabolism were acetate, formate, succinate, and lactate. These results showed that carbohydrates could be removed from media in which amino acids constituted the basic nutrients. Moreover, glucose added to growth media composed of a mixture of amino acids is not utilized until the other metabolites are significantly depleted (185, 188). These results are contrary to the conclusion of Tomb et al. (280) from genomic analysis that the glycolysis-gluconeogenesis metabolic axis constitutes the backbone of energy production in the bacterium.

Nonetheless, other comparisons between experimental results and molecular data from the genome show good correlations, even before some pathways have been completely elucidated. For example, the catabolism of alanine, arginine, aspartate, asparagine, glutamate, glutamine, and serine is dependent on the presence of several enzymes, whose genes have been identified. Indeed, the genes coding for alanine dehydrogenase (ald, HP1398), aspartate-ammonia lyase (aspA, HP0649), L-asparaginase II (asnB, HP0723), glutamate dehydrogenase (gdhA, HP0380), and L-serine deaminase (sda, HP0132) were found by sequence similarities.

The conversion of arginine to urea and ornithine described by Mendz and Hazell (186) is very interesting because it offers some insight into the nitrogen metabolism of H. pylori. The authors inferred the presence of an arginase necessary to catalyze this reaction, and the kinetic parameters, specificity, effects of cations, pH profile, and inhibition of this enzyme activity were studied in vivo and in situ (123, 191). In agreement with these results, Tomb et al. (280) identified ORF HP1399, with similarity to the rocF gene from Bacillus subtilis encoding arginase, and ORF HP1017, with similarity to the rocE gene encoding an arginine transporter. Moreover, an aliphatic amidase which hydrolyzes short-chain amides to produce carboxylic acid and ammonia has been identified in H. pylori (261). The gene encoding this enzyme was characterized and found to have high similarity (75%) to the amiE gene from Pseudomonas aeruginosa. This aliphatic amidase activity has been previously found only in environmental bacteria. These observations led to the suggestion that the amiE gene was acquired by horizontal transfer. Two paralogous genes, HP0294 and HP1238, seem to be present in H. pylori DNA.

Analysis of the genome sequence supports the experimental data concerning the synthesis pathways of the amino acids and explains the in vitro requirement for some of them by the lack of genes encoding enzymes involved in those synthesis pathways. However, more experiments are needed to fully understand amino acid catabolism in the bacterium, and these investigations should be guided by the genomic data.

In E. coli, fatty acid degradation occurs by cyclic -oxidation and thiolytic cleavage, yielding acetyl-CoA (62). The first step of the degradation is the activation of the free fatty acids to an acyl-CoA thioester by acyl-CoA synthetase. This -oxidation requires four enzymes: acetyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. Only the orthologous genes HP1045 and HP0690, corresponding to acoE encoding acyl-CoA synthetase and fadA encoding the 3-ketoacyl-CoA thiolase, respectively, have been identified by genome sequence analysis.

Concerning phospholipid degradation, several studies have reported phospholipase activities in H. pylori. Various strains have been shown to express phospholipase A₁ (160), A₂ (227), or C (33, 291); however, in the genome, only ORF HP0499 showed similarity to phospholipase A₁ gene from E. coli. It has to be concluded that the other genes may not have sufficient sequence similarity to be identified as such.

The biosynthesis of lipids and phospholipids by H. pylori is an area of investigation which requires further basic research. In particular, the biosynthetic pathways of fatty acids have not been completely elucidated, although some features have been established. The mechanism of fatty acid synthesis is conserved in prokaryotes and eukaryotes and proceeds in two stages, initiation and cyclic elongation (63).

The first committed step in fatty acid biosynthesis is the formation of malonyl-CoA from acetyl-CoA catalyzed by acetyl carboxylase (Fig. 3). The prokaryotic form of this enzyme, exemplified by the E. coli enzyme, consists of a complex of three individual proteins: biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase ( and  subunits). The enzyme complex of H. pylori has been characterized by Burns et al. (37), and the genes encoding the proteins in the complex, HP0370 (accC), HP0371 (fabE), HP0557 (accA), and HP0950 (accD), have been found in H. pylori DNA. The next steps in the initiation of fatty acid biosynthesis involve the attachment of the acyl carrier protein (ACP) to the acetyl and malonyl moieties. The genes encoding ACP and the two subunits of the holo-ACP synthase, HP0559, HP0962 (acpP), and HP0808 (acpS), respectively, are also present in the genome. The transfer reactions of CoA-bearing acyl chains to ACP are catalyzed by acetyl-CoA:ACP transacylase and malonyl-CoA:ACP transacylase (Fig. 3A). The gene encoding acetyl-CoA:ACP transacylase was not identified in the H. pylori genome. However, this function could be performed by a thiolase product of HP0690 (fadA). Malonyl-CoA is converted to malonyl-CoA:ACP by the malonyl-CoA:ACP transacylase encoded by HP0090 (fabD).

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Initiation (A) and elongation (B) phases of fatty acid biosynthesis. Initiation: accA, carboxyltransferase ( subunit); accD, carboxyltransferase ( subunit); fadA, thiolase; fabD, malonyl-CoA:ACP transacylase. Elongation: fabF, 3-ketoacyl-ACP synthase; fabH, 3-ketoacyl-ACP synthase; fabI, enoyl-ACP reductase; fabG, 3-ketoacyl-ACP-reductase; fabZ, (3R)-hydroxymyristoyl-ACP dehydratase.

Five genes, whose corresponding proteins are involved in elongation reactions of fatty acid synthesis, are present in the DNA sequence (Fig. 3B): HP0558 (fabF) and HP0202 (fabH), encoding 3-ketoacyl-ACP synthases; HP0561 (fabG), encoding 3-ketoacyl-ACP reductase; HP0195 (fabI), encoding enoyl-ACP reductase; and HP1376 (fabZ), encoding (3R)-hydroxymyristoyl-ACP dehydratase. In E. coli, a specific dehydrase enzyme, 3-hydroxydecanoyl-ACP dehydrase, catalyzes a key reaction at the point at which the biosyntheses of unsaturated and saturated fatty acids diverge (31). The fabZ gene might function in a similar manner in H. pylori, since the major fatty acids in the bacterium are tetradecanoic acid (14:0) and 19-carbon cyclopropane (19:0). Detection of the ORF HP0416, with similarity to the cfa gene from E. coli, explains the presence of cyclopropane fatty acids in H. pylori, in common with many other bacteria (103, 104).

There is only a small proportion of neutral lipids in the lipid composition of H. pylori. The most abundant phospholipids are phosphatidylethanolamine (PE), cardiolipin, and phosphatidylglycerol (PG); this composition is similar to that in many other gram-negative bacteria (119). The biosynthesis pathway for phospholipids utilizes sn-glycerol-3-phosphate (Glp), which can also serve as a substrate for glycolysis or gluconeogenesis in prokaryotes. The key phospholipid synthetic intermediate, phosphatidic acid, is formed in two steps (Fig. 4). Glp is acylated first by the glycerol-3-phosphate acyltransferase, using the acyl-ACP products of fatty acid synthesis, and then a second fatty acid is added by a 1-acyl-Glp acyltransferase to form phosphatidic acid (251). Both enzymes seem to be present in H. pylori, since the corresponding genes, HP0201 (plsX) and HP1348 (plsC), respectively, have been identified. Phosphatidic acid is converted to CDP-diglycerol. This reaction is catalyzed by CDP-diglycerol synthase. CDP-diglycerol reacts with serine to form phosphatidylserine (PS) or with Glp to form phosphatidylglycerolphosphate (PGP). The decarboxylation of PS by PS decarboxylase yields PE, and the dephosphorylation of PGP by PGP phosphatase yields PG. Analysis of the H. pylori DNA sequence led to the identification of three ORFs, HP0215, HP1016, and HP1357, with similarity to the cdsA, pgsA, and psd genes, respectively, of E. coli, whose products are required for the enzymatic reactions listed above (Fig. 4). The PS synthase and its corresponding gene (pssA) have been described by Ge and Taylor (93). However, no sequence similarity to a gene encoding PGP phosphatase, which allows the synthesis of PG, or with the gene encoding the cardiolipin synthase was identified in H. pylori.

View larger version (9K): [in this window] [in a new window]   FIG. 4.   Synthesis of phosphatidic acid, PS, PE, PGP, PG, and cardiolipin. plsX, glyceraldehyde-3-phosphate (G3P) acyltransferase; plsC, 1-acyl-G1P acyltransferase; cdsA, CDP-diglycerol synthase; pssA, PS synthase; psd, PS decarboxylase; pgsA, PGP synthase; pgp, PGP phosphatase; cls, cardiolipin synthase. Asterisks denote enzymes for which no genes were identified in the H. pylori sequence.

A unique characteristic of the lipid composition of this bacterium is the presence of three cholesteryl glucosides synthesized de novo, which account for about 25% (wt/wt) of the total lipids (110, 119). Cholesteryl glucosides are synthesized in plants and fungi but are rare in higher animals and bacteria. The cholesteryl glucosides found in H. pylori have a glycosidic linkage, which is unusual for natural glycosides, and one has a phosphate-linked glycoside that has not yet been found in prokaryotes or eukaryotes (119). UDP-glucose:sterol glucosyltransferase is the enzyme catalyzing the synthesis of cholesteryl glucosides in plants and fungi. No ORF has been found in the genome of H. pylori to account for this enzymatic activity.

The results of many of these analyses have not yet been backed up by experimental results. Elucidation of the role of a number of different genes identified in the lipid metabolism of the bacterium must await further study.

In general, the surface-exposed lipopolysaccharide (LPS) molecules play an important role in the interaction between gram-negative bacteria and their hosts. They are efficient immunomodulators and potent stimulators of the immune system (208). LPS is composed of three parts: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide region, and the core polysaccharide region, which connects the other two.

The lipid A portion is the component responsible for the immunological and endotoxic properties. Several groups have studied the relationship between the lipid A structure, LPS endotoxic properties, and immunobiological activities (14, 143, 209, 272); the H. pylori LPS is remarkable in its low toxicity. The polysaccharide moiety of H. pylori LPS contains Lewis^x and Lewis^y antigenic motifs that mimic Lewis antigens present on parietal cells of the human gastric mucosa (6, 7, 259).

At least 27 genes likely to be involved in LPS biosynthesis have been found in H. pylori (280). In contrast to the situation in other bacteria, LPS biosynthesis genes in H. pylori are scattered throughout the genome, not clustered at one locus. A homologue of GDP-D-mannose dehydratase from Vibrio cholerae and homologues of galactosyltransferases from Klebsiella pneumoniae have been suggested to be involved in O-antigen synthesis. The genes HP0379 and HP0651, coding for two 1,3-fucosyltransferases, have been identified; these transferases catalyze 1,3-glycosidic linkages implicated in the expression of sialyl-Lewis^x antigens in humans. DNA motifs evoking phase variation have been found near the 5' end of the 1,3-fucosyltransferase genes. The relevance of these motifs in vivo is examined in more detail below in the section on the diversity of H. pylori. Berg et al. (22) pointed out the presence of gene HP0326, an orthologue of neuA, that codes for CMP-N-acetylneuraminic acid synthetase. This enzyme is the glycosyl donor of sialyl groups and thus could be involved in the sialyl-Lewis^x antigen synthesis which has been reported in some strains (296). ORFs similar to genes encoding lipid A biosynthetic enzymes, namely, HP1375 for UDP-N-acetylglucosamine acyltransferase (lpxA), HP0867 for lipid A disaccharide synthetase (lpxB), HP0196 for UDP-3-O-(3-hydroxymyristoyl)glucosamine-N-acyltransferase (lpxD), and HP1052 for UDP-3-O-acyl-N-acetylglucosamine deacetylase (envA), were found in the H. pylori sequence. Orthologues of genes involved in core region synthesis were also identified: HP0858 (rfaE), HP1191 (rfaF), HP0859 (rfaD), and HP0279 (rfaC) for the heptose region of the inner core; HP0003 (kdsA), HP0230 (kdsB), and HP1386 (rpe) for the 3-deoxy-D-mannooctulosonic acid (KDO) residue region of the inner core; and HP0159, HP0208, HP1416 (three copies of rfaJ), HP1166 (pgi), and HP0646 (galU) for the outer core. KDO transferase, which catalyzes the attachment of KDO groups to lipid A, was also found to be encoded by the genes HP0742, HP1166, and HP0646, which are orthologues of kdsA. The identification of HP0159, HP0208, and HP1416 as homologues of the 1,2-glucosyltransferase gene (rfaJ) is confusing, since no 1,2-linked glucose has been found in H. pylori LPS (22). The authors suggested that these genes may encode 1,4-galactosyltransferase and/or 1,3-N-acetylglucosaminyltransferase functions, for which no orthologous genes have been found in the H. pylori sequence. The gene encoding 1,2-fucosyltransferase is also missing; it seems to be truncated in the genome of the sequenced strain (22).

In conclusion, genomic analysis allowed the identification of genes involved in LPS synthesis. However, in a few cases, further information is required to elucidate the relationship between functions deduced from the sequence analysis and those determined experimentally.

De novo synthesis of UTP and CTP. The pathway responsible for the de novo synthesis of UTP and CTP, two precursors of RNA, comprises nine enzymes (Fig. 5A). Carbamoylphosphate is a substrate for the first reaction of the pathway and is formed by a synthetase which catalyzes the reaction from glutamine or ammonium, bicarbonate, and ATP. Analysis of the H. pylori DNA sequence identified two ORFs, HP1237 and HP0919, with similarities to the pyrAa gene from Salmonella cholerasuis and the pyrAb gene from Bacillus caldolyticus (280), which encode carbamoyl-phosphate synthetases.

View larger version (12K): [in this window] [in a new window]   FIG. 5.   De novo synthesis of UTP and CTP (A) and ATP and GTP (B). (A) pyrA, carbamoyl-phosphate synthase; pyrB, aspartate transcarbamoylase; pyrC, dihydroorotase; pyrD, dihydroorotase dehydrogenase; pyrE, orotate phosphoribosyltransferase; pyrF, OMP decarboxylase; pyrH, UMP kinase; ndk, nucleoside diphosphokinase; pyrG, CTP synthetase. (B) guaB, IMP dehydrogenase; guaA, GMP synthase; gmk, GMP kinase; purA, adenylosuccinate synthetase; purB, adenylosuccinate lyase; adk, adenylate kinase.

De novo synthesis of ATP and GTP. Inosinate (IMP) is the first purine ribonucleotide formed de novo in the pathway (Fig. 5B). AMP and GMP are synthesized from IMP via separate branches of the pathway. From these monophosphates, ATP and GTP are generated by phosphorylations, with the intermediate formation of ADP and GDP (218).

Nucleotide salvage pathways. Salvage pathways enable cells to scavenge preformed nucleic bases and nucleosides for nucleotide synthesis or to reutilize bases and nucleosides produced endogenously as a result of nucleotide turnover, allowing circumvention of de novo pathways. Utilization of preformed pyrimidine bases is limited in H. pylori, with significant uptake of orotate and a lesser incorporation of uracil into the cells (192), but no gene similar to uraA, which encodes a cytoplasmic membrane protein required for uracil uptake in E. coli, was found in the H. pylori genome. Phosphoribosyltransferases are important for the salvage of free nucleic bases for biosynthesis, and enzyme activities for both orotate (OPRTase) and uracil (UPRTase) were observed in situ in H. pylori (192). In E. coli, the upp gene, coding for UPRTase, is the first gene of a bicistronic operon, with uraA as the second gene. In H. pylori, the gene HP1257, with similarity to pyrE coding for T. aquaticus OPRTase (part of the de novo pathway described above), was identified but no gene orthologous to upp was found. HP1180 has similarity to nupC, coding for a pyrimidine nucleoside transport protein in B. subtilis; however, the bacterium incorporates only very small quantities of uridine (192). No other genes orthologous to those encoding enzymes required for pyrimidine salvage in E. coli, such as deoA and tdk, coding for thymidine phosphorylase and thymidine kinase, respectively, or udp and udk, coding for uridine phosphorylase and uridine kinase, respectively, have been identified in H. pylori DNA.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Purine salvage pathways. deoD, purine nucleoside phosphorylase; apt, adenine phosphoribosyltransferase; gpt, xanthine/guanine phosphoribosyltransferase. Circles denote enzymes whose enzymatic activities have not been observed but whose corresponding genes were identified. Dashed arrows show steps given in detail in Fig. 5B.

Deoxyribonucleotide biosynthesis. In cells, the ratio of RNA to DNA is between 5:1 and 10:1; thus, most of the carbon which flows through the nucleotide biosynthesis pathways is directed to the rNTP pools. Nonetheless, the relatively small fraction that is directed to synthesizing dNTPs is of fundamental importance for the life of the cell, since dNTPs are used almost exclusively in the biosynthesis of DNA. In most organisms, the first step specific for deoxyribonucleotide synthesis is catalyzed by rNDP reductase (rNDPase), which converts all four rNDPs to the corresponding 2'-deoxyribonucleoside diphosphates. In H. pylori, the ORFs HP0680 and HP0364 have similarities to the nrdA gene from E. coli, encoding the  subunit of this enzyme, and the nrdB gene from Synechocystis sp., encoding the  subunit, respectively.

Thymine nucleotides are deoxy compounds with no ribonucleotide counterparts and therefore cannot arise simply through the action of rNDPase. The biosynthesis of dTTP occurs partly from the dUDP produced by rNDPase and partly from deoxycytidine nucleotides. In E. coli, dTTP synthesis requires four additional steps catalyzed by dUTPase (dUTPdUMP) encoded by dut, thymidylate synthase (dUMPdTMP) encoded by thyA, dTMP kinase (dTMPdTDP) encoded by tmk, and nucleoside diphosphokinase (dTDPdTTP) encoded by ndk. In H. pylori DNA, the genes HP0865, HP1474, and HP0198 are orthologous to dut, tmk, and ndk, respectively; but no gene orthologous to thyA was found (Fig.

View larger version (9K): [in this window] [in a new window]   FIG. 7.   Deoxyribonucleotide biosynthesis. nrd, ribonucleoside diphosphate reductase; ndk, nucleoside diphosphokinase; dcd, dCTP deaminase; dut, deoxyuridinetriphosphatase; thyA, thymidylate synthase; tmk, thymidylate kinase. The asterisk denotes an enzyme for which no corresponding gene was identified. N can be A, C, and U.

Many substrate oxidations are exploited by cells to generate metabolic energy. A central goal of respiration is to obtain reducing power to energize proton-translocating systems. Bacterial respiratory chains have a modular character, comprising dehydrogenase complexes, quinone pools, and terminal oxidoreductases (95). The terminal respiratory acceptor can be oxygen (aerobic respiration) or other substrates (anaerobic respiration). Besides their bioenergetic function (generation of proton motive force), respiratory systems also participate in the maintenance of intracellular redox balance (regeneration of NAD⁺) and the control of dioxygen concentration (scavenging of oxygen).

H. pylori has genes coding for proteins involved in both types of respiration. The genes for an NADH-quinone oxidoreductase respiratory chain have been found in an operon coding for the NDH-1 complex, HP1260 to HP1263 and HP1266 to HP1273. In bacteria, this complex is made up of 14 protein subunits whose genes are arranged in the same order with few absences in different NDH-1 operons (302). The H. pylori NDH-1 operon has genes encoding 12 subunits of the complex, and they are in the same order as their homologues in other bacterial NDH-1 operons, but it lacks the genes coding for two subunits of the complex which are involved in binding and oxidizing NADH. These genes are not found anywhere else in the H. pylori genome. In place of the two missing genes, the operon has HP1264 and HP1265, which lack binding motifs for NADH, flavin mononucleotide, and an FeS cluster, suggesting that H. pylori NDH-1 is a quinone reductase and a proton pump but not an NADH dehydrogenase (80). In the H. pylori genome, there is no gene encoding the NDH-2 complex, another type of bacterial NADH-quinone reductase which serves as an entry point to the respiratory chain of electrons donated by NADH but which is not a coupling site and does not translocate protons across the membrane (95). Since NADPH oxidase activity has been observed in membrane preparations (44) and crude bacterial supernatants (264), the hypothesis that NADPH was the electron donor for the H. pylori NDH-1 complex was investigated by Finel (80), who concluded that it was unlikely.

The ORFs HP0265, HP0378, HP0147, HP0144, HP0145, HP1539, HP1538, HP1540, HP0146, HP1461, and HP1227 are similar to genes encoding enzymes required for the synthesis of cytochrome c, cytochrome bc₁ complex, and cbb₃-type cytochrome c oxidase. In agreement with this observation, Marcelli et al. (170) reported that H. pylori cells and membranes contain b- and c-type cytochromes but not terminal oxidases of the a or d type. Also, the quinol oxidases, cb-type and cbb₃-type cytochrome oxidases, appear to act as a terminal oxidases in the respiratory chain of H. pylori (1, 211).

In addition to the NADH-ubiquinone oxidoreductase complex, three other respiratory electron-generating dehydrogenases have been identified in the genome of H. pylori. The cytoplasmic membrane Ni hydrogenase of W. succinogenes consists of three polypeptides, HydA, HydB, and HydC, and contains cytochrome b. The hydrogenase genes are arranged in the order hydA, hydB, and hydC, with the transcription start site in front of hydA. An intergenic region of 69 nucleotides separates hydC from at least two more ORFs of unknown function (68). The sequential genes HP0631, HP0632, and HP0633 of the H. pylori genome are orthologous to the hydABC operon and are followed by ORF HP0634, which is similar to the W. succinogenes hydD, suggesting that H. pylori encodes a quinone-reactive Ni/Fe-hydrogenase. In the chromosome of Rhodobacter capsulatus B10, there is a sequence of 20 hup and hyp genes encoding hydrogenase-specific products which bear significant structural identity to the hydrogenase gene products from other bacteria (50). In the genome of H. pylori, the five ORFs HP0869, HP0900, HP0899, HP0898, and HP0047 are orthologues of the hyp genes, whose proteins are involved in hydrogenase expression and synthesis in several bacteria. In particular, ORF HP0048 has similarity to the hypF gene of R. capsulatus, whose product, HypF, is involved in the regulation of hydrogenase synthesis (49). Maier et al. (166) observed hydrogen uptake hydrogenase activity coupled to whole-cell respiration and identified Ni-Fe hydrogenase activity, which was shown to be subject to anaerobic activation.

H. pylori HP0961 and HP0666 seem to encode two distinct sn-glycerol-3-phosphate dehydrogenases, a so-called aerobic enzyme and an anaerobic enzyme. These enzymes serve as electron donors by converting glycerol-3-phosphate to dihydroxyacetone phosphate. In E. coli, the level of each enzyme is modulated by the growth conditions (32). A putative D-lactate dehydrogenase encoded by HP1222 (dld), which may serve as an electron donor, was found in H. pylori. D-Amino acid dehydrogenases are also potential electron donors, and the gene HP0943, an orthologue of dadA, which encodes the smaller subunit of the D-amino acid dehydrogenase in E. coli, is present in the H. pylori sequence; however, a gene for the larger subunit was not found. Recently, Evans et al. (76) identified a 27-kDa hydrophobic protein whose N-terminal sequence is similar to the  subunit of the formate dehydrogenase of Desulfovibrio vulgaris. The genes encoding the  and  subunits of this enzyme were not found by Tomb et al. (280), but the gene for the  subunit was located. In D. vulgaris, formate dehydrogenase and cytochrome c₅₅₃ form an oxidoreduction complex. In H. pylori, the polypeptide encoded by the ORF HP1227 was identified as an orthologue of cytochrome c₅₅₃ from D. vulgaris, but Evans et al. (76) were not able to detect enzymatic activity of H. pylori formate dehydrogenase, leaving its contribution to energy production uncertain.

Other components that function as oxidoreduction mediators among the donor and acceptor complexes are quinones and b-type cytochromes (see above). Quinones are thought to function as mobile carriers which deliver reducing equivalents from dehydrogenases to terminal oxidoreductases in respiratory chains. Marcelli et al. (170) showed that the major isoprenoid quinone in H. pylori is menaquinone-6, with traces of menaquinone-4. The midpoint potential of the menaquinone/menaquinol couple in the membrane is about 190 mV lower than for the ubiquinone/ubiquinol couple; thus, menaquinones are better suited for a respiratory chain with lower potential electron acceptors; this could be a reason why these naphthoquinones are more commonly used for anaerobic respiration (95). The presence of menaquinones in H. pylori leads to the assumption that the anaerobic respiratory chain is used more often than the aerobic chain (275). This conclusion, however, must be drawn cautiously, because the specificity of a particular quinone for a given respiratory chain may also reflect particular structural features of quinones (95). Present in H. pylori genome are the genes HP0929 and HP0240, orthologues of the H. influenzae ispA gene, encoding geranyl pyrophosphate synthetase, and the E. coli ispB gene, encoding octaprenyl pyrophosphate synthetase, respectively. These are two enzymes of the biosynthesis pathway of the octaprenyl side chain of ubiquinones. HP1360 is an orthologue of the E. coli ubiA gene, which is involved in the second step of ubiquinone biosynthesis.

Fumarate may serve as an electron acceptor in anaerobic respiration. The existence of a fumarate transport system and an active fumarate catabolism via fumarate reductase in H. pylori was demonstrated in situ (184, 189, 191). The presence of fumarate reductase activity suggests the possibility of ATP generation via anaerobic respiration in the bacterium, in a manner similar to that in other anaerobic or facultative bacteria. The cloning and functional characterization of the H. pylori fumarate reductase operon showed that the FrdA and FrdB subunits are the catalytic dimers and that the FrdC subunit serves as a membrane anchor. This protein has a striking degree of sequence identity to W. succinogenes FrdC, which is a cytochrome b with two heme groups (25, 92). It has been proposed that the hydrogenase and fumarate reductase of H. pylori may be two components of an anaerobic respiratory chain which utilizes fumarate as the terminal electron acceptor (111).

The ability of H. pylori to use other terminal electron acceptors for anaerobic respiration seems to be limited, since no orthologues of genes coding for reductases of trimethylamine N-oxide, dimethyl sulfoxide, tetrathionate, or sulfite have been found in H. pylori. HP0642 and HP0954 exhibit similarity to a putative NAD(P)H nitroreductase gene from H. influenzae, and Ziebarth et al. (308) suggested that H. pylori possesses nitrate reductase and/or nitrosating enzymes such as cytochrome cd-1 nitrite reductase. Goodwin et al. (105) have shown that mutation in the rdxA gene encoding an oxygen-insensitive NADPH nitroreductase is associated with metronidazole resistance. HP1572 is a homologue of dniR, whose product in E. coli has a positive regulatory action inducing the synthesis of the nitrite reductase (cytochrome c₅₅₂) when nitrate is used as a terminal electron acceptor in anaerobic respiration (138). Interestingly, there is no evidence of nitrate reductase activity in H. pylori or of a homologue of nitrite reductase gene in the genome. However, the HP0313 gene is an orthologue of B. subtilis narK, which encodes a nitrite extrusion protein, thus providing a mechanism for exporting nitrite from H. pylori cells.

The conundrum of the presence of an operative aerobic respiratory chain in H. pylori, together with the anaerobic respiration used by the bacterium at the low oxygen tensions, has not been resolved. A complete understanding of the physiology of this microaerophile requires the solution of this apparent paradox.

There appear to be three principal mechanisms which enable H. pylori to resist oxidative damage, and they are catalyzed by the enzymes superoxide dismutase, catalase, and alkylhydroperoxide reductase (Ahp) (112, 167, 224, 233, 266). Inflammation within the gastric mucosa leads to an increase in toxic oxygen metabolites (66, 213). The superoxide anion, a highly reactive oxygen species formed as part of the oxidative burst of polymorphonuclear leukocytes, is dismutated to H₂O₂ by superoxide dismutase (207). Hydrogen peroxide is in turn converted to oxygen and water by catalase. Alkylhydroperoxide reductase catalyzes the reduction of alkyl hydroperoxide to the corresponding alcohol (233). In most bacteria, alkylhydroperoxide reductase is a two-component system consisting of the proteins AhpF and AhpC. AhpC is responsible for the peroxide reductase activity, while the accessory flavoenzyme, AhpF, possesses NADH or NADPH oxidase activity. In S. typhimurium, NADH oxidase or NADH oxidase-like activity coupled to AhpC is sufficient to generate active alkylhydroperoxide reductase (219, 220). The H. pylori gene HP1563 (tsaA) is an orthologue of ahpC (240, 241), but a homologue of ahpF was not identified in the H. pylori genome. However, there is evidence of the presence of NADH oxidase activity (264) in the bacterium. Catalase appears to be expressed in the cytoplasm and the periplasmic space and at the cell surface (112, 236, 242). It is a "classical" catalase, lacking peroxidase activity. These catalases usually are found in mammalian cells and contain a heme prosthetic group and NADPH binding activity (81). Examination of the H. pylori catalase gene katA (HP0875) product reveals a GXGXXG motif commonly associated with NADH rather than NADPH binding proteins (167). The H. pylori ORF HP0485 has similarity to the catalase-like gene cysR of Synechococcus spp., but at present its function is unclear.

Genomic and experimental data concerning enzymes involved in protection against oxidative damage agree, but the functions of some genes identified by sequence analysis with potential involvement in defence mechanisms require further elucidation.

Nitrogen is essential for the growth of all living organisms. Genome analysis indicates that H. pylori is able to use several substrates, including urea, ammonia, and some amino acids, as nitrogen sources (182, 280). Ammonia can be produced by the activity of urease (293), which allows access to urea nitrogen in the form of ammonium ions. The availability of energy and nitrogen determines the participation of glutamine synthetase (GSase), glutamate synthase (GOGATase), and glutamate dehydrogenase (GDHase) in ammonia assimilation. In energy-poor, nitrogen-rich (ammonia-containing) media, GDHase is important for glutamate synthesis and ammonia assimilation; in energy-rich, nitrogen-rich (ammonia-containing) media, GDHase levels are high and ammonia can be assimilated via GSase (244). The presence of the HP0512 gene in the H. pylori genome, similar to the glnA gene encoding GSase in E. coli, shows that assimilation of ammonia can be achieved via this route by converting glutamate to glutamine. This hypothesis was confirmed by the results obtained by Garner et al. (91). The failure to detect a gene orthologous to gltB, which encodes GOGATase in E. coli, suggests that -ketoglutarate is transformed into glutamate by GDHase, whose gene, HP0380, an orthologue of gdhA, is in the H. pylori sequence (Fig. 8). The presence of glnA and gdhA and the absence of gltB suggest that H. pylori is adapted to an ammon