* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download L-Serine, D- and L-proline and alanine as respiratory substrates of
Survey
Document related concepts
Peptide synthesis wikipedia , lookup
Butyric acid wikipedia , lookup
Metalloprotein wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
Point mutation wikipedia , lookup
Proteolysis wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
15-Hydroxyeicosatetraenoic acid wikipedia , lookup
Protein structure prediction wikipedia , lookup
Citric acid cycle wikipedia , lookup
Fatty acid metabolism wikipedia , lookup
Specialized pro-resolving mediators wikipedia , lookup
Genetic code wikipedia , lookup
Biosynthesis wikipedia , lookup
Transcript
Microbiology (2003), 149, 2023–2030 DOI 10.1099/mic.0.26203-0 L-Serine, D- and L-proline and alanine as respiratory substrates of Helicobacter pylori: correlation between in vitro and in vivo amino acid levels Kumiko Nagata,1 Yoko Nagata,2 Tadashi Sato,3 Masayuki A. Fujino,3 Kazuhiko Nakajima1 and Toshihide Tamura1 1 Department of Bacteriology, Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501, Japan Correspondence Kumiko Nagata 2 [email protected] Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda, Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan 3 First Department of Medicine, Yamanashi Medical University, Kofu, Yamanashi, 409-3898, Japan Received 23 December 2002 Revised 10 April 2003 Accepted 22 April 2003 Helicobacter pylori whole cells showed high rates of oxygen uptake with L-serine and L-proline as respiratory substrates, and somewhat lower rates with D-alanine and D-proline. These respiratory activities were inhibited by rotenone and antimycin A at low concentrations. Since pyruvate was produced from L-serine and D- and L-alanine in whole cells, the respiratory activities with these amino acids as substrates occurred via pyruvate. Whole cells showed 2,6-dichlorophenolindophenol (DCIP)-reducing activities with D- and L-proline and D-alanine as substrates, suggesting that hydrogen removed from these amino acids also participated in oxygen uptake by the whole cells. High amounts of L-proline, D- and L-alanine, and L-serine were present in H. pylori cells, and these amino acids also predominated in samples of human gastric juice. H. pylori seems to utilize D- and L-proline, D-alanine and L-serine as important energy sources in its habitat of the mucous layer of the stomach. INTRODUCTION Helicobacter pylori is a Gram-negative bacterium associated with gastric inflammation and peptic ulcer disease and is a risk factor for gastric cancer (Blaser, 1990; Parsonnet et al., 1991, 1994; Rabeneck & Ransohoff, 1991). The natural habitat of H. pylori is the mucous layer of the human gastric epithelium, where populations are considered to persist for the lifetime of the host. H. pylori is a microaerophilic bacterium exhibiting a strict respiratory form of metabolism and oxidizing organic acids as an energy source. The carbohydrate utilized by H. pylori as the energy source has been reported to be only glucose (Mendz et al., 1993). More recently the whole-genome analysis of H. pylori has supported these findings (Tomb et al., 1997). However, the incorporation of glucose from the culture medium was not influenced by inhibitors known to affect other bacterial glucose-related enzymes, such as glucose permease, and the Km value of its glucose transport was reported to be rather high, 4?8 mM (Burns & Mendz, 2001). In addition, glucose added to the culture medium composed of a mixture of amino acids was not utilized until the amino acids were Abbreviation: DCPIP, 2,6-dichlorophenolindophenol. 0002-6203 G 2003 SGM significantly depleted (Mendz et al., 1993; Mendz & Hazell, 1995). These results suggested that glucose is not a preferred energy substrate of H. pylori. Candidates for the substrates of energy metabolism in this organism are thought to be organic acids such as pyruvate, D-lactate and succinate. Chang et al. (1995) reported that lower concentrations (25 mM) of pyruvate, D-lactate and succinate were rapidly oxidized, and the respiration rates were relatively high, suggesting that H. pylori cells may have adapted to utilizing these substrates in vivo. However, the oxygen uptake during lactate and pyruvate oxidation was insufficient for their complete oxidation to CO2 and H2O via the citric acid cycle (Chang et al., 1995; Kelly et al., 2001). Thus, instead of organic acids, amino acids are considered to be other potential respiratory substrates and energy sources in H. pylori cells. Little study has been done on amino acids as respiratory substrates of H. pylori. H. pylori has genes encoding D-amino acid dehydrogenase (dadA), L-alanine dehydrogenase (ald) and L-serine deaminase (sdaB) (Tomb et al., 1997). These three enzymes produce pyruvate, which is the main respiratory substrate of H. pylori cells. In addition, H. pylori has a gene encoding proline dehydrogenase (putA), which is Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:41:11 Printed in Great Britain 2023 K. Nagata and others associated with the respiratory chain employing molecular oxygen as terminal electron acceptor in Escherichia coli and Salmonella typhimurium (Scarpulla & Soffer, 1978; Menzel & Roth, 1981). In this context, we have investigated the respiratory activity of intact whole H. pylori cells with alanine, serine and proline, and other amino acids of which both D- and L-isomers serve as respiratory substrates. In this report, we describe: (1) a high rate of utilization of L-serine and L-proline, followed by D-alanine and D-proline, as respiratory substrates; (2) an unusually high content of L-proline in H. pylori cells; and (3) the composition of free amino acids including D-isomers in gastric juice from patients infected with H. pylori and those from uninfected persons. These findings revealed an interesting relationship between some amino acids utilized as respiratory substrates and the composition of amino acids in gastric juice. METHODS Materials. Rotenone, antimycin A, D-cycloserine and DL-penicillamine were from Sigma. Other inhibitors, substrates of respiration and chemicals for enzyme assay were from Wako Pure Chemical Industries. The strain of H. pylori used, NCTC 11637, was cultured on Brucella agar plates (Becton Dickinson) containing 5 % newborn calf serum (Sigma) under 10 % CO2 in an incubator at 37 uC for 20 h. Cultured cells were harvested by centrifugation at 10 000 g for 15 min at 4 uC, and were suspended in phosphate-buffered saline (pH 7?2) for immediate assays of respiratory and enzymic activities. Respiration assay of H. pylori whole cells. Respiration of whole cells (5–106108 cells ml21) was monitored polarographically with a Clark-type oxygen electrode (YSI Inc.) in a semi-closed vessel containing a medium of 10 mM HEPES buffer (pH 7?0) and 0?9 % NaCl at 37 uC as described previously (Nagata et al., 2001). The medium was bubbled with nitrogen gas to bring the oxygen concentration to 75 mM. Various kinds of amino acids and pyruvate with a final concentration of 10 mM and inhibitors such as rotenone and antimycin A were introduced into the oxygen electrode vessel with a syringe. Respiratory activity [oxygen uptake min21 (mg protein)21] was determined based on polarographic traces of the oxygen electrode as described previously (Nagata et al., 2001). Enzyme assay and protein determination. Pyruvate production from amino acids was assayed as described previously (Nagata et al., 1988). Briefly, H. pylori cells were added to a reaction mixture containing 50 mM sodium phosphate buffer (pH 7?0), 10 mM NaN3 and 10 mM of amino acids. After 10 min incubation at 37 uC, 2,4-dinitrophenylhydrazine was added and the mixture kept at room temperature for 10 min. After addition of NaOH, hydrazone formation was measured by reading the A445 using a Beckman DU 640 spectrophotometer. The amount of pyruvate was calculated based on the A445 value of pure pyruvate as a standard. 2,6-Dichlorophenolindophenol (DCIP)-reducing activity was measured as follows. The cells were added to the reaction mixture containing 50 mM sodium phosphate buffer (pH 7?0), 10 mM of an amino acid and 0?5 mM DCIP. After 10 min incubation at 37 uC, the cells were removed by centrifugation at 10 000 g for 10 min and then the A600 of the supernatant was measured. An e600 of 21?6 mM21 cm21 was used. Ammonia production was measured in the same reaction mixture as used for the assay of pyruvate production except without addition of NaN3. After incubation at 37 uC, the cells were removed by centrifugation at 10 000 g for 10 min, and the amounts of ammonia in the supernatant were measured by indophenol formation using the 2024 ammonia test reagent kit (Wako Pure Chemical Industry) as described previously (Nagata et al., 1993). The A630 was measured. Activities of pyruvate and ammonia production and of DCIP reduction were obtained by subtracting the activities without amino acids from those with amino acids. The amount of protein of the whole H. pylori cells was determined by a modification of the Lowry procedure (Markwell et al., 1981). Determination of free D- and L-isomers of amino acids in human gastric juice and H. pylori cells. Gastric juice was col- lected from patients suspected of having gastroduodenal diseases. After pharyngeal anaesthesia with 1 % lidocaine hydrochloride, a disinfected endoscope was inserted into the stomach and gastric juice was collected through the aspiration channel of the endoscope. The status of H. pylori infection was determined from the results of culture and an immunological rapid urease test described previously (Sato et al., 2000). A patient was considered to be H. pylori-positive if the culture was positive. H. pylori-negative patients were defined as those with negative results for both tests. Gastroscopy was performed to evaluate the possibility of H. pylori infection. Informed consent was obtained from each patient prior to inclusion in the study. To prepare the gastric juice sample, 30 % trichloroacetic acid solution was added to the juice to a final concentration of 5 % (w/v). Sample preparation from H. pylori cells has been described previously (Nagata et al., 1998). The supernatant of the trichloroacetic-acidtreated sample was passed through a Dowex 50W68 (H+ form) column and eluted with 4 M NH4OH after washing with distilled water to obtain purified free amino acids. The eluate was evaporated to dryness in vacuo in a centrifugal evaporator (Taitec) below 4 uC. The determination of amino acids of each enantiomer was performed as described previously (Nagata et al., 1992). The free amino acids were treated with FDAA (Marfey’s reagent, Pierce) (Marfey, 1984) to form diastereomers of amino acids. The FDAA derivatives were separated on a Silica Gel 60 plate (Merck) by two-dimensional thin-layer chromatography. FDAA amino acids recovered from the plate were analysed by HPLC for the resolution of D- and L-enantiomers, using a reversed-phase column, Nova-Pak C18 (15063?9 mm i.d., Waters), and a Tosoh or Jasco gradient HPLC system. The amounts of D- and L-enantiomers of the amino acids were calculated based on the peak areas of the elution patterns as obtained by a Chromato-Integrator (D-2500, Hitachi, Tokyo). Known amounts of authentic D- and L-enantiomers of each amino acid examined were added to the samples as internal controls, and subsequently hydrolysed and analysed as described. RESULTS Respiratory activity of whole H. pylori cells with amino acids as substrates We polarographically measured oxygen uptake of whole cells using a Clark-type oxygen electrode in a vessel containing medium bubbled with nitrogen gas to create a low concentration of oxygen. Under these conditions, the respiratory activity of endogenous substrate in cells was low [about 3 nmol oxygen min21 (mg cellular protein)21]. Oxygen uptake of the whole H. pylori cells increased when pyruvate and D- and L-isomers of alanine, serine and proline were added to the cell suspension. Of the six amino acids, L-serine was found to be the most effective for increasing oxygen uptake, followed by L-proline, D-alanine and D-proline (Table 1). The respiratory activity on L-serine was comparable Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:41:11 Microbiology 149 Amino acid respiration in H. pylori Table 1. Respiration of H. pylori cells with D- and L-isomers of alanine, serine, proline and pyruvate as substrates, and the effect of respiratory inhibitors About 109 cells were added to an oxygen electrode vessel containing the medium with a low concentration of oxygen (75 mM). After 2?5 min, D-and L-isomers of alanine, serine or proline and pyruvate with a final concentration of 10 mM were added; various concentrations of rotenone or antimycin A were added after about 5 min. Data are means of duplicates from a representative experiment. Substrate Respiratory activity* Activity with inhibitors (% of control) Rotenone 5 mM D-Alanine Antimycin A 10 mM 20 mM 16?32 10?21 53?0 46?9 43?2 28?6 9?0 18?5 5?17 32?72 47?7 18?7 25?3 10?1 7?7 9?8 L-Proline 16?34 29?00 26?3 61?6 18?6 47?6 12?9 9?8 Pyruvate 34?22 58?7 26?2 26?2 L-Alanine D-Serine L-Serine D-Proline *Respiratory activity [nmol oxygen min21 (mg cellular protein)21] determined from polarographic traces of the oxygen electrode. to that on pyruvate. Cells incubated with D- and L-isomers of glutamate and aspartate, L-arginine and glycine did not show significant respiratory activity (data not shown). To determine whether these respiratory activities are associated with the respiratory chain, we examined the inhibitory activity of rotenone, which is a potent inhibitor of NADH-quinone oxidoreductase, and antimycin A, which is an inhibitor of the cytochrome bc1 complex. As shown in Table 1, the respiratory activities were inhibited by rotenone and antimycin A at low concentrations. The IC50 value (concentration giving 50 % inhibition) for rotenone against the respiration of D-alanine was about 8 mM, which was close to the IC50, 3–8 mM, for rotenone against the respiration of pyruvate (data not shown). These results suggested that D- and L-isomers of alanine, serine and proline were metabolized via the respiratory chain in H. pylori cells. D-alanine increased time-dependently as shown in Fig. 1. Amounts of ammonia similar to those of pyruvate were produced time-dependently from D-alanine, and the rates of production of pyruvate and ammonia were the same (Fig. 1). The apparent Km value for D-alanine in the pyruvate production was 0?14 mM (data not shown). Although the presence of the gene encoding D-alanine aminotransferase has not been reported for H. pylori, Pyruvate and ammonia production from D- and of amino acids in H. pylori cells L-isomers H. pylori has the genes dadA, ald and sdaB (Tomb et al., 1997), encoding enzymes that produce pyruvate and ammonia from D- and L-alanine and from L-serine, respectively. Thus, respiratory activities with these amino acids as substrates presumably occurred via pyruvate. We assayed pyruvateand ammonia-producing activities from these amino acids. However, whole H. pylori cells quickly utilized pyruvate as the respiratory substrate. To prevent this consumption of pyruvate, we added various amounts of NaN3 to the reaction mixture; the maximum pyruvate production from D-alanine was obtained at 10 mM NaN3 (data not shown). In the presence of 10 mM NaN3, the pyruvate production from http://mic.sgmjournals.org Fig. 1. Time-course of production of pyruvate (#) and ammonia (n) from D-alanine in H. pylori cells. Assays of pyruvate and ammonia production are described in Methods. Data are means of duplicates from a representative experiment. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:41:11 2025 K. Nagata and others an alanine aminotransferase may produce pyruvate and ammonia. The activity of D-alanine aminotransferase is dependent on pyridoxal 59-phosphate as a cofactor and is inhibited strongly by specific inhibitors of pyridoxal 59phosphate, such as D-cycloserine, DL-penicillamine, NH3OH and 3-methyl-2-benzothiazoline hydrazone hydrochloride (Yonaha et al., 1975). Since the activity of the pyruvate production from D-alanine was not affected significantly by those inhibitors (data not shown), pyruvate was probably not produced via the catalytic function of this enzyme in our experiments. Pyruvate production from L-alanine added to whole cells of H. pylori was about 50 % of that from D-alanine (Table 2). Ammonia production was also about half that for Dalanine, showing a specific activity of 2?46 nmol min21 (mg protein)21. These results indicated that pyruvate production from L-alanine was due to L-alanine dehydrogenase, suggesting that this enzyme activity was weak compared to the D-amino acid dehydrogenase activity on D-alanine. These facts were consistent with the results that the respiratory activity on L-alanine was lower than that on D-alanine, as shown in Table 1. High pyruvate production from L-serine was found in whole H. pylori cells (Table 2). The specific activity of ammonia production from L-serine was 6?28 nmol min21 (mg protein)21, which was similar to that for pyruvate production. Since the respiration with L-serine as substrate was inhibited by rotenone and antimycin A as for D-alanine (Table 1), the oxygen uptake may have been caused by pyruvate produced by L-serine deaminase. DCIP-reducing activity of whole H. pylori cells with D- and L-isomers of alanine, serine and proline as substrate D-Amino acid dehydrogenase and L-alanine dehydrogenase show DCIP-reducing activity with concomitant production of pyruvate and ammonia (Olsiewski et al., 1980). We examined the DCIP-reducing activity of whole H. pylori cells Table 2. Activities of pyruvate production and DCIP reduction by H. pylori cells with D- and L-isomers of alanine, serine and proline as substrates Data are means of duplicates from a representative experiment. Substrate Pyruvate production* DCIP reductionD D-Alanine 4?40 2?10 3?81 0?74 0?13 8?67 0?72 0?68 1?46 0?48 4?26 7?14 L-Alanine D-Serine L-Serine D-Proline L-Proline *nmol pyruvate min21 (mg cellular protein)21. Dnmol DCIP min21 (mg cellular protein)21. 2026 using D- and L-isomers of alanine. Since H. pylori cells have strong activity of cytochrome c oxidase (Nagata et al., 1996), the reduced DCIP is considered to be oxidized immediately. To prevent DCIP oxidation, we added various amounts of NaN3 to the reaction mixture used for the assay of DCIP reduction by D-alanine; the maximum DCIP reduction was obtained at 10 mM NaN3 (data not shown). In the presence of 10 mM NaN3, the specific activity of the DCIP reduction with D-alanine was close to that of pyruvate production with D-alanine (Table 2). L-Alanine and D- and L-serine added to whole cells of H. pylori led to low DCIP-reducing activities (Table 2). On the other hand, high activities of DCIP reduction appeared with D- and L-proline, although the activities of pyruvate production from these amino acids were low (Table 2). Free amino acid composition of human gastric juice and H. pylori cells Table 3 shows the contents of free D- and L-isomers of alanine, serine, proline, aspartate and glutamate in human gastric juices from H. pylori-infected and uninfected subjects. The content of L-proline in infected specimens was significantly higher than that in uninfected ones. The contents of D- and L-alanine and L-serine were also high. No significant difference was observed in amino acid composition, except for L-proline, between infected and uninfected specimens. As shown in Table 4, H. pylori cells contained extremely large amounts of L-proline and considerable amounts of D- and L-alanine, L-serine and D-aspartate. The content of glutamate was low. DISCUSSION Although blood- or serum-containing media are commonly used for routine culture of H. pylori, the nature of the carbon and energy sources used in the host is unknown. H. pylori is very limited in its use of oxidizable carbon substrates (Doig et al., 1999). In fact, glucose does not seem to be the main energy source for H. pylori (Kelly et al., 2001); instead of glucose, organic acids and amino acids are considered to serve as potential respiratory substrates and energy sources. Utilization of amino acids by H. pylori has been investigated under various culture conditions (Mendz & Hazell, 1995; Reynolds & Penn, 1994; Stark et al., 1997). Stark et al. (1997) reported that amino acids such as alanine, arginine, asparagine, aspartate, glutamine, glutamate, proline and serine were mainly consumed from continuous culture medium by H. pylori cells, and suggested that the products of metabolism of these amino acids can be further metabolized as energy sources by enzymes of the TCA cycle (Stark et al., 1997). Chang et al. (1995) did not detect oxygen uptake when acetate, glycerol, L-lactate, oxaloacetate and amino acids such as aspartate and glutamate were added to H. pylori cells. However, they did not study the utilization of other amino acids, including the D-isomers, as respiratory substrates. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:41:11 Microbiology 149 Amino acid respiration in H. pylori Table 3. Free D- and L-isomers of amino acids in human gastric juices from subjects infected with H. pylori and from uninfected normal subjects Statistical analyses were carried out using Student’s t-test. Gastric juice* Amino acid (nmol per ml gastric juice) Ala D Ser Pro L D L 1 4?5 2 4?2 3 4?6 4 52?0 5 3?4 6 57?8 Mean±SE 21?1±10?7 12?6 12?5 57?4 49?3 38?1 101?8 45?3±13?6 0?3 0?0 0?0 0?0 0?3 1?1 0?3±0?2 3?0 3?3 78?8 37?1 9?3 67?5 33?2±13?7 7 8 9 10 11 12 Mean±SE 64?0 0?0 781?8 226?0 0?0 92?1 349?1 0?0 357?5 28?8 0?0 21?5 55?8 0?0 12?3 42?4 0?0 10?1 127?7±53?2 0?0±0?0 212?6±126?1 18?8 13?2 60?4 3?3 5?5 1?9 17?2±9?0 Asp L** D D L 0?0 189?8 0?0 0?0 180?7 0?4 9?6 109?8 3?9 90?4 241?6 0?8 1?0 11?4 0?1 3?2 16?8 0?0 17?4±14?5 125?0±39?0 0?9±0?6 3?5 1?2 6?0 0?1 2?7 0?6 2?4±0?9 13?9 8?2 18?0 8?5 8?3 2?7 10?1±2?1 Glu D 0?0 0?9 43?1 14?4 2?1 0?0 10?1±7?0 L 0?0 0?3 0?0 2?3 0?1 18?7 0?5 12?9 0?2 22?3 1?4 40?3 0?5±0?4 16?1±6?0 1?2 109?0 0?1 0?0 10?1 0?0 0?0 28?2 0?0 2?3 15?1 0?5 0?1 3?6 0?5 0?1 0?7 0?0 0?5±0?4 27?8±16?7 0?2±0?1 7?3 0?9 1?9 22?0 10?5 1?8 7?4±3?3 *Gastric juices nos 1–6 were from persons infected with H. pylori and nos 7–12 from uninfected persons. **P<0?05, specimens 1–6 compared with specimens 7–12. In this report, we have presented evidence that H. pylori cells predominantly utilized D- and L-alanine and proline and also L-serine as respiratory substrates as well as pyruvate. The respiratory activities with these amino acids as substrate were inhibited by the respiratory inhibitors rotenone and antimycin A at low concentrations (Table 1), suggesting the respiratory activities to be coupled with ATP production. Although D-alanine as well as D-glutamate contributes to the synthesis of the cell wall of bacteria, other physiological functions and the metabolic pathways of these D-amino acids are equivocal in H. pylori cells. In the present study, we demonstrated that whole H. pylori cells utilized D-alanine but not D-glutamate as a respiratory substrate. As genome sequence analysis shows that H. pylori has the dadA gene, the reaction is considered to be the result of D-amino acid dehydrogenase function. Thus, oxygen uptake with D-alanine as substrate seems to have occurred via pyruvate, which is the main respiratory substrate in H. pylori cells (Mendz et al., 1994). Considerably high activities of oxygen uptake and pyruvate production were found when L-serine was added Table 4. Free D- and L-isomers of amino acids in H. pylori cells H. pylori cells were cultured on Brucella agar plates containing 5 % newborn calf serum under 10 % CO2 in an incubator at 37 uC for 2 days. Harvested cells were washed once with phosphate-buffered saline (pH 7?2). Sample of cells Amino acid (nmol per g wet weight of cells) Ala D Ser L D Pro L D Asp L D Glu L D L 1 2?9 4?9 0?1 3?5 6?1 587?5 1?0 0?5 0?0 0?0 2 106?3 90?4 0?0 146?7 5?6 441?7 14?2 23?4 0?6 22?2 3 196?9 203?8 12?5 93?1 8?1 672?5 328?8 20?6 21?8 1?4 4 195?6 168?1 17?5 144?4 5?8 350?0 179?3 22?5 0?0 0?0 5 150?8 144?3 23?9 136?9 8?9 520?5 118?7 8?3 0?0 0?0 6 294?4 266?7 29?1 310?6 7?5 597?3 176?6 11?3 0?0 0?0 Mean±SE 157?8±40?1 146?3±37?1 13?9±5?0 139?2±40?8 7?0±0?6 528?3±47?8 136?4±49?7 14?4±3?8 3?7±3?6 3?9±3?7 http://mic.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:41:11 2027 K. Nagata and others to whole cells of H. pylori (Tables 1 and 2). As H. pylori has a sdaA gene, this activity seems likely to be due to serine deaminase. The presence of D-amino acid dehydrogenase has been reported in Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhimurium and Escherichia coli (Jones & Venables, 1983; Magor & Venables, 1987; Olsiewski et al., 1980; Tsukada et al., 1966; Wild et al., 1974). However, its metabolic function has not been clarified. D-Amino acid dehydrogenase and L-alanine dehydrogenase remove hydrogen from substrates of amino acids in the process of producing pyruvate and ammonia. Although the substances reduced by this hydrogen have not yet been identified in H. pylori cells, this hydrogen may be transferred to oxygen via the electron-transfer system leading to oxygen uptake by whole cells. However, it has not been clarified to what extent this hydrogen, besides pyruvate, participates in the oxygen uptake by D- and L-alanine in H. pylori cells. In H. pylori, pyruvate is dehydrogenated by different dehydrogenase systems from those in the usual proteobacteria producing NADH, which is the major electron donor in the respiratory chain (Hughes et al., 1995; Kelly, 1998). Twostep systems generating NADPH seem to be present in H. pylori; pyruvate may be oxidized by flavodoxin, and the reduced flavodoxin reduces NADP+. NADPH-menaquinone oxidoreductase oxidized NADPH, and the menaquinone may be oxidized by the cytochrome bc1 complex of the respiratory chain. Thus, the reducing system in pyruvate may be different from that in the hydrogen removed from D- and L-alanine. Moreover, H. pylori showed marked activities of oxygen uptake with L- and D-proline as substrate, which were also inhibited by respiratory inhibitors (Table 2). E. coli, S. typhimurium and P. aeruginosa have the gene putA, whose product, PutA, shows L-proline dehydrogenase activity (Ling et al., 1994; Vinod et al., 2002). Recently, Satomura et al. (2002) reported that the hyperthermophilic archaeon Pyrobaculum islandicum has a dye-linked D-proline dehydrogenase which is different from D-amino acid dehydrogenase. Both the L- and D-proline dehydrogenases have been reported to show DCIP reduction activity by the respective amino acids (Abrahamson et al., 1983; Satomura et al., 2002). In the present experiments, we demonstrated that H. pylori whole cells showed DCIP-reducing activity with D- and L-proline as substrate (Table 2). H. pylori has the genes putP and putA, which encode proline permease and L-proline dehydrogenase, respectively (Tomb et al., 1997). However, the gene corresponding to D-proline dehydrogenase has not been reported. From the genome sequence database, H. pylori dadA encoding D-amino acid dehydrogenase shows low DNA similarity (24 %) with Pb. islandicum dye-linked D-proline dehydrogenase (Satomura et al., 2002). Since the substrate specificity of D-amino acid dehydrogenase may not be strict, this dehydrogenase may metabolize D-proline as a substrate for DCIP-reducing activity as well as D-alanine. The hydrogen atoms removed 2028 from D- and L-proline by H. pylori whole cells may reduce a flavoprotein-like substance, and electrons produced concomitantly reduce oxygen via the electron-transport chain. Thus, different dehydrogenases and electron-transport pathways seem to participate in the oxidation of D- and L-isomers of alanine and proline. As shown in Table 2, the inhibitory action of rotenone and antimycin A against respiratory activities varied considerably among respiratory substrates, being from 10 to 48 % of the control in the case of rotenone. These results may be due to the participation of different dehydrogenases and electron pathways depending on respiratory substrates. Olson & Maier (2002) reported recently that H. pylori cells use molecular hydrogen as an energy source in mice. In the stomach of mice many kinds of anaerobic bacteria are present, in high density, and the molecular hydrogen derived from these anaerobic bacteria seems to be dominant. On the other hand, there are few bacteria in the human stomach, where amino acids are dominant owing to the degradation products of food, including L-serine derived from degradation of mucin. Since H. pylori resides in the lower layers of the gastric mucus, it is considered to use various amino acids diffused from the gastric juice as an energy source, although we cannot exclude the possibility that molecular hydrogen is also used by H. pylori in the human stomach as discussed by Olson & Maier (2002). Previously, we reported the occurrence of free D-amino acids in various bacteria including H. pylori (Nagata et al., 1998). In the present study, we showed that of the amino acids contained in H. pylori cells, L-proline was present at the highest level, followed by D- and L-alanine, L-serine and D-aspartate (Table 4). All these amino acids except D-aspartate were used predominantly as respiratory substrates by H. pylori whole cells as described above. We also analysed the L- and D-amino acid content of human gastric juice, which to the best of our knowledge is the first report of such measurement. We found considerable amounts of L- and D-isomers of alanine and proline, and of L-serine, in the gastric juice (Table 3). Again, these amino acids coincided with the respiratory substrates used predominantly by H. pylori. Specimens from patients with H. pylori showed a significantly higher level of L-proline than those from uninfected subjects, although there was no significant difference in the contents of other free amino acids between the patients and the uninfected control group (Table 3). There is the possibility that the free amino acids in gastric juice infected with H. pylori cells were derived from H. pylori cells since this organism contains a large amount of L-proline. H. pylori cells possess alanine racemase activity that converts L-alanine to D-alanine (unpublished observation). All these results, together with an apparently low Km value for the pyruvate-producing reaction from D-alanine, suggest that H. pylori cells utilize D-alanine, L-serine, and D- and L-proline as the main energy sources in their habitat of the mucous layer. H. pylori cells possess high levels of urease. The hydrolysis of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:41:11 Microbiology 149 Amino acid respiration in H. pylori urea produces ammonia, which protects the organism from the highly acidic environment of the stomach. Not only urease but also enzymes that produce ammonia from amino acids, such as D-amino acid and L-alanine dehydrogenase, and L-serine deaminase, may contribute to protecting H. pylori cells from their acidic environment. Thus, the present study on the utilization of L-serine and D- and L-isomers of alanine in H. pylori cells offers important information on H. pylori metabolism, not only related to the energy source but also to protection measures against an acidic environment. These findings should be helpful for developing new anti-H. pylori therapies. and effect of solubilization on its properties. Biochemie 69, 63–69. Marfey, P. (1984). Determination of D-amino acids. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res Commun 49, 591–596. Markwell, M. A. K., Haas, S. M., Tolbert, N. E. & Bieber, L. L. (1981). Protein determination in membrane and lipoprotein samples; manuals and automated procedures. Methods Enzymol 72, 1920–1931. Mendz, G. L. & Hazell, S. L. (1993). Glucose phosphorylation in Helicobacter pylori. Arch Biochem Biophys 300, 522–525. Mendz, G. L. & Hazell, S. L. (1995). Amimo acid utilization by Helicobacter pylori. Int J Biochem Cell Biol 27, 1085–1093. Mendz, G. L., Hazell, S. L. & Burns, B. P. (1993). Glucose utilization and lactate production by Helicobacter pylori. J Gen Microbiol 139, 3023–3028. ACKNOWLEDGEMENTS Mendz, G. L., Hazell, S. L. & van Gorkom, L. (1994). Pyruvate We are indebted to Dr Nobuhito Sone of the Department of Biological Engineering, Kyushu Institute of Technology, for his valuable discussion. metabolism in Helicobacter pylori. Arch Microbiol 162, 187–192. Menzel, R. & Roth, J. (1981). Enzymatic properties of the purified put A protein from Salmonella typhimurium. J Biol Chem 256, 9762–9766. Nagata, Y., Shimojo, T. & Akino, T. (1988). Two spectrophotometric REFERENCES Abrahamson, J. L. A., Baker, L. G., Stephenson, J. T. & Wood, J. M. (1983). Proline dehydrogenase from Escherichia coli K12. Properties for the membrane-associated enzyme. Eur J Biochem 134, 77–82. Blaser, M. J. (1990). Helicobacter pylori and the pathogenesis of gastroduodenal inflammation. J Infect Dis 161, 626–633. Burns, B. P. & Mendz, G. L. (2001). Metabolic transport. In Helicobacter pylori: Physiology and Genetics, pp. 207–217. Edited by H. L. T. Mobley, G. L. Mendz & S. L. Hazell. Washington, DC: American Society for Microbiology. Chang, H. T., Marcelli, S. W., Davison, A. A., Chalk, P. A., Poole, R. K. & Miles, R. J. (1995). Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori. FEMS Microbiol Lett 129, 33–38. Doig, P., Jonge, B. L., Alm, R. A. & 10 other authors (1999). Helicobacter pylori physiology predicted from genomic comparison of two strains. Microbiol Mol Biol Rev 63, 675–707. Hughes, N. J., Chalk, P. A., Clayton, C. L. & Kelly, D. J. (1995). Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate : flavodoxin oxidoreductase from Helicobacter pylori. J Bacteriol 177, 3953–3959. Jones, H. & Venables, W. A. (1983). Effects of solubilization on assays for D-amino acid oxidase: the study of distribution patterns. Int J Biochem 20, 1235–1238. Nagata, K., Satoh, H., Iwahi, T., Shimoyama, T. & Tamura, T. (1993). Potent inhibitory action of the gastric proton pump inhibitor lansoprazole against urease activity of Helicobacter pylori: unique action selective for H. pylori cells. Antimicrob Agents Chemother 37, 769–774. Nagata, K., Tsukita, S., Tamura, T. & Sone, N. (1996). A cb-type cytochrome-c oxidase terminates the respiratory Helicobacter pylori. Microbiology 142, 1757–1763. chain in Nagata, Y., Fujiwara, T., Kawaguchi-Nagata, K., Fukumori, Y. & Yamanaka, T. (1998). Occurrence of peptidyl D-amino acids in soluble fractions of several eubacteria, archaea and eukaryotes. Biochim Biophys Acta 1379, 76–82. Nagata, Y., Yamamoto, K. & Shimojo, T. (1992). Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography. J Chromatogr 575, 147–152. Nagata, K., Sone, N. & Tamura, T. (2001). Inhibitory activities of lansoprazole against respiration in Helicobacter pylori. Antimicrob Agents Chemother 45, 1522–1527. Olsiewski. , Kaczorowski, G. J. & Walsh, C. (1980). Purification and properties of D-amino acid dehydrogenase, an inducible membranebound iron-sulfur flavoenzyme from Escherichia coli B. J Biol Chem 255, 4487–4494. some properties of the membrane-bound respiratory enzyme acid dehydrogenase of Escherichia coli. FEBS Lett 151, 189–192. Olson, J. W. & Maier, R. J. (2002). Molecular hydrogen as an energy Kelly, D. J. (1998). The physiology and metabolism of the Parsonnet, J., Friedman, G. D. Vandersteen D. P., Chang, Y., Vogelman, J. H., Orentreich, N. & Sibley, R. K. (1991). Helicobacter D-amino human gastric pathogen Helicobacter pylori. Adv Microb Physiol 40, 136–189. Kelly, D. J., Hughes, N. J. & Poole, R. K. (2001). Microaerobic source for Helicobacter pylori. Science 298, 1788–1790. pylori infection and the risk of gastric carcinoma. N Engl J Med 325, 1127–1131. physiology: aerobic respiration, anaerobic respiration, and carbon dioxide metabolism. In Helicobacter pylori: Physiology and Genetics, pp. 113–126. Edited by H. L. T. Mobley, G. L. Mendz & S. L. Hazell. Washington, DC: American Society for Microbiology. Parsonnet, J., Hansen, S., Rodriguez, L., Gelb, A. B., Warnke, R. K., Jellum, E., Orentrich, N., Vogelman, J. H. & Friedman, G. D. (1994). Ling, M., Allen, S. W. & Wood, J. M. (1994). Sequence analysis identifies the proline dehydrogenase and d1–pyrroline-5-carboxylate Rabeneck, L. & Ransohoff, D. F. (1991). Is Helicobacter pylori a cause of duodenal ulcer? A methodologic critique of current evidence. Am J Med 91, 566–572. dehydrogenase domains of the multifunctional Escherichia coli PutA protein. J Mol Biol 243, 950–956. Magor, A. M. & Venables, W. A. (1987). Solubilization, purifica- tion of D-amino acid dehydrogenase from Pseudomonas aeruginosa http://mic.sgmjournals.org Helicobacter pylori infection and gastric lymphoma. N Engl J Med 330, 1267–1271. Reynolds, D. J. & Penn, C. W. (1994). Characteristics of Helicobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology 140, 2649–2656. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:41:11 2029 K. Nagata and others Sato, T., Fujino, M. A., Koijima, Y., Kitahara, F., Morozumi, A. Nagata K., Nakamura, M. & Hosaka, H. (2000). Evaluation of immunological rapid urease testing for detection of Helicobacter pylori. Eur J Microbiol Infect Dis 19, 438–442. Tomb, J. F., White, O., Kerlavage, A. & 39 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539–547. Tsukada, K. (1966). D-Amino acid dehydrogenase of Pseudomonas Satomura, T., Kawakami, R., Sakuraba, H. & Ohshima, T. (2002). fluorescens. J Biol Chem 241, 4522–4528. Dye-linked D-proline dehydrogenase from hyperthermophilic archaeon Pyrobaculum islandicum is a novel FAD-dependent amino acid dehydrogenase. J Biol Chem 277, 12861–12867. Vinod, M. P., Bellur, P. & Becker, D. F. (2002). Electrochemical and Scarpulla, R. C. & Soffer, R. J. (1978). Membrane-bound proline Wild, J., Walczak, W., Krajewaka-Crynkiewicz, K. & Klopotowski, T. (1974). D-Amino acid dehydrogenase: the enzyme of the first dehydrogenase from Escherichia coli. Solubilization, purification, and characterization. J Biol Chem 253, 5997–6001. Stark, R. M., Suleiman, M. S., Hassan, I. J., Greenman, J. & Millar, M. R. (1997). Amino acid utilization and deamination of glutamine and asparagine by Helicobacter pylori. J Med Microbiol 46, 793–800. 2030 functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli. Biochemistry 41, 6525–6532. step of D-histidine and D-methionine racemization in Salmonella typhimurium. Mol Gen Genet 128, 131–146. Yonaha, K., Misono, H., Yamamoto, T. & Soda, K. (1975). D-Amino acid aminotransferase of Bacillus sphaericus. J Biol Chem 250, 6983–6989. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:41:11 Microbiology 149