Aminoacid utilization by Helicobacter pylori

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J. Biochem.

1357-2725(!B)OOO6!W

Aminoacid Utilization GEORGE L. MENDZ,‘”

Vol. 27, No. 10, pp. 1085-1093, 1995 Copyright 0 1995Elsevier ScienceLtd Printed in Great Britain. All rights reserved 1357-2725ps $9.50+ 0.00

Cdl Bid.

by Helicobacter pylori

STUART

L. HAZELL*

‘Schools of Biochemistry and Molecular Genetics, and ‘Microbiology University of New South Wales, Sydney, NSW 2052, Australia

and Immunology,

The

Utilization of aminoacidsduring growth by laboratory adapted and wild type Helirobacter pylori strains was investigated employing nuclear magnetic resonance spectroscopy and aminoacid analysis. All H. pylori strains tested showed growth rates with doubling thnes of approx. 11.5 hr in liquid cultures with semi-definedmedia or with de&d an&acid broth without carbohydrates. Fast utilization of severalaminoacids at rates between I30and 250 pM/hr was ohservedin culture broths hmculated with approx. 10’ cells/ml, and acetate, formate and succhate accmmdated as catabolic products in the growth media. Suspensionsof bacterial cells and lysates in isotonic solutions converted arginine, asparagine, aspartate, glotine, and serine used as sole substrates at significant rates; and under these conditions the principal metabolic products observed were acetate, formate, succinate and lactate. The findings of the study hwlicated that H. pylori can surviveemploying amhmacids as the basic nutrients, and suggestedsomeof thesemetabolites were utiliied via fermentative pathways with common characteristics to those found in anaerobes. Keywords: H. pyfori

Aminoacids NMR spectroscopy

Int. J. Biochem. Cell Biol. (1995) 27, 1085-1093

INTRODUCTION

For a long time microbiologists considered conditions in the stomach too harsh for permanent bacterial colonization; notable exceptions were acid-tolerant lactobacilli and yeasts found in the gastric mucosa of rodents (Savage, 1977). The isolation of Helicobacter pylori from humans and its association with gastroduodenal disease revived interest in bacteria in the stomach, which led to the discovery of similar organisms colonizing gastric surfaces in many animals including cats, cheetahs, dogs, ferrets, mice, monkeys and rats (Lee, 1989). Until recently, little was known of the metabolism of this important group of microorganisms, although some studies had been carried out on the urease, catalase (Hazel1 et al., 1991) and other enzymes of the bacterium as well as on the nutrient requirements for growth (Hazel1 et al., 1989a,b). The general objective of these studies on H. pylori is to obtain an understanding of the physiology of the bacterium with the specific aim ‘To whom all correspondence should be addressed. Received 16 December 1994; accepted 1 May 1995.

of identifying potential targets for therapeutic intervention. It was established that unlike other Campylobacter-like bacteria and contrary to the prevailing opinion accepted in the literature, H . pylori has the capacity to take up and metabolize glucose via the pentose phosfate and Entner-Doudoroff pathways and possibly other metabolic networks as yet not identified (Mendz and Haze& 1991, 1993a; Me& et al., 1993, 1994a). Study of biosynthetic functions revealed the existence of de novo pyrimidine and salvage purine nucleotide synthesis pathways (Mendz et al., 1994b,c). Specific aspects of the bioenergetits of H. pylori were explored including studies on fumarate and pyruvate metabolism (Mendz and Hazell, 1993b; Mendz et al., 1994d), which indicated that these intermediates are catabolized via fermentative pathways. Growth culture techniques, aminoacid analyses and nuclear magnetic resonance (NMR) spectroscopy were applied to study the utilization of aminoacids by H. pylori, and to establish th e metabolic fates of some of these basic growth factors. Since fermentative processes appear to be important in the transformation of other basic nutrients and intermediates to

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supply the biosynthetic and bioenergetic needs of the cells, it was important to investigate the pathways of aminoacid assimilation. Fermentation products accumulated in incubations of H. pylori cells with a defined aminoacid broth, and fast degradation rates were measured for several aminoacids. These processes showed similarities with the metabolism characteristic of anaerobiosis. MATERIALS

AND

METHODS

Materials

Blood Agar Base No. 2, Columbia Agar Base, Isosensitest Broth and horse serum were from Oxoid (Basingstoke, U.K.). Bovine serum albumin Fraction V, bovine liver catalase, polymyxin B, and trimethoprim were from Sigma (St Louis, MO); amphotericin B (Fungizone) from Squibb (Princeton, N.J.); and vancomycin from Eli Lilly (West Ryde, NSW, Australia). Sterile 0.2 pm Minisart filters were from Sartorius (Giittingen, Germany), and self-vented tissue culture flasks and sterile microtiter trays from Corning (Corning, N.Y.). All other reagents were of analytical grade. Bacterial cultures and preparation H. pylori laboratory adapted strains NCTC 11639, UNSW PlO and UNSW RU 1, and wild type strains UNSW 920023, UNSW 920042, UNSW 920106 and UNSW 10593/5 were grown on Blood Agar Base No. 2 plates supplemented with 5% (v/v) horse blood, polymyxin B (1.25 U/l), trimethoprim (5 mg/l), vancomycin

(10 mg/l) and amphotericin B (2.5 mg/l). Cultures were passaged every 32-38 hr and incubated in a Forma Stericult incubator (Marietta, Ohio) in an atmosphere of 10% CO, in air, 95% humidity at 37C. Cells were harvested in log phase (approx. 24 hr) in sterile NaCl (150 mM), checked for purity under phase contrast microscopy, and tested for positive urease and catalase activity. Cells were washed 3 times by centrifuging at 17,OOOg(6°C 3 min), discarding the supernatant, collecting the pellet and resuspending it in sterile saline. After the final wash, packed cells were resuspended in sterile saline and employed as inoculum for liquid cultures or aminoacid metabolism experiments at a concentration of approx. 108-lo9 cells/ml. Bacterial lysates were prepared by harvesting cells in sterile KC1 (150 mM) and centrifuging them at 17,OOOg(6°C 8 min), the supernatant was discarded and the pellet was collected and resuspended in KCl. The procedure was repeated 3 times. Following the final wash, packed cells were resuspended to a concentration of approx. 108-IO9 cells/ml in sterile KCI. Lysates were prepared by twice freezing in liquid nitrogen and thawing packed cell suspensions; phase contrast microscopy indicated that more than 99% of the cells were lysed. Cells were grown in liquid cultures of semidefined Isosensitest Broth or defined aminoacid broth supplemented with BSA (0.5% w/v) and catalase (0.1% w/v). The aminoacid broth has a similar composition to the Isosensitest Broth, but with no saccharides and the hydrolyzed casein and peptones substituted by the aminoacid mixture given in Table 1. Cultures

Table 1. Aminoacid content of defined aminoacid broth (0 hr) and after 19 hr incubation in liquid cultures of H. pylon’ strain NCTC 11639 Concentration (mM) Change Aminoacid 0 hr 19hr WV Alanine 8.99 6.27 -2.72 Arginine 3.16 0.26 -2.90 Asparagine and Aspartate 14.51 Il.89 -2.62 Cysteine 0.46 0.39 -0.07 Glutamate and Glutamine 37.08 27.90 -9.18 Cilycine 13.38 10.18 -3.20 Isoleucine 6.97 6.61 -0.36 Leucine 9.10 8.92 -0.18 Lysine 2.30 2.39 +0.39 Methionine 3.35 3.22 -0.13 Ornithine 0.59 3.21 +2.62 Phenylalanine 5.29 5.10 -0.19 Proline 28.01 25.47 -2.74 Serine 5.34 3.83 -1.51 Threonine 5.05 4.47 -0.58 Tyrosine 1.05 0.98 -0.07 Valine 9.03 6.51 -2.52 Ammonia 0.81 1.52 +0.71

Aminoacid utilization by H. pylori

were incubated in a Forma Stericult incubator in an atmosphere of 10% CO* in air, 95% humidity at 37°C. Bacterial growth was monitored by the changes in the absorbance of cell suspensions measured at 600 nm, and bacterial viability was measured by the number of colonies formed using the method of Miles and Misra (1938). At regular intervals samples were taken from the cultures, their absorbance measured and inoculated onto blood agar plates (Blood Agar Base No. 2---Qxoid, plus 5% horse serum) at dilutions between 10’ and 10m6. Incubation products were separated by centrifuging samples at 17,000g (6”C, 8 min), collecting the supernatants, and filtering through membranes with a molecular weight cutoff of 1000 (YM-1, Amicon, VIC, Australia).

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free-induction decays were averaged over 2048 memory locations. Contour maps of 2048 x 2048 data points were obtained from 1024 individual experiments by zero filling in the evolution time domain prior to Fourier transformation. The plots are absolute-value mode, with square sine-bell apodization and a shift for sine-bell window of n/2 along the acquisition and the evolution time domains. ‘H chemical shifts are quoted relative to sodium 2,2 dimethyl-2-silapentane-5-sulfonate at 0 ppm. Aminoacid metabolism

For studies of aminoacid metabolism employing ‘H-NMR spectroscopy bacterial lysates prepared in KC1 (150 mM) were placed into 5 mm tubes (Wilmad, Buena, N.J.), and the appropriate substrates added to start the reacAminoacid analyses tions. Free induction decays were collected using a Bruker AM-500 spectrometer, operating Samples were taken from liquid cultures and in the pulsed Fourier transform mode with the cells separated by centrifugation (17,OOOg, quadrature detection. Measurements were car6”C, 8 min). Supernatants were collected, ried out at 37°C. One-dimensional proton specfiltered through membranes with a molecular tra were acquired with presaturation of the weight cutoff of 1000 and diluted 1: 100 (v/v) water resonance. The instrumental parameters with Li-S Beckman diluent. Aminoacid concenwere spectral width: 5434.78 Hz, memory size trations were determined in triplicates using a 8 K, acquisition time 0.754 set, number of tranBeckman 6300 aminoacid analyzer (North sients 144256 and relaxation delays with solRyde, NSW, Australia). vent presaturation 1.253-2.745 sec. Exponential Identification of metabolic products filtering of 1 Hz was applied prior to Fourier Two-dimensional nuclear magnetic reson- transformation. Chemical shifts are quoted relative to sodium 4,4-dimethyl-4-silapentane- lance (NMR) { ‘H-“C} heteronuclear multiple quantum coherence (HMQC) experiments were sulfonate at 0 ppm. The time-evolution of the substrate and acquired in a Bruker DRX-500 spectrometer products was followed by acquiring sequential using a standard programme. The acquisition spectra of the reactions. Progress curves were parameters were: ‘H spectral width 4496.4 Hz, obtained by measuring the integrals of sub13C spectral width 28277.64 Hz, recycling time strate and product resonances at each point in 2.228 set, and 32 free-induction decays were time. Maximal rates were calculated from good averaged over 2048 memory locations. Contour maps of 2048 x 1024 data points were fits (correlation coefficients 2 0.99) of the data obtained from 512 individual experiments by to straight lines for 30-60 min of the incubations. Calibrations of the peaks arising from zero filling in the evolution time domain before Fourier transformation. The plots are substrates were performed by extrapolating the phase-sensitive mode, with sine-bell squared resonance intensity data to zero time and assignapodization and shift for sine-bell window of ing to this intensity the appropriate concenn/2 along the acquisition and evolution time tration value. The intensity of resonances domains. 13Cchemical shifts are quoted relative corresponding to products were calibrated by adding the appropriate metabolite to cell susto bicarbonate at 160 ppm. pensions and constructing standard concenTwo-dimensional double quantum-filtered ‘H tration curves. homonuclear correlated (DQF-COSY) experiments with presaturation of the solvent resonRESULTS ance were acquired employing a standard Bacterial growth in aminoacid broth programme in a Bruker AM-500 spectrometer. The acquisition parameters were: spectral width Long-term growth and survival of 4310.34 Hz, 2.238 set recycling time, and 32 H. pyfori in blood-free liquid cultures has been

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demonstrated for semi-defined media consisting aminoacid analyses. The concentrations of these of Isosensitest Broth supplemented with BSA metabolites and ammonia in the media at the and catalase (Hazel1 et al., 1989b). No signifi- beginning and after 19 hr of incubation of strain cant differences were observed in the growth NCTC 11639 are given in Table 1. The highest and number of colony forming units/ml between consumption rate was that of glutamate and bacteria grown in liquid cultures of Isosensitest glutamine (0.483 mMjhr), followed by those of Broth or the aminoacid broth, both media glycine (0.168 mM/hr), arginine (0.153 mM/hr), supplemented with BSA and catalase. Figure 1 aspartate and asparagine (0.138 mM/hr), prodepicts growth and viability data of strain line (0.134 mM/hr), valine (0.133 mM/hr) and NCTC 11639 in Isosensitest Broth; the log serine (0.079 mM/hr). Ornithine and ammonia (cfu/ml) increased 0.05 f 0.01 U hr ‘, with a accumulated in the incubation media at rates of linear increase during 15-20 hr. Considerable 0.138 and 0.037 mM/hr, respectively. variations in growth were observed between strains. IdentiJication of metabolic products

Aminoacid utilization

Utilization of aminoacids in liquid cultures of the defined aminoacid broth was monitored by measuring their concentrations employing 0.27

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Fig. I. H. pylori cell growth as a function of time presented as the absorbance of the suspensions measured at 600nm (top), and cell viability as the log number of viable cells/ml (bottom). The number of viable cells was determined by the number of colonies formed/ml by the method of Miles and Misra (1938). Liquid Isosensitest Broth cultures were inoculated with approx. 107cells/ml and incubated in an atmosphere of 10% CO, in air, 95% humidity at 37°C.

Proton NMR spectra of ultrafiltrates of the aminoacid growth medium at the beginning and end of an incubation of H. pylori cells are shown in Fig. 2. Changes in the resonances of the spectrum of the ultrafiltrate after 32 hr. incubation were observed at the spectral positions of 1.310, 1.462, 1.895, 2.382 and 8.442ppm. The four peaks located between 0.8 and 4.3 ppm, in the aliphatic region of the spectrum, are marked in Fig. 2. Two-dimensional NMR techniques were employed to identify the metabolites from which these resonances arose. The DQF-COSY contour map of growth media after 32 hr incubation indicated that the protons at 1.310 and 1.462 ppm showed connectivities to resonances at 4.258 and 4.150 ppm, respectively, whereas the protons giving rise to the other three resonances did not show connectivities. The aliphatic region of the DQF-COSY contour map is shown in Fig. 3. One-bond correlations between protons and carbon atoms were obtained from an HMQC contour map of ultrafiltrates from a 32 hr incubation. The aliphatic region of this contour map is given in Fig. 4 showing the correlations between the resonances of protons at 1.310, 1.462, 1.895, 2.382, 4.258 and 4.150 ppm with the carbon peaks at 19.71, 16.62, 24.54, 35.09, 67.03 and 51.65ppm, respectively. The connectivities revealed by the homonuclear correlation plot together with the proton and carbon chemical shifts obtained from the heteronuclear correlations, allowed the unique assignments of these resonances to lactate, alanine, acetate and succinate, respectively. The proton resonance at 8.442 ppm was correlated to a carbon peak at 182.36 ppm, the lack of connectivities in the DQF-COSY map and the ‘H and 13C chemical shifts indicated that these resonances corresponded to formate. Thus, bacterial growth in the aminoacid broth

Aminoacid utilization by H. pylori

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PPM Fig. 2. ‘H-NMR spectra of ultrafiltrates of the defined aminoacid growth medium collected at the beginning (bottom) and after 32 hr incubation (top). H. pylori liquid cultures were started with approx. lO’cells/ml and incubated in an atmosphere of 10% CO, in air, 95% humidity at 37°C. The arrowheads indicate some of the changes observed in the resonances of the spectrum of the ultrafiltrate after 32 hr incubation.

accumulated acetate, alanine, formate, lactate and succinate as metabolic products. Aminoacid metabolism

Employing ‘H-NMR spectroscopy it was observed that several of the aminoacids that H. pylori cells utilized at higher rates, underwent fast transformation when they were added as the sole substrate to suspensions of packed bacterial cells in sterile NaCl (150 mM) or lysates in sterile KC1 (150 mM). Addition of L-arginine, L-asparagine, L-aspartate, L-glutamate, L-glutamine or L-serine, showed the disappearance of the aminoacid and concomitant appearance of metabolic products. The products were identified by adding the appropriate metabolite to the suspensions and observing the overlap of their resonances with those of the incubation products. For cells and lysates the main products observed were: from alanine, succinate and acetate; from arginine, omithine and urea; from aspartate, fumarate, pyruvate, succinate, acetate, and for-mate; from asparagine, aspartate and all the products derived from it; from glutamate, succinate, acetate and formate; from glutamine, glutamate and all the products derived from it; and from serine, pyruvate, acetate, formate, lactate and alanine.

Fumarate and pyruvate are metabolic intermediaries in H. pylori and are converted in different proportions to succinate, acetate, lactate, formate and alanine; the latter being an intermediary to succinate and acetate (Mendz and Hazel& 1993b; Mendz et al., 1994d). Figure 5 shows spectra from a time course of the metabolism of asparagine by bacterial lysates. Enzyme activities were identified by the products formed employing single aminoacids as sole substrates. Aspartate was transformed initially to fumarate, and serine to pyruvate, indicating the presence of aspartase and serine dehydratase, respectively. Although commonly microbial biodegradative serine dehydratases show activity also with threonine, the H. pylori enzyme appeared to be specific for serine because no threonine deamination was observed with intact cell or lysate preparations. The first step in the catabolism of asparagine and glutamine was their deamination to aspartate and glutamate, respectively, indicating the presence of asparaginase and glutaminase activities. Arginine was converted to ornithine and urea showing the existence of arginase in the bacterium. To utilize exogenous metabolites bacteria require first to transport them across the cell wall.

George L. Mendz and Stuart L. Hazel1

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Fig. 3. Two-dimensional ‘H homonuclear double-quantum filter correlated (DQF-COSY) contour plot of ultrafiltrates from aminoacid growth media of H. pylori cells incubated for 32 hr in liquid culture. The peaks corresponding to the proton resonances of acetate (Ace) and succinate (sipe) are indicated on the main diagonal. Dashed lines show the connectivities between the methine and methyl resonances of alanine and lactate, and the corresponding cross-peaks for alanine (Aia) and lactate (Lac) are indicated. The one-dimensional ‘H spectrum is shown on the left-hand side and on the top.

Comparison of rates of utilization by cells and lysates showed that there were no significant differences under the experimental conditions employed. Maximal rates of conversion of these aminoacids by wild type strain UNSW 10593/5 lysates determined using ‘H-NMR spectroscopy are shown in Table 2. The results showed that the catabolism of the five aminoacids by H. pylori was initiated via deamination reactions and yielded the same fermentation products observed to accumulate in growth media.

DWCUSSION

In vitro H. pylori cells grew and were viable in a defined medium made up of a mixture of aminoacids (Table 1) supplemented with adenine, guanine, uracil, ions and vitamins. The concentrations of the nucleotide precursors and the types and concentrations of ions and vitamins are the same as in the Isosensitest Broth.

Although the minimal requirements for the growth of the bacterium are not known, the results showed that carbohydrates could be removed from media in which aminoacids constituted basic nutrients. Anaerobic bacteria degrade aminoacids by pathways involving always oxidations and reductions. Within the limitations imposed by the absence of molecular oxygen or other high potential oxidants, the oxidation reactions in anaerobes are similar or identical to the corresponding ones catalized by aerobes. More distinctive of anaerobiosis are the reduction reactions carried out in aminoacid catabolism, because each organism needs to generate electron acceptors of suitable potential from the aminoacids it can metabolize. For instance, Group II Clostridia utilize gfutamate, glycine, arginine, aspartate and serine (Mead, 1971). Reduction end products from anaerobic aminoacid fermentations inelude su&+rate, short-chain fatty acids, and other metabolites

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Aminoacid utilization by H. pylori

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1H (PPm Fig. 4. Aliphatic region of the two-dimensional {‘H-I%} heteronuclear multiple quantum coherence (HMQC) contour map of ultrafiltrates from aminoacid growth media of H. pylori cells incubated for 32 hr in liquid culture. The resonances arising from lactate (Lac), alanine (Ah), succinate (Sue) and acetate (Ace) are indicated on the one-dimensional ‘H spectrum shown on the top. The natural abundance 13Cspectrum is shown on the left-hand side. The correlations between the resonances in the ‘H spectrum and the ‘rC spectrum are indicated by dashed lines.

(Barker, 1981). Microorganisms of the genera Escherichia, Salmonella and Shigella carry out mixed acid fermentations with acetate, formate, lactate, succinate, carbon dioxide and molecular hydrogen as end products (Gottschalk, 1986). The utilization of aminoacids by H. pylori cells in liquid cultures measured by carrying out analyses of the incubation media at different timepoints (Table 1) showed rapid utilization of several aminoacids. ‘H-NMR spectroscopy was employed to investigate the initial steps of the degradation of aminoacids, and the rates of conversion of arginine, asparagine, aspartate, glutamine and serine (Table 2) indicated that H. pyfori utilized these growth factors rapidly. The main metabolic products formed in liquid cultures of H. pylori cells in the aminoacid broth were acetate, formate, lactate and succinate (Fig. 2). Identification of the catabolic products

in incubations with a single aminoacid as the sole substrate showed the final degradation products were the same products observed to accumulate in growth media. The formation of these metabolites indicated that fermentation was an important mode of aminoacid utilization by the bacterium, and suggested a similarity between this aspect of the physiology of H. pyfori and that of anaerobes. H. pylori fermented aspartate to fumarate, in common with many aerobes, facultative and obligate anaerobes (Barker, 1961). Aspartate and asparagine are readily fermented by the microaerophile Campylobacter sp. under aerobic and anaerobic conditions. The initial step in the degradation of aspartate is catalyzed by aspartase, and the end products are acetate, succinate, carbon dioxide and ammonia (Laanbroek et al., 1978a). Campylobacter sp. partly oxidizes fumarate to acetate, succinate

George L. Mendz and Stuart L. Hazel1

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Fig. 5. ‘H nuclear magnetic resonance spectra of a time-course of the utilization of L-asparagine by H. strain NCTC 11639 lysates. The total substrate concentration was 60 mM. Lysates were suspended in sterile KC1 and incubated at 37°C. The resonances corresponding to the substrate L-asparagine (Arm) and the products formate (For), fumarate (Fum), aspartate (Asp), succinate (Sue), acetate (Ace) and lactate (Lae) am indicated on the figure. The time at which each spectrum was acquired is shown on the right-hand side.

pylori

and carbon dioxide, and partly reduces fumarate to succinate; and formate is a substrate which increases growth rates under anaerobic conditions, and aerobically supports bacterial growth with acetate as carbon source (Laanbroek et al., 1978b). The reaction catalyzed by fumarate reductase is fundamental to Table 2. Activitities of Several Deam.inases in H. pylori Strain UNSW 10593/5 Lysates Maximal rates of utilization’ Enzyme (nmol/min/mg protein) Arginase 23.6 Asparaginase 29.9 Aspartase 151.9 Glutaminase 192.2 Serine dehvdratase 86.6 ‘Maximal rates were calculated from good fits (corm&ion coeflicients > 0.99) of the data to straight lines for 30-60 min of the incubations. Calibrations of the peaks arising from substrates were performed by extrapolating the resonance intensity data to zero time and assigning to this intensity the appropriate conceutration value. Protein estimation of bacterial lysates suspensions was made by the bicinchoninic acid method employihg a microtiter protocol (Pierce, Rockford, ill).

anaerobic respiration, a central process of the bioenergetics of most anaerobic or facultative bacteria (Kroger et al., 1992). No dismutation of fumarate has been observed in H. pylori, but only reduction to succinate (Mendz and Wazell, 1993b), and formate was formed as an end catabolic product. Thus, the catabolism of aspartate by H. pylon’ appeared to be closer to the fermentative pathways employed by some anaerobes, than to the fermentation carried out by another microaerophile, Campylobacter sp. Two classes of serine dehydratase are known in Escherichia coli, a biosynthetic dehydratase involved in the synthesis of isoleucine, and a biodegradative dehydratase involved in anaerobic energy production by supplying or-ketoacids (Davis and Metzler, 1972). A specific serine dehydratase with little activity towards threonine is Functional in the fermentation of uric acid by the anaerobe Clostridirm acidiwici (Sagers and Carter, 1971). An important route for the utili@on of serine by H. pyLnoriwas the conversion of the amino&d to pyruvate by a biodegradative serine dehydratase which

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Aminoacid utilization by H. pylori

showed no activity with threonine. Subsequent catabolism to acetate, formate, lactate and succinate suggested the presence of mixed acid fermentation of pyruvate in the bacterium (Mendz et al., 1994d). A characteristic enzyme of this fermentative pathway is pyruvateformate lyase which is rapidly inactivated by oxygen (Gottschalk, 1986). The presence in H. pylori of a particular type of biodegradative serine dehydratase and a mixed acid fermentative pathway were metabolic characteristics that underlined the similarities between the catabolism of aminoacids by the bacterium and many anaerobes. The design of chemotherapeutic agents targeted against specific biochemical pathways of H. pylori requires to understand the essential metabolism of the bacterium, and in particular its mode of utilization of basic nutrients and adaptability to obtain metabolites needed for survival. The study indicated that H. pylori can grow employing aminoacids as biosynthetic precursors, and suggested that some of them were utilized via fermentative pathways characteristic of anaerobiosis, although oxygen is essential for the growth of the microorganism. Acknowledgements-This

support of the National Council of Australia.

work was made possible by the Health and Medical Research

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