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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
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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
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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
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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.
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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.
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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
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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
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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.
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