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Mobley HLT, Mendz GL, Hazell SL, editors. Helicobacter pylori: Physiology and Genetics. Washington (DC): ASM Press; 2001.

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Helicobacter pylori: Physiology and Genetics.

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Chapter 12The Citric Acid Cycle and Fatty Acid Biosynthesis

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Diversity and Multiple Roles of the Citric Acid Cycle in Prokaryotes

The citric acid cycle (CAC) is arguably the most important central metabolic pathway in living cells. It has long been appreciated that this pathway often serves a dual role in energy conservation and in the biosynthesis of key cellular intermediates for anabolic reactions; for example, 2-oxoglutarate and oxaloacetate for amino acid biosynthesis, and succinyl-coenzyme A (CoA) for heme synthesis in some bacteria. The latter function is particularly important under anaerobic conditions, where the usual complete "aerobic" CAC is converted into two branches, an oxidative, or C6 branch, from citrate to 2-oxoglutarate, and a reductive, or C4 branch, from oxaloacetate to succinate. This occurs largely as the result of the anaerobic repression of the 2-oxoglutarate dehydrogenase multi-enzyme complex and succinate dehydrogenase, and the induction of fumarate reductase. The regulatory mechanisms by which this occurs in some bacteria are well known. In Escherichia coli, for example, they involve the oxygen-sensing activities of Fnr and the ArcA/ArcB system (51).

However, not all bacteria conform to this pattern. With the advent of genome sequencing it has become apparent that there are many variations on the basic theme of the "textbook" CAC, and a surprisingly large number of bacteria, particularly pathogens, are turning out to have incomplete or otherwise unusual CAC, which deviate from the E. coli paradigm (10, 24). Indeed, in analyses of the CAC predicted from 19 completely sequenced genomes, Huynen et al. (24) concluded that in the majority of species the cycle is incomplete or even absent, reflecting particular adaptations to the metabolic lifestyle of that particular organism. While this may be the case, one must be cautious about "missing" enzymes from genome analysis alone; actual biochemical experiments can reveal activities for which no gene appears to exist. More sensitive similarity searching may "find" the gene, or nonhomologous gene displacement with unexpected enzymes performing the expected function may be responsible (10, 24). With this background, the available biochemical and genomic data are employed to assess the nature of the CAC in Helicobacter pylori. Also described in this chapter is fatty acid biosynthesis, linked to the cycle through the utilization of acetyl-CoA as its starting point.

The CAC in H. pylori

Predictions from the Genome Sequence

Table 1 lists the open reading frames (ORFs) in strains 26695 (53) and J99 (1) encoding products with homology to CAC enzymes. In both strains, genes encoding citrate synthase, aconitase, isocitrate dehydrogenase, fumarase, fumarate reductase, and fumarase are clearly present. The first unusual feature to note is that genes encoding the usual pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multi-enzyme complexes, found in most aerobic bacteria, are absent. Instead, H. pylori utilizes 2-oxoacid:acceptor oxidoreductases specific for pyruvate (POR) and 2-oxoglutarate (OOR) to carry out the same biochemical reactions, a clear example of nonhomologous gene displacement. There are no genes encoding homologs of succinate dehydrogenase, succinyl-CoA synthetase (SCS), or NAD-linked malate dehydrogenase (MDH). There is also no evidence for genes encoding glyoxylate shunt enzymes (isocitrate lyase and malate synthase). From simple genomic predictions alone, the conclusion would be that the CAC is incomplete, owing to the absence of SCS and MDH.

Table 1. Genomic and biochemical evidence for CAC enzymes in H. pyloria.

Table 1

Genomic and biochemical evidence for CAC enzymes in H. pyloria.

Experimental Evidence for CAC Reactions

There have been conflicting biochemical data published on the overall activity of the CAC in H. pylori. Several studies of H. pylori metabolism in intact cells have indicated the lack of an active oxidative cycle. The amount of oxygen uptake during respiration of various carbon substrates was considered by Chang et al. (8) to be insufficient for complete oxidation through the CAC. In nuclear magnetic resonance (NMR) studies (7), the accumulation of significant concentrations of acetate from pyruvate metabolism under aerobic incubation conditions strongly suggests a major diversion of the acetyl-CoA resulting from the POR reaction toward acetate production rather than oxidation through the citric acid cycle. This could occur via phosphotransacetylase and acetate kinase, genes for which have been identified in strains 26695 (53) and J99 (1), and would yield one ATP per mole of acetyl-CoA.

In contrast, the operation of a complete oxidative CAC with a glyoxylate bypass has been inferred from the results of spectrophotometric enzyme assays by Hoffman et al. (21). However, SCS was not assayed in this study, and other workers have been unable to detect this activity (11, 46), consistent with the absence of the cognate genes in the genome sequences. It is also likely that the succinate dehydrogenase activity reported by Hoffman et al. (21) is in fact due to fumarate reductase. Using both spectrophotometric assays and NMR spectroscopy, Pitson et al. (46) concluded that H. pylori possessed a noncyclic branched pathway, as in anaerobes, with fumarate reductase but no succinate dehydrogenase or SCS. They found no evidence for an operational glyoxylate or gamma-aminobutyrate shunt, although malate synthase activity was detected. The differences between these studies may be due to differences in the methods used to assay the enzymes. A number of experimental problems exist in assaying many CAC enzymes, and particular caution is needed where low activities are measured with assays prone to interference (e.g., isocitrate lyase) or high background rates, as such activities may not be physiologically significant. Pitson et al. (46) have tried to avoid some of the problems with spectrophotometric assays by using NMR analyses of substrates and products. Nevertheless, any assay with crude cell-free extracts can be misleading owing to additional or unsuspected metabolism of the substrates, cofactors, or products, and it should be emphasized that the kinetic properties of an enzyme can only be reliably determined by initial rate measurements with the pure protein, although the in vivo properties of enzymes may be modulated by interactions with other cell components. Unfortunately, only a few H. pylori CAC enzymes have yet been purified. The enzyme activities that have been detected are discussed in more detail below.

Properties of Individual CAC Enzymes

Pyruvate:flavodoxin oxidoreductase

The oxidative decarboxylation of pyruvate is an important reaction in archaea, bacteria, and eukaryotes alike, generating acetyl-CoA necessary for CAC reactions, fatty acid biosynthesis, and many other reactions requiring acyl-CoA. In many aerobic bacteria and also mammalian systems this reaction is catalyzed by the pyruvate dehydrogenase multi-enzyme complex (43). Under anaerobiosis in bacteria capable of mixed acid fermentation, the generation of acetyl-CoA from pyruvate is catalyzed by pyruvate:formate lyase (31). Biochemical and genomic data indicate that both of these enzymes are absent in H. pylori, and instead this reaction is catalyzed by an unusual four-subunit pyruvate:flavodoxin oxidoreductase (POR) enzyme (22, 23). POR enzymes are generally associated with an anaerobic-type metabolism, and there has been growing interest recently in this class of enzymes as targets for the development of novel anti-anaerobe compounds (48). Indeed, the first crystal structure of a POR enzyme has recently been published (9). These enzymes can be broadly grouped into three types: single-subunit POR, found for example in Clostridium sp. (37), Anabeana sp. and Klebsiella sp. (49), Desulfovibrio africanus (45), and some anaerobic protozoa (14, 48, 55); two-subunit POR, so far identified only in the aerobic archaeon, Halobacterium halobium (29); and the four-subunit POR commonly associated with the members of the archaea and hyperthermophilic eubacteria (4, 5, 52). POR has been purified from H. pylori and shown to belong to this ancestral, four-subunit group of enzymes (22), and was the first example of a four-subunit POR found in a mesophilic eubacterium. Characteristically, these enzymes catalyze the reduction of low-potential electron acceptors in vivo that are commonly either ferredoxin or flavodoxin proteins. There is evidence that the in vivo electron acceptor of the H. pylori POR enzyme is a flavodoxin (FldA; HP1161/JHP1088) (22, 23, 26). The molecular masses of the subunits are as follows: PorA, 47 kDa; PorB, 36 kDa; PorC, 24 kDa; and PorD, 14 kDa (22). The structural genes for POR are located adjacent to each other in the chromosomes of both strains 26695 and J99 in the gene order porCDAB (HP1108–1111/JHP1035–1038). The molecular mass of the native POR was estimated to be 240 kDa; however, the stoichiometry of the native complex is unknown. The PorB subunit displays the conserved amino acid binding motif for thiamine pyrophosphate (TPP) in common with other POR enzymes (23). The PorD subunit is strikingly similar to bacterial ferredoxins, displaying the characteristic cysteine-rich Fe-S binding motifs. Unlike pyruvate dehydrogenases, POR enzymes do not utilize either lipoic acid or flavin adenine dinucleotide (FAD) as cofactors, as the oxidative decarboxylation reaction involves a free-radical mechanism (30, 44, 50). The H. pylori POR was found to be highly oxygen labile, and the presence of this enzyme, along with the related oxygen-labile OOR enzyme (see below), may play an important role in the micro-aerophily of H. pylori (23, 28). Insertion-inactivation of the porB gene was unsuccessful, implying that POR is essential for the growth of H. pylori (23).

It is not known how the POR-reduced flavodoxin is reoxidized in H. pylori, but in cell-free extracts a POR-dependent reduction of NADP (but not NAD) can be observed, which would be consistent with reduced flavodoxin reducing NADP via a hypothetical flavodoxin:NADP oxidoreductase (23, 28). Alternatively, reoxidation of FldA may involve other electron acceptors, including the electron transport chain.

Finally, there is indirect evidence for in vivo expression of POR, through identification in the sera of H. pylori-infected individuals of the immunogenic PorA subunit (36).

Citrate synthase

Citrate synthase (EC 4.1.3.7) catalyzes the first step in the oxidative branch of the CAC in which acetyl-CoA and oxaloacetate are condensed to generate citrate and CoA. Citrate synthases in other bacteria are encoded by gltA, and a single copy of a gltA homolog is present in both H. pylori 26695 (HP0026) and J99 (JHP0022) strains. The purification of the H. pylori citrate synthase enzyme has been described (15). In common with other gram-negative citrate synthase enzymes, the native enzyme exists as a hexamer, with an approximate molecular mass of 300 to 340 kDa and a subunit size of 50 kDa. However, regulation of the H. pylori enzyme is more characteristic of citrate synthases from gram-positive bacteria and eukaryotes. For example, unlike citrate synthases from gram-negative bacteria, the H. pylori enzyme is not inhibited by NADH (46). This has been related to the role of the CAC in H. pylori being directed toward biosynthesis rather than energy generation. Also, 2-oxoglutarate, succinyl-CoA, AMP, and ADP, which inhibit other bacterial citrate synthases, do not inhibit the H. pylori enzyme. However, ATP was found to act as a strong competitive inhibitor with respect to acetyl-CoA (46).

Aconitase

The next step in the oxidative branch of the cycle is carried out by aconitase (EC 4.2.1.3), which catalyzes the reversible isomerization of citrate to generate isocitrate via cis-aconitate. Unlike E. coli, which contains two differentially regulated aconitase enzymes, AcnA and AcnB (12), only a single copy of an acnB homolog has been identified in the H. pylori genomes (HP0779/JHP0716). Aconitase activity has been detected in the cytosolic fraction of H. pylori cells both by NMR and spectrophotometric assays (46). However, full activity was only detected if the cytosolic fraction was recombined with the cell membrane fraction, indicating that interaction with the cell envelope is required for optimum activity.

Isocitrate dehydrogenase

Isocitrate dehydrogenase (EC 1.1.1.42) catalyzes the NAD(P)-dependent oxidative decarboxylation of isocitrate to generate 2-oxoglutarate and CO2. In E. coli this enzyme acts as a critical branch point between the CAC reactions and the glyoxylate bypass during growth on C2 compounds like acetate. Regulation is achieved through reversible phosphorylation, leading to inactivation of this enzyme by the AceK protein, an isocitrate dehydrogenase kinase/phosphatase (33). The gene icd (HP0027/JHP0023), encoding isocitrate dehydrogenase, is located immediately downstream of the citrate synthase gene in H. pylori (1, 52). It is unlikely that the H. pylori isocitrate dehydrogenase enzyme acts as a branch point in isocitrate metabolism for two reasons: H. pylori probably lacks a functional glyoxylate bypass (see discussion below), and no homologs of aceK have been identified in the genome sequence. Pitson et al. (46) have reported that the H. pylori enzyme is NADP-specific and cannot utilize NAD as the electron acceptor, whereas Hoffman et al. (21) detected low levels of NAD-dependent activity. Sigmoidal kinetics with isocitrate and NADP indicates that H. pylori isocitrate dehydrogenase may be subject to allosteric regulation, and the activity is slightly stimulated by AMP (45).

2-Oxoglutarate metabolism

The next step in the oxidative branch of the conventional CAC is the oxidative decarboxylation of 2-oxoglutarate in the presence of CoA to generate succinyl-CoA with the release of CO2. This reaction is commonly catalyzed by the 2-oxoglutarate dehydrogenase multi-enzyme complex. Both enzymatic data (11, 21, 23, 46) and genome sequence analysis (1, 53) indicate that H. pylori lacks this enzyme complex. Instead, oxidation of 2-oxoglutarate is carried out by an oxygen-labile 2-oxoglutarate:acceptor oxidoreductase enzyme (23), which shares a number of biochemical and structural features in common with the POR enzyme described above. The OOR enzyme has been purified from H. pylori (23) and is composed of four subunits, OorA (43 kDa), OorB (33 kDa), OorC (21 kDa), and OorD (10 kDa). In the chromosome, the structural genes for the OOR enzyme are located together in the order oorDABC (23) and correspond to gene numbers HP0588–0591 and JHP0536–0539 in H. pylori strains 26695 and J99, respectively (1, 53). OorB and OorD display sequence motifs for TPP and Fe-S cluster binding in common with POR and related enzymes. Interestingly, however, the pair-wise amino acid identity of the corresponding POR and OOR polypeptides is relatively low (25% or less), and in contrast to POR, the OorA and OorB subunits are more similar to the two-subunit puruvate:ferredoxin oxidoreductase of H. halobium than to the four-subunit enzymes commonly found in extremophiles. This strongly suggests independent evolutionary origins for these oxidoreductases in H. pylori. Mutants in oorA could not be generated, suggesting that OOR is essential for H. pylori (23).

Corthesy-Theulaz et al. (11) concluded that succinyl-CoA was not generated via CAC reactions in H. pylori owing to the absence of the 2-oxoglutarate dehydrogenase complex, but it is now clear that OOR effectively substitutes for this enzyme. The product of the OOR reaction is succinyl-CoA, and Hughes et al. (23) showed that the activity of the purified enzyme was strictly 2-oxoglutarate and CoA-dependent with Km values of 0.3 and 13 μM, respectively. However, Pitson et al. (46) studied 2-oxoglutarate oxidation in H. pylori cell extracts using 1H-NMR spectroscopy and reported an FAD or benzyl viologen-dependent 2-oxoglutarate "oxidase" activity not absolutely dependent on, but strongly stimulated by, the addition of CoA in an apparently allosteric manner. Unusual for an allosteric activator, 2-mM CoA increased the apparent Km for 2-oxoglutarate and FAD to nonphysiological values (26 and 28 mM, respectively) despite the Vmax also being raised. The product of this reaction was succinate, whereas succinyl-CoA production could not be detected. These results are difficult to reconcile with the properties of purified OOR reported by Hughes et al. (23) but may be due to the use of cell extracts in the work of Pitson et al. (46).

Interconversion of succinate and SCS

SCS catalyzes the sole reaction of the CAC in which a nucleotide triphosphate is generated from the conversion of succinyl-CoA to succinate and CoA. The enzyme can also operate in the reverse direction to generate succinyl-CoA. There is no biochemical evidence for the presence of SCS activity in H. pylori (11, 46), and no homologs of the sucCD genes have been identified in the either of the H. pylori genome sequences. The absence of this enzyme would be consistent with other evidence that the CAC in H. pylori consists of a reductive branch ending in succinate and an oxidative branch ending in succinyl-CoA, with no physiological need to connect the two branches. A succinyl-CoA:acetoacetate CoA-transferase (SCOT) enzyme has been identified by Corthesy-Theulaz et al. (11), which in principle could substitute for SCS (10), although this would be dependent on acetoacetate or acetoacetyl-CoA. The role of this enzyme is unclear and is further considered below.

Fumarate reductase

In E. coli, the interconversion of fumarate and succinate can be carried out by two enzymes: succinate dehydrogenase, which is expressed under aerobic conditions, or fumarate reductase, which is induced under anaerobiosis (25, 51). In the absence of oxygen, fumarate can be used as an alternative terminal electron acceptor for the proton-translocating electron transport chain and is important in ATP generation in many anaerobic bacteria (32). H. pylori lacks succinate dehydrogenase (1, 46, 53), whereas the presence of a fumarate reductase has been confirmed by biochemical analysis (3, 38, 46) and sequence data (1, 17, 53). H. pylori fumarate reductase is closely related to that of the related anaerobe Wolinella succinogenes and is encoded by three genes, frdCAB (HP0193–0191/jhp179–177), encoding polypeptides of 27, 81, and 31 kDa, respectively (17). FrdA and FrdB display the amino acid motifs for FAD and Fe-S binding, respectively, in common with other fumarate reductases and also succinate dehydrogenases. The frdC gene encodes a hydrophobic, di-heme cytochrome b, which may serve as a membrane anchor for FrdA and B. Like the fumarate reductase of W. succinogenes, activity was localized in the membrane fraction (3, 17). Mutants in frdA have been generated, indicating this enzyme is not essential for H. pylori. However, the mutants demonstrated a prolonged lag phase on standard growth medium under microaerobic conditions (17). FrdA is also immunogenic, and was recognized in 55% of serum samples from H. pylori-infected individuals (3). Expression of fumarate reductase activity appears to be constitutive in H. pylori, as unlike E. coli, levels of activity did not markedly change in cells grown under varying O2 concentrations (13). Also, although the presence of fumarate reductase provides evidence of anaerobic-type respiration, H. pylori has not been successfully cultured under anaerobic conditions, even in the presence of additional fumarate as a terminal electron acceptor. Mendz et al. (40) found that the H. pylori enzyme was inhibited by three known inhibitors of fumarate reductase, morantel, oxantel, and thiabendazole, although high MICs would preclude their use as chemotherapeutic agents.

Fumarase

Fumarase (EC 4.2.1.2) catalyzes the reversible generation of malate from fumarate, and homologs of the structural gene for a type II fumarase, fumC, have been identified in both of the H. pylori genome sequences (HP1325/JHP1245). Fumarase activity has been investigated by Pitson et al. (46). The Km and Vmax values strongly indicate that the enzyme functions preferentially in the reductive direction, i.e., in the formation of fumarate from malate (fumarate Km, 121 mM; Vmax, 4.1 μmol min−1 mg of protein −1; malate Km, 7.3 mM; Vmax, 10.8 μmol min−1 mg of protein−1). Again, this is consistent with fumarase operating in a reductive CAC arm leading to succinate via fumarate reductase.

Malate dehydrogenase (NAD-linked) and malate quinone oxidoreductase

Only very low specific activities (less than 10 nmol min−1 mg of protein−1) of NADH-dependent malate dehydrogenase were detected in H. pylori by Hoffman et al. (21), but much higher activities (around 100 nmol min−1 mg of protein−1 assayed in the reductive direction) were measured by Pitson et al. (46). It is difficult to reconcile these values except as strain or assay differences. No homologs of a typical NAD-MDH have been identified in either of the H. pylori genome sequences, suggesting the enzyme responsible for the activity is very divergent or otherwise novel.

Davison et al. (13) reported the presence in H. pylori of a loosely membrane-bound dye-linked (NAD-independent) malate dehydrogenase activity, and l-malate-dependent cytochrome reduction. The enzyme responsible was predicted by Kelly (28) to be a flavoprotein malate:quinone reductase (Mqo), which functions as an electron donor to the quinone pool, analogous to the better known d-lactate dehydrogenase (41). Kather et al. (27) have confirmed this and have shown that the HP0086 ORF encodes a protein with Mqo activity that is only distantly related to known Mqo enzymes. Why should H. pylori possess two types of malate-oxidizing enzymes? It is likely that these function differentially; the Mqo is involved in the use of malate as a respiratory chain electron donor, whereas the NAD-MDH could function in the opposite (reductive) direction to synthesize malate as part of the reductive C4 branch of the CAC pathway.

The Anaplerotic Formation of Oxaloacetate

If a branched CAC exists in H. pylori, a source of oxaloacetate is needed to fuel both branches. One of the puzzling features of H. pylori carbon metabolism is the apparent lack of anaplerotic CO2 fixation enzymes that could form oxaloacetate from pyruvate or phosphoenolpyruvate (PEP). PEP carboxykinase, PEP carboxylase, and pyruvate carboxylase could not be detected in strain 11637 by Hughes et al. (22), although very low activities were reported in other strains by Hoffman et al. (21). In view of the fact that there are no candidate genes for any of these enzymes in strains 26695 or J99 (the assertion of Cordwell [10] that HP0370 is a pyruvate carboxylase is incorrect; it is a subunit of acetyl-CoA carboxylase), and no biotinylated protein in cell-free extracts of the expected size for a pyruvate carboxylase (6, 22), the mechanism of anaplerotic oxaloacetate formation remains unclear. However, for the reductive arm of the CAC, a supply of fumarate for fumarate reductase-associated electron transport may be obtained from extracellular aspartate via aspartase (see below).

Other Enzymes Which May Have a Role in the CAC

Aspartase

The assimilation of amino acids may play an important role in the provision of biosynthetic intermediates and energy conservation in H. pylori in vivo, as the bacterium is capable of growth in defined media supplemented with only amino acids as the sole carbon source (39). H. pylori was found to metabolize aspartate rapidly, and the initial product of aspartate metabolism was identified as fumarae (39), indicating a role for aspartase in fumarate formation. Aspartase is encoded by the aspA gene, and homologs have been identified in both genome sequences (HP0649/jhp594).

Enzymes of the glyoxylate bypass

Malate synthase and isocitrate lyase together catalyze the two reactions of the glyoxylate bypass, which is an anaplerotic mechanism for C4 dicarboxylic acid synthesis that specifically operates during growth on C2 compounds as sole carbon source. Malate synthase catalyzes the conversion of glyoxylate and acetyl-CoA to malate and CoA, and is encoded by aceB in E. coli. Although significant malate synthase activity has been detected in H. pylori (21, 46), no homologs of aceB have been identified in the genome sequences, possibly suggesting that the H. pylori enzyme may have diverged from other malate synthases. Hoffman et al. (21) have reported very low isocitrate lyase activity in some strains whereas Pitson et al. (46) could not detect this enzyme activity either spectrophotometrically or by 1H-NMR. As no homologs of isocitrate lyase genes have been identified in the genome sequence, and the phenylhydrazine assay is prone to interference from many carbonyl-containing compounds, it seems unlikely that a functional glyoxylate bypass is operative in H. pylori.

Conclusions: H. pylori Has a Branched Incomplete CAC

Figure 1 shows the likely arrangement of CAC reactions in H. pylori. The evidence supporting the operation of separate reductive (C4) and oxidative (C6) branches can be summarized as follows: (i) the use of fumarate reductase rather than succinate dehydrogenase in the C4 branch; (ii) a fumarase with kinetics strongly favoring fumarate formation; (iii) the absence of SCS; (iv) lack of allosteric inhibition of citrate synthase by NADH, supporting a role in biosynthesis rather than energy conservation; and (v) NMR and oxygen uptake studies with intact cells that show significant acetate excretion from pyruvate and insufficient oxygen uptake during respiration of several substrates for a complete oxidative CAC to be operational. There are also some unusual features of the CAC reactions in H. pylori: (i) the use of the oxygen-labile POR and OOR in place of the corresponding 2-oxoacid dehydrogenases; (ii) the presence of NAD-linked MDH activity and also malate synthase without obvious genes encoding them; (iii) the malate: quinone oxidoreductase, which should not really be considered part of the CAC but as a mechanism by which electrons from malate can be used for respiration; and (iv) the apparent absence of anaplerotic reactions for oxaloacetate synthesis. The function of the C6-branch CAC reactions in H. pylori is clearly biased toward biosynthesis of 2-oxoglutarate and succinyl-CoA, the key enzyme being OOR, whereas the C4 branch is concerned with fumarate respiration to allow redox balancing and energy conservation. The key enzyme here is fumarate reductase.

Figure 1

Figure 1

. Citric acid cycle and related reactions in H. pylori. Enzymes are denoted by numbers. 1, pyruvate:flavodoxin oxidoreductase; 2, phosphotransacetylase; 3, acetate kinase; 4, citrate synthase; 5, aconitase; 6, isocitrate dehydrogenase; 7, 2-oxoglutarate:acceptor (more...)

Fatty Acid Biosynthesis in H. pylori

Fatty Acid Composition of H. pylori

The composition of fatty acids varies significantly between different bacteria and can be a useful chemotaxonomic marker to aid identification. The study of the lipid and fatty acid profiles of eight Helicobacter species has revealed some characteristic features of the Helicobacter genus (18). Helicobacter species can be differentiated into two groups on the basis of fatty acid profiles. Group A, which contains mostly gastric colonizers including H. pylori, contains a high percentage of tetradecanoic (14:0) fatty acids and 19-carbon cyclopropane (19:0cyc) fatty acids, and a low proportion of octadecanoic (18:1) fatty acids. Haque et al. (18) also reported the presence of unusual cholesteryl glucosides in 11 out of 13 Helicobacter species studied to date. These lipids may be a useful chemotaxonomic marker to differentiate Helicobacter species from the closely related Campylobacter and Wolinella species, which apparently lack cholesteryl glucosides. Three different cholesteryl glucosides were identified in H. pylori, which together comprise approximately 25% of the total lipid content of the bacterium (20). These glycolipids were found to contain an alpha-glycosidic linkage, which is unusual for natural glycosides, and, furthermore, one was a newly identified phosphate-linked glycoside. In common with other gram-negative bacteria, the predominant phospholipids identified in H. pylori were phosphatidylethanolamine, cardiolipin, and phosphatidylglycerol. Phosphatidylserine was also detected, but at lower abundance (20).

Biosynthesis of Fatty Acids

Indications from the genome sequence

Few studies have examined the synthesis of fatty acids in H. pylori, and most of our understanding has come from analysis of the genome sequences (1, 53). H. pylori utilizes the type II or dissociated fatty acid synthesis pathway, typical of most bacteria and plants, in which discrete proteins catalyze individual steps in the pathway. Table 2 lists the relevant genes in strains 26695 and J99, and Figure 2 shows the predicted pathway for fatty acid biosynthesis in H. pylori, largely based on the E. coli model for fatty acid biosynthesis (reviewed in reference 47).

Table 2. Summary of genes associated with fatty acid synthesis in H. pylori.

Table 2

Summary of genes associated with fatty acid synthesis in H. pylori.

Figure 2

Figure 2

. Predicted pathways for fatty acid and phospholipid biosynthesis in H. pylori. During the initiation phase of fatty acid biosynthesis, acetyl-CoA is carboxylated to generate malonyl-CoA, which is then converted to malonyl-ACP. Malonyl-ACP is also required (more...)

The first committed step of fatty acid biosynthesis, in which malonyl-CoA is generated from acetyl-CoA, is catalyzed by acetyl-CoA carboxylase (ACC) (see below). Malonyl-CoA reacts with the acyl-carrier protein (ACP) to generate malonyl-ACP, which is catalyzed by the fabD gene product, malonyl-CoA:ACP transacylase. Malonyl-ACP is required not only for initiation of fatty acid biosynthesis, but also for each subsequent round of elongation of the fatty acid chain. ACP is a small, soluble protein that plays a critical role in fatty acid biosynthesis in most bacteria by covalently binding the intermediates of the pathway. The structural gene for ACP (acpP) has been identified in both genome sequences. Interestingly, there is an additional ACP homolog found only in H. pylori strain 26695 (HP0962), which encodes a larger protein, extended at the N terminus. To function in fatty acid biosynthesis, the apo-ACP protein must first be activated by transfer of the 4′-phospho-pantotheine from CoA, and this reaction is predicted to be catalyzed by holo-ACP synthase, encoded by acpS in H. pylori.

To initiate fatty acid synthesis, acetoacetyl-ACP is generated from malonyl-ACP, and in E. coli there are three potential pathways leading to the formation of acetoacetyl-ACP (47). First, malonyl-ACP can undergo condensation with acetyl-CoA to generate acetoacetyl-ACP. In E. coli, this reaction is catalyzed by β-ketoacyl-ACP synthase III (FabH), which also catalyzes an alternate pathway in which acetyl-CoA is converted to acetyl-ACP. Acetyl-ACP is then condensed with malonyl-ACP to generate acetoacetyl-ACP, which in E. coli is catalyzed by β-ketoacyl-ACP synthase I (FabB) and II (FabF). FabB can also function to decarboxylate malonyl-ACP to generate acetyl-ACP, which then undergoes condensation with a second malonyl-ACP molecule. In the case of H. pylori, fabF and fabH homologs are present, whereas fabB has not been identified. Further biochemical studies are required to confirm the function, and to ascertain the substrates and side reactions associated with these enzymes in H. pylori.

Acetoacetyl-ACP is converted to β-hydroxyacyl-ACP by the β-ketoacyl-ACP reductase enzyme (FabG), and the product of this reaction is then dehydrated. In E. coli, this dehydration reaction can be carried out by two enzymes, FabA and FabZ. However, only FabA catalyzes the isomerization of trans-2 decenoyl-ACP to cis-3-decenoyl-ACP, the intermediate for unsaturated fatty acid synthesis in E. coli. A homolog of fabZ is present in the H. pylori genome sequences, whereas fabA has not been identified. It is currently unknown whether the H. pylori FabZ has a similar role to E. coli FabA, as the branch point between the synthesis of saturated and unsaturated fatty acids. FadR, which has a dual role in E. coli as a positive activator for transcription of fabA and a repressor of fatty acid degradation, has not been identified in H. pylori.

The final step in the cycle is catalyzed by enoyl-ACP reductase (FabI) to generate acyl-ACP, which can then enter another round of elongation or, alternatively, enter the pathway for membrane phospholipid synthesis. The first step of phospholipid biosynthesis is the transfer of the acyl chain to the 1-position of glycerol-3-phosphate, the scaffold for this synthesis, by glycerol-3-phosphate acyltransferase (PlsB) (19). A plsB homolog has not been identified in the H. pylori genome. However, a homolog of the gene encoding PlsC, which catalyzes the transfer of a second acyl group to glycerol-3-phosphate, is present. H. pylori also contains a homolog of plsX, which by mutational analysis plays an as yet poorly defined role in phospholipid biosynthesis in E. coli (34). Genes associated with the production of CDP-diglycerol (cdsA), phosphatidylserine (pssA), and phosphatidylethanolamine (psd) from phosphatidic acid have been identified in H. pylori, and Ge and Taylor (16) have found that insertion-inactivation mutants of pssA could not be generated, indicating this enzyme is essential in H. pylori. A homolog of pgsA encoding the enzyme for production of phosphatidylglycerophosphate is present, whereas the genes encoding the subsequent enzymes of this pathway, leading to the production of phosphatidylglycerol and cardiolipin (pgp and cls, respectively), have not been identified.

As discussed above, H. pylori contains cyclopropane fatty acids, which in other bacteria are generated through modification of membrane phospholipids. This modification involves methylenation of the phospholipid by S-adenosylmethionine and is predicted to be catalyzed by the product of the H. pylori cfa gene. H. pylori also contains unusual cholesteryl glucosides, which are synthesized though the action of UDP-glucose:sterol glucosyl-transferases in other organisms. No sequence similarity to genes encoding these enzymes has been identified in H. pylori.

Enzymes of Fatty Acid Biosynthesis

ACC

H. pylori ACC has been characterized by Burns et al. (6), and the possible role of this enzyme in the capnophilic phenotype of H. pylori is discussed elsewhere (see chapter 10). Genome sequence data (1, 53) indicate that H. pylori ACC is typical of other prokaryotic enzymes, in that it is composed of four individual proteins: biotin carboxylase, biotin carboxyl carrier protein, and two subunits constituting the carboxytransferase. NMR spectroscopy has confirmed that the end product of the ACC reaction is malonyl-CoA, and that the reaction is reversible (6).

SCOT and thiolase

Several enzymes have been identified in H. pylori that may contribute to the production of acetyl-CoA in the bacterium. Corthesy-Theulaz et al. (11) have identified a SCOT enzyme in H. pylori, which catalyzes the reversible transfer of CoA from succinyl-CoA to acetoacetate to generate acetoacetyl-CoA. H. pylori SCOT is composed of two subunits of molecular masses of 26 kDa (encoded by HP0691/JHP0637) and 24 kDa (HP0692/JHP0636). SCOT may play a role in the metabolism of acetoacetate in H. pylori, forming acetyl-CoA from acetoacetyl-CoA through the action of a thiolase enzyme, encoded by fadA (HP0690/JHP0638), which lies immediately upstream of the SCOT (scoAB) genes. However, the precise physiological role of SCOT in H. pylori has not been clearly defined, and a role in succinate/succinyl-CoA interconversion has also been suggested (10, 11).

Fatty Acid Degradation

There is currently little information about the metabolism of lipids by H. pylori. The initial step in the oxidation of fatty acids is catalyzed by acyl-CoA synthetase (AcoE), and the corresponding gene has been identified only in the genome sequence of strain 26695 (HP1045). β-oxidation of fatty acids then proceeds in a cyclical fashion, via four enzyme activities, acyl-CoA dehydrogenase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and thiolase. Apart from thiolase, no other genes have been identified for fatty acid degradation. H. pylori possesses lipolytic activity, and phospholipase A1 (35), A2 (42), and C (54) activities have been detected in different strains. However, only one gene with homology to A1 phospholipase has been identified in the genome sequences of H. pylori 26695 (HP0449) and J99 (jhp451).

Effects of Fatty Acids on Host Cells

Several studies have indicated that H. pylori fatty acids directly cause damage to the host. Both 19:0cyc and 14:0 fatty acids may have an antisecretory action in vivo, as at high concentrations they were found to inhibit H+/K(+) ATPase activity in parietal cells, and exhibit detergent action at the apical parietal cell membrane (2). Also, H. pylori cholesteryl glucosides may damage gastric epithelial cells in vivo, as they demonstrate hemolytic activity in vitro (20).

Acknowledgments

Work on H. pylori in Professor Kelly's laboratory has been funded by the U.K. Biotechnology and Biological Sciences Research Council, The Wellcome Trust, and Glaxo-Wellcome.

References

1.
Alm R. A., Lee L.-S., Moir D. T., King B. L., Brown E. D., Doig P. C., Smith D. R., Noonan B., Guild B. C., de Jonge B. L., Carmel G., Tummino P. J., Caruso A., Uria-Nickelson M., Mills D. M., Ives C., Gibson R., Merberg D., Mills S. D., Jiang Q., Taylor D. E., Vovis G. F., Trust T. J. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature. 1999;397:176–180. [PubMed: 9923682]
2.
Beil W., Birkholz C., Wagner S., Sewing K. F. Interaction of Helicobacter pylori and its fatty acids with parietal cells and gastric H+/K(+)-ATPase. Gut. 1994;35:1176–1180. [PMC free article: PMC1375690] [PubMed: 7959221]
3.
Birkholz S., Knipp U., Lemma E., Kroger A., Opferkuch W. Fumarate reductase of Helicobacter pylori—an immunogenic protein. J. Med. Microbiol. 1994;41:56–62. [PubMed: 8006945]
4.
Blamey J. M., Adams M. W. Purification and characterization of pyruvate:ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus. Biochim. Biophys. Acta. 1993;1161:19–27. [PubMed: 8380721]
5.
Blamey J. M., Adams M. W. Characterization of an ancestral type of pyruvate:ferredoxin oxidoreductase from the hyperthermophilic bacteriumThermotoga maritima. Biochemistry. 1994;33:1000–1007. [PubMed: 8305426]
6.
Burns B. P., Hazell S. L., Mendz G. L. Acetyl-CoA carboxylase in Helicobacter pylori and the requirement for increased CO2 for growth. Microbiology. 1995;141:3113–3118. [PubMed: 8574404]
7.
Chalk P. A., Roberts A. D., Blows W. M. Metabolism of pyruvate and glucose by intact cells of H. pylori studied by 13C-NMR spectroscopy. Microbiology. 1994;140:2085–2092. [PubMed: 7921258]
8.
Chang H. T., Marcelli S. W., Davison A. A., Chalk P. A., Poole R. K., Miles R. J. Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori. FEMS Microbiol. Lett. 1995;129:33–38. [PubMed: 7781988]
9.
Charbriere E., Charon M., Volbeda A., Pieulle L., Hatchikian E., Fontecilla-Camps J. Crystal structure of the key anaerobic enzyme pyruvate:ferredoxin oxidoreductase, free and in complex with pyruvate. Nat. Struct. Biol. 1999;6:182–190. [PubMed: 10048931]
10.
Cordwell S. J. Microbial genomes and "missing" enzymes: redefining biochemical pathways. Arch. Microbiol. 1999;172:269–279. [PubMed: 10550468]
11.
Corthesy-Theulaz I. E., Bergonzelli G. E., Hemry H., Bachmann D., Schorderet D. F., Blum A. L., Ornston L. N. Cloning and characterization of Helicobacter pylori succinyl CoA: acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family. J. Biol. Chem. 1997;272:25659–25667. [PubMed: 9325289]
12.
Cunningham L., Gruer M. J., Guest J. R. Transcriptional regulation of the aconitase genes (acnA and acnB) of Escherichia coli. Microbiology. 1997;143:3795–3805. [PubMed: 9421904]
13.
Davison A. A., Kelly D. J., White P. J., Chalk P. A. Citric-acid cycle enzymes and respiratory metabolism in H. pylori. Acta Gastro-Enterol. Belg. 1993;56S:96.
14.
Docampo R., Moreno S. N. J., Mason R. P. Free radical intermediates in the reaction of pyruvate:ferredoxin oxidoreductase in Tritrichomonas foetus hydrogenosomes. J. Biol. Chem. 1987;262:12417–12420. [PubMed: 3040744]
15.
Dunkley M. L., Harris S. J., McCoy R. J., Musicka M. J., Eyers F. M., Beagley L. G., Lumley P. J., Beagley K. W., Clancy R. L. Protection against Helicobacter pylori infection by intestinal immunization with a 50/52-kDa subunit protein. FEMS Immunol. Med. Microbiol. 1999;24:221–225. [PubMed: 10378424]
16.
Ge Z. Q., Taylor D. E. The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis. J. Bacteriol. 1997;179:4970–4976. [PMC free article: PMC179351] [PubMed: 9260935]
17.
Ge Z., Jiang Q., Kalisiak M. S., Taylor D. E. Cloning and functional characterization of Helicobacter pylori fumarate reductase operon comprising three structural genes coding for subunits C, A and B. Gene. 1997;204:227–234. [PubMed: 9434188]
18.
Haque M., Hirai Y., Yokota K., Mori N., Jahan I., Ito H., Hotta H., Yano I., Kanemasa Y., Oguma K. Lipid profile of Helicobacter spp.: presence of cholesteryl glucoside as a characteristic feature. J. Bacteriol. 1996;178:2065–2070. [PMC free article: PMC177906] [PubMed: 8606185]
19.
Heath R. J., Rock C. O. A missense mutation accounts for the defect in the glycerol-3-phosphate acyltransferase expressed in the plsB26 mutant. J. Bacteriol. 1999;181:1944–1946. [PMC free article: PMC93600] [PubMed: 10074094]
20.
Hirai Y., Haque M., Yoshida T., Yokota K., Yasuda T., Oguma K. Unique cholesteryl glucosides in Helicobacter pylori: composition and structural analysis. J. Bacteriol. 1995;177:5327–5333. [PMC free article: PMC177327] [PubMed: 7665522]
21.
Hoffman P. S., Goodwin A., Johnsen J., Magee K., Veldhuzyen van Zanten S. J. O. Metabolic activities of metronidazole-sensitive and resistant strains of Helicobacter pylori: repression of pyruvate oxidoreductase and expression of isocitrate lyase activity correlate with resistance. J. Bacteriol. 1996;178:4822–4829. [PMC free article: PMC178263] [PubMed: 8759844]
22.
Hughes N. J., Chalk P. A., Clayton C. L., Kelly D. J. Identification of carboxylation enzymes and characterization of a novel four subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori. J. Bacteriol. 1995;177:3953–3959. [PMC free article: PMC177123] [PubMed: 7608066]
23.
Hughes N. J., Clayton C. L., Chalk P. A., Kelly D. J. Helicobacter pylori porCDAB and oorDABC genes encode distinct pyruvate:flavodoxin and 2-oxoglutarate:acceptor oxidoreductases which mediate electron transport to NADP. J. Bacteriol. 1998;180:1119–1128. [PMC free article: PMC106998] [PubMed: 9495749]
24.
Huynen M. A., Dandekar T., Bork P. Variation and evolution of the citric-acid cycle: a genomic perspective. Trends Microbiol. 1999;7:281–291. [PubMed: 10390638]
25.
Ingeldew W. J., Poole R. K. The respiratory chains of Escherichia coli. Microbiol. Rev. 1984;48:222–271. [PMC free article: PMC373010] [PubMed: 6387427]
26.
Kaihovaara P., Hook-Nikanne J., Uusi-Oukari M., Kosunen T. U., Salaspuro M. Flavodoxin-dependent pyruvate oxidation, acetate production and metronidazole reduction by Helicobacter pylori. J. Antimicrob. Chemother. 1998;41:171–177. [PubMed: 9533458]
27.
Kather B., Stingl K., van der Rest M. E., Altendorf K., Molenaar D. Another unusual type of citric-acid cycle enzyme in Helicobacter pylori: the malate:quinone oxidoreductase. J. Bacteriol. 2000;182:3204–3209. [PMC free article: PMC94508] [PubMed: 10809701]
28.
Kelly D. J. The physiology and metabolism of the human gastric pathogen Helicobacter pylori. Adv. Microb. Physiol. 1998;40:137–189. [PubMed: 9889978]
29.
Kerscher L., Oesterhelt D. Purification and properties of two 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. Eur. J. Biochem. 1981;116:587–594. [PubMed: 6266826]
30.
Kerscher L., Oesterhelt D. The catalytic mechanism of 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. Eur. J. Biochem. 1981;116:595–600. [PubMed: 6266827]
31.
Knappe J., Sawers G. A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli. FEMS Microbiol. Rev. 1990;6:383–398. [PubMed: 2248795]
32.
Kroger A., Geisler V., Lemma E., Theis F., Lenger R. Bacterial fumarate respiration. Arch. Microbiol. 1992;158:311–314.
33.
LaPorte D. C., Chung T. A single gene codes for the kinase and phosphatase which regulates isocitrate dehydrogenase. J. Biol. Chem. 1985;260:15291–15297. [PubMed: 2999109]
34.
Larson T. J., Ludtke D. N., Bell R. M. sn-Glycerol-3-phosphate auxotrophy of plsB strains of E. coli: evidence that a second mutation, plsX, is required. J. Bacteriol. 1984;160:711–717. [PMC free article: PMC214795] [PubMed: 6094487]
35.
Lichtenberger L. M., Hazell S. L., Ramero J. J., Graham D. Y. Helicobacter pylori hydrolysis of artificial lipid monolayers: insight into a potential mechanism of mucosal injury. Gastroenterology. 1990;98:A78.
36.
McAtee C. P., Fry K. E., Berg D. E. Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by "proteome" technologies. Helicobacter. 1998;3:163–169. [PubMed: 9731985]
37.
Meinecke B., Bertram J., Gottschalk G. Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum. Arch. Microbiol. 1989;152:244–250. [PubMed: 2774799]
38.
Mendz G. L., Hazell S. L. Fumarate catabolism in Helicobacter pylori. Biochem. Mol. Biol. Int. 1993;31:325–332. [PubMed: 8275020]
39.
Mendz G. L., Hazell S. L. Aminoacid utilization by Helicobacter pylori. Int. J. Biochem. Cell. Biol. 1995;27:1085–1093. [PubMed: 7496998]
40.
Mendz G. L., Hazell S. L., Srinivasan S. Fumarate reductase: a target for therapeutic intervention against Helicobacter pylori. Arch. Biochem. Biophys. 1995;321:153–159. [PubMed: 7639515]
41.
Narindrasorasak S., Goldie A. H., Sanwal B. D. Characteristics and regulation of a phospholipid-activated malate oxidase from Escherichia coli. J. Biol. Chem. 1979;254:1540–1545. [PubMed: 368072]
42.
Ottlecz A., Romero J. J., Hazell S. L., Graham D. Y., Lichtenberger L. M. Phospholipase activity of Helicobacter pylori and its inhibition by bismuth salts. Biochem. Biophys. Res. Commun. 1993;38:2071–2080. [PubMed: 8223083]
43.
Patel M. S., Roche T. E. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J. 1990;4:3224–3233. [PubMed: 2227213]
44.
Pieulle L., Charon M. H., Bianco P., Bonicel J., Petillot Y., Hatchikian E. C. Structural and kinetic studies of the pyruvate-ferredoxin oxidoreductase/ferredoxin complex from Desulfovibrio africanus. Eur. J. Biochem. 1999;264:500–508. [PubMed: 10491097]
45.
Pieulle L., Guigliarelli B., Asso M., Dole F., Bernadec A., Hatchikian E. C. Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate reducing bacteria Desulfovibrio africanus. Biochim. Biophys. Acta. 1995;1250:49–59. [PubMed: 7612653]
46.
Pitson S. M., Mendz G. L., Srinivasan S., Hazell S. L. The tricarboxylic acid cycle of Helicobacter pylori. Eur. J. Biochem. 1999;260:258–267. [PubMed: 10091606]
47.
Rock C. O., Cronan J. E. Escherichia coli as a model for the regulation of dissociable (type II) fatty acid biosynthesis. Biochim. Biophys. Acta. 1996;1302:1–16. [PubMed: 8695652]
48.
Rosenthal B., Mai Z., Caplivski D., Ghosh S., de la Vega H., Graf T., Samuelson J. Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica. J. Bacteriol. 1997;179:3736–3745. [PMC free article: PMC179172] [PubMed: 9171424]
49.
Shah V. K., Stacey G., Brill W. J. Electron transport to nitrogenase: purification and characterization of pyruvate: flavodoxin oxidoreductase, the nifJ gene product. J. Biol. Chem. 1983;258:12064–12068. [PubMed: 6352705]
50.
Smith E. T., Blamey J. M., Adams M. W. Pyruvate ferredoxin oxidoreductases of the hyperthermophilic archaeon, Pyrococcus furiosus and the hyperthermophilic bacterium Thermatoga maritima have different catalytic mechanisms. Biochemistry. 1994;33:1008–1016. [PubMed: 8305427]
51.
Spiro S., Guest J. R. Adaptive responses to oxygen limitation in Escherichia coli. Trends Biochem. Sci. 1991;61:310–314. [PubMed: 1957353]
52.
Tersteegen A., Linder D., Thauer R. K., Hedderich R. Structures and functions of four anabolic 2-oxoacid oxidoreductases in Methanobacterium thermoautotrophicum. Eur. J. Biochem. 1997;244:862–868. [PubMed: 9108258]
53.
Tomb J.-F., White O., Kerlavage A. R., Clayton R. A., Sutton G. G., Fleishmann R. D., Ketchum K. A., Klenk H. P., Gill S., Dougherty B., Nelson K., Quackenbush J., Zhou L., Kirkness E. F., Peterson S., Loftus B., Richardson D., Dodson R., Khalak H. G., Glodek A., McKenney K., Fitzegerald L. M., Lee N., Adams M. D., Hickey E., Berg D. E., Gocayne J. D., Utterback T. R., Peterson J. D., Kelley J. M., Cotton M. D., Weidman J. M., Fujii C., Bowman C., Watthey L., Wallin E., Hayes W. S., Borodovsky M., Karp P. D., Smith H. O., Fraser C. M., Venter J. C. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 1997;388:539–547. [PubMed: 9252185]
54.
Weitkamp H. J. H., Perez-Perez G. I., Bode G., Malfertheiner P., Blaser M. J. Identification and characterization of Helicobacter pylori phospholipase C activity. Int. J. Med. Microbiol. Virol. Parisitol. Infect. Dis. 1993;280:11–27. [PubMed: 8280931]
55.
Williams K., Lowe P. N., Leadlay P. F. Purification and characterization of pyruvate ferredoxin oxidoreductase from the anaerobic protozoan Trichomonas vaginalis. Biochem. J. 1987;246:529–536. [PMC free article: PMC1148305] [PubMed: 3500709]
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