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Journal of General Microbiology (1984), 130, 1787-1793. Printed in Great Britain 1787 The Acid End-products of Glucose Metabolism of Oral and Other Haemophili By J . E. T U Y A U , ' W. S I M S 1 * t A N D R . A. D . W I L L I A M S 2 Department of Microbiology and Preventive Dentistry, Royal Dental Hospital, Leicester Square, London WC2H 7LJ, UK Department of Biochemistry, London Hospital Medical College, Turner Street, London El 2AD, UK (Received 5 December I983 ;revised 28 February 1984) The acids produced in broth culture by various species of oral haemophili and by stock strains of capsulated and other haemophili were identified and measured by gas-liquid chromatography. Succinic acid was the major acid end-product of all strains, with acetic acid also being regularly produced but in smaller amounts. A stock strain, Haemophilus paraintftuenzae NCTC 4101, produced less succinic acid than other strains of this species and produced much more oxalacetic and pyruvic acid than all the other strains of haemophili. Strain NCTC 4101 possessed all the enzymes of the tricarboxylic acid cycle, as previously reported, but in the other haemophili examined only succinic dehydrogenase, fumarase and malate dehydrogenase could be detected. No other enzymes of the tricarboxylic acid cycle were detected and isocitrate lyase, malate synthase and pyruvate carboxylase were also absent. Phosphoenolpyruvate-carboxylase was present in all strains. A partial tricarboxylic acid cycle and marked malate dehydrogenase activity appear to be characteristic of haemophili. The pathway to succinate in haemophili appears to be via carboxylation of phosphoenolpyruvate to oxalacetate and thence via malate and fumarate. The results of tracer studies on a single oral strain of H . parain8uenzae using various labelled substrates were in keeping with this proposed metabolic pathway. INTRODUCTION Haemophili form part of the normal oral flora (Sims, 1970; Kilian, 1976) and are commonly found in specimens from infections of the teeth and jaws in which they are occasionally the predominant organisms (Sims, 1974). The haemophili are usually of the V-factor (NAD)requiring species with Haemophilus influenzae being isolated infrequently and capsulated forms of this species never. The contribution which haemophili make to these oral infections, which invariably involve a variety of other bacteria, is difficult to determine but we have been examining the endotoxins, enzymes and various other aspects of these oral haemophili and comparing them with the established pathogenic capsulated species in the hope of elucidation. We report here on the acid end-products of metabolism of haemophili and, because the findings seemed to lack accord with the small amount of published work in this field, some preliminary studies of enzymes, the incorporation of radioactive substrates into cell constituents and the conversion of radioactive glucose into acid end-products. METHODS Organisms. These were Haemophilus injluenzae, capsulated types a to f, respectively NCTC 8465, 7279, 8469, 8470, 8472 and 8473, H . parainjluenzae NCTC4101 and 10665; and, isolated from various oral sources, H . injluenzae (non-capsulated) ( 3 strains), H . haemolyticus (3 strains), H . parainfluenzae (4 strains), H . parahaemolyticus (8 strains), H . aphrophilus (3 strains) and H , paraphrophilus (5 strains). t Present address: The Dental School, UCL School of Medicine, Mortimer Market, London WClE 6JD, UK. 0022-1287/84/0001-1631$02.00 0 1984 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 17:37:19 1788 I . E . T U Y A U , W . SIMS A N D R . A . D . WILLIAMS Growth of haemophili.All strains were maintained on heated blood agar incubated at 37 "C in air supplemented with 5% (v/v) C o t . Haemophili were grown in broth containing per litre: neopeptone (Oxoid), 15 g; yeast extract (Difco) 5 g; NaC1, 2.5 g; cysteine hydrochloride, 1 g; 5 ml salts solution A (0.5 g each of ZnS0,.4H20, MnSO, .4H20and FeSO,. 7H20, and 5 mlO.1 M-H2S04per litre); and 0.1 ml salts solution B (1 g MgSO,. 7H20, 1 g (NH4)2S04and 5 g sodium citrate per litre). When required, glucose was added to give a final concentration of 0.5% (w/v). For some experiments, pyruvate, malate, fumarate or 2-oxoglutarate was added to a final concentration of 10 mM and the salts solutions were omitted. Each litre of medium was supplemented with 0.1 ml of a 1% (w/v) solution of NAD and 0.2 ml of a 1% (w/v) solution of haematin. Estimation of acid end-products. The general methods, preparation and incorporation of standards were essentially as described by Holdeman et al. (1977). Broth (5 ml) was inoculated with one drop of an overnight culture and incubated at 37 "C for 48 h. It was then centrifuged and the supernatant removed. Volatile acids were extracted by mixing 0.2 ml 50% (v/v) H2S04 with 2.0 ml supernatant, adding 0.4 g NaC1, mixing gently with 1.0 ml diethyl ether, briefly centrifuging to break any emulsion, freezing at - 20 "C and removing the upper ether layer. The non-volatile acids were extracted by mixing 1a 0 ml supernatant with 0.4 ml50% (v/v) H2S04 and 2.0 ml methanol and heating the mixture for 30 min at 56 "C. The methylated acids were extracted in 0.5 ml chloroform. The acids were identified and measured by comparison with similarly processed solutions of standards using GLC. Uninoculated broths were processed as described and the values obtained were subtracted from the cultures of haemophili. For estimating succinate production from various substrates, the controls were broths without substrate but inoculated with haemophili and incubated. Volatile acids were separated on 5 % free fatty acid phase Chromosorb G (WS DHCS 80-100 mesh) columns and the non-volatile acids on 20% diethylene glycol succinate on Chromosorb W (HMDS 80-100 mesh) columns at 135 "C. The instrument was a Perkin Elmer F17 using flame ionization detectors, N2 (30 1 min-l) carrier gas and H2 and air at 16 and 22 lbf in-2 (110.4 and 151.8 kPa), respectively, for combustion. All of the strains listed were examined on at least three separate occasions - many other strains of haemophili have been routinely examined once when isolated from oral infections. Enzyme assays. Haemophili were grown on brain heart infusion heated blood agar for 24 h at 37 "C, harvested into PBS [Oxoid Dulbecco A pH 7.3 containing (g 1-l): NaCl, 8.0; KC1 0.2; Na2HP04, 1.15 and KH2P04,0.21, washed and the packed cells resuspended in water to a concentration of about 7% (v/v). The organisms were disrupted at room temperature by 30 s bursts of sonication, centrifuged at lOOOOg and 4 "C for 30 min and the supernatant used for the assays. Enzyme activity was determined in 1 cm light path cuvettes containing 3 ml reaction volumes using a recording spectrophotometer. The methods of Reeves et al. (1971) were used for assaying citrate synthase (EC 4.1 .3.7), aconitase (EC 4.2.1 .3), isocitrate dehydrogenase (NAD+) (EC 1 . 1. 1.42), 2oxoglutarate dehydrogenase (EC 1 .2.4.2), fumarase (EC 4.2.1 .2), malate dehydrogenase (EC 1 . 1 . 1.37), isocitrate lyase (EC 4.1 .3,1)and malate synthase (EC 4.1 .3.2). The method of Jurtshuk et al. (1969) was used for determining succinic dehydrogenase (EC 1.3.99.1) and fumarate reductase (NADH) (EC 1 .3.1.6) was measured by the method of Kroger et al. (1971). Phosphoenolpyruvate carboxylase (EC 4.1.1.31) and pyruvate carboxylase (EC 6.4.1.1) were assayed as described by Scrutton (1971). Protein was measured by the Lowry method using crystalline bovine albumin (Sigma) for the standard. Incorporation of14Cfrom various substrates. Each substrate [lo pCi (0.37 MBq) U14C] was dissolved to a final concentration of 2 mM in 150 ml broth which was then sterilized by filtration, inoculated and incubated at 37 "C for 48 h. After centrifuging at 4 "C and 6000 g for 30 min the cells were washed in distilled water and fractionated by the method of Roberts et al. (1955). The hydrolysed residue was dried under vacuum over phosphorus pentoxide and redissolved in 1.0 ml pyridine (0.25 M)/acetate buffer (pH 5.2). Amounts of 25 p1 were separated by electrophoresis at 700 V and 80 mA for 2 h on Whatman 3MM paper saturated with pyridine/acetate buffer. After drying, the paper was cut into strips 1.0 cm wide and placed in vials to which 1.0 ml distilled water and 8 ml NE 250 scintillation fluid were added. A Packard Tri-Carb model 3000 liquid scintillation spectrophotometer was used for counting. Glucose metabolism. Strain S15 of H. parainjluenzae was grown on brain heart infusion heated blood agar, harvested into 10 mM-phosphate buffer (pH 7.5) containing 2 mM-ammonium sulphate, washed and resuspended in the same buffer to an optical density giving about 5 x lo9 organisms per ml. Samples of 1.0 ml were distributed in vials which were warmed to 37 "C then 0.5 ml 1 .OmM-glucose solution, containing 1 pCi [U-14C]glucose,was added to each vial and mixed. Vials were kept at 37 "C and removed at 15, 30,45, 60, 75, 105, 120 and 130 min, acidified with 50 p1 1 M-HCland cooled in melting ice. After centrifuging at lOOOOg for 10 min the Supernatant was removed and stored at - 20 "C. Organic acids were separated by ascending chromatography on cellulose TLC (85 : 1 : 14, by vol.) as solvent. This solvent was used because it gave sheets using butan-1-ol/diethylamine/water better separation of succinate from acetate and lactate than the other solvents tried [propan-1-ol/ammonia (70 :30, w/v) and butan-1-ol/acetic acid/water (12 :3 : 5 , by vol.)] although it failed to separate acetate and lactate. The sheets were dried at 60 "C, cooled, lightly sprayed with 0.02% ninhydrin in acetone warmed to visualize the ammonium salts of the acids, which were identified by their R, values. Radioactivity was determined in each sample by cutting the sheets into 1 x 1 cm squares and counting with 5 ml scintillation fluid as above. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 17:37:19 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 17:37:19 (capsulated) H . infiuenzae H . haemolyticus H . parainfiuenzae H . parainfuenzae (NCTC 4101) H . parahaemolyticus H. aphrophilus H . paraphrophilus H . infuenzae Species 3 5 0-0.19 04.05 04-23 0.48-2'26 0.99-7.1 3 3-68-5.00 1-02 0-367.32 4.70-7.57 3.58-7-64 0 0-0.07 04-22 1.35 1*60-2-45 6 8 04-05 Succinic Oxalacetic 04-14 04-59 (rl.52 0 04.11 04-51 0 0 Lactic Non-volatile acids (mM) No. of strains I 04.30 0 04.29 0 0 04-08 0.74 0 Pyruvic 1 I 04.16 0 4 . 31 04.13 0.48466 0.18-1.77 0-20-2-31 0-95 0.99-2.85 0.22- 1+08 0.18-1-23 04.39 0-37-2.20 0-0.06 0 0 0 Propionic Acetic 04-06 04-17 0-0-02 0 0 0-0.0 1 04.07 04-08 04.15 04-15 04-05 0 Valeric 0 4 . 01 04.18 04.15 0 iso- or n- iso- or nButyric Volatile acids (mM) Table 1. Acids produced by haemophili when grown in broth containing 0.5% (wIu) glucose for 48 h at 37 "C \ 3 s. 5 % ;s- s 9 'a 9 3 s b I 2 is: 1790 J . E . T U Y A U , W . SIMS A N D R . A . D . WILLIAMS RESULTS A N D DISCUSSION The results of a single batch of estimations of the acid end-products of the Haemophilus strains are shown in Table 1. Succinic acid was consistently the principal acid produced from glucose in normal broth culture. Acetic acid was also invariably produced but in smaller quantities. Small amounts of other acids were produced variably from strain to strain. The results obtained following incubation under strictly anaerobic conditions were essentially the same. Mechanically aerated cultures were considered irrelevant to in vivo conditions and were not studied. The acids produced by the individual species were not sufficiently dissimilar to be of value in classification but the general production of succinate and acetate does seem to characterize the genus. In this respect haemophili resemble Bacteroides, a genus which, interestingly, has DNA G + C compositions extending over a similar range (Kilian, 1976; Holdeman et al., 1977), requires haemin for growth and has endotoxins which do not contain 3-keto-deoxyoctonic acid (Hofstad, 1974; Tuyau & Sims, 1983). Table 1 shows that H.parainfluenza NCTC 4101 produced much less succinate than the other strains of H . parainfluenzae and was the only strain of Haemophilus to form significant amounts of oxalacetate and pyruvate, the former being its major product. Hollander (1976) found all the enzymes of the tricarboxylic acid cycle present in this strain and thought the cycle to be functional in strain Bossy No. 7 Leidy of H . parainfluenzae which was studied by White (1966). This strain could not be obtained for study but Hollander (1976) thought it atypical because of its rapid utilization of glucose with consequent high growth yield and its feeble malate dehydrogenase activity. It seemed likely that the differences between Table 2. Enzyme activities of extracts of various haemophili Enzyme activity [nmol substrate used (mg protein)-' min-l at 25 "C] I Strain 1 Succinic dehydrogenase Fumarase Malate dehydrogenase Phosphoenol pyruvate carboxylase 6.6 8.8 543 62.9 19.9 0.44 28 1 48.2 617 22.4 2040 18.5 H . influenzae (capsulated NCTC 7279) H. influeltzae (oral strain S9) H. parahaemolyticus (oral strain S43) H. parainfluenzae (NCTC 10665) H . parainfluenzae (oral strain S15) H. parainfluenzae (oral strain S24) H. parainfluenzae (NCTC 4101)* 38.7 6.3 33.0 0.43 34.8 30.4 3128 20.2 10.5 22.4 2095 16.4 27.2 26-8 251 1 18.9 * This strain only had the following enzyme activities also (see text): citrate synthase, 3.18; aconitase, 17.4; isocitrate dehydrogenase (NAD+), 12.0; 2-oxoglutarate dehydrogenase, 22.3 ; fumarate reductase, 38.9. Table 3. Succinate produced from various substrates by strains of haemophili Succinate (mM) produced in 48 h from 10 mM substrate Strain Substrate H . influenzae (capsulated NCTC 7279) H. parahaemolyticus (oral strain S43) H . parainfluenzae (oral strain S15) H . parainfluenzae (NCTC 4101) . .. I 1 Pyruvate Malate Fumarate 1*06 0.89 1*40 0 0.69 1.14 0.82 0 0.53 1.32 1.01 0 0.60 0.60 3.50 2-0 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 17:37:19 2-Oxoglutarate Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 17:37:19 x Total c.p.m. incorporated into cells Percentage of total c.p.m. incorporated into: cold TCA-soluble ethanol-soluble ether/ethanol-soluble hot TCA-soluble hydrolysed residue x Total c.p.m. in hydrolysate residue Percentage of c.p.m. of hydrolysate incorporated into: lysine glucosamine neutral compounds glutamate aspartate 46-7 4.7 18-8 6.9 75-0 5.5 40.0 1-8 2-7 26.0 3.4 - 0-5 3.8 - 3-8 1-6 5-0 14.5 75.1 114 7-4 9.9 1-8 8-2 72.8 28-3 11.1 30.9 2-9 31.7 23-4 65-8 152 Aspartate 38-9 Glutamate 28 1 Glucose 0-5 4.4 85.4 0.3 0.1 0-9 7-3 69-5 84-5 m.5 1-7 121 Alanine A Substrate (2 m ~ ) -, No counts higher than background detected. f 55-9 1-4 7.3 - 1.9 8-1 21-5 2-4 23-2 44-6 13-3 29-8 Lactate 46-9 2-1 13-2 - 1.3 7.2 56-5 2-4 13-2 20.7 17.0 32-3 Malate Table 4. Fractionation of H . paramjluenzae S15 after growth in broth containing I4C-labelled substrates 0-5 90.7 0-2 0.7 - 3.1 88.7 0-3 2.3 5.5 12-7 229 \ Acetate %s 4 3- b 5 B kY 9 3 b 5 1792 J . E . T U Y A U , W . SIMS A N D R . A . D . WILLIAMS strain NCTC 4101 and the other haemophili studied would involve enzymes of the tricarboxylic acid cycle. The enzyme activities of some strains of haemophili are shown in Table 2. Although we were able to confirm the finding of Hollander (1976) that strain NCTC 4101 has all the enzymes of the tricarboxylic acid cycle, the strains examined by us had only four enzymes of the central metabolic pathways related to the cycle, namely, succinic dehydrogenase, fumarase, malate dehydrogenase and phosphoenolpyruvate-carboxylase. A partial tricarboxylic acid cycle and high malate dehydrogenase activity seem to be characteristic attributes of most haemophili. All of the haemophili tested lacked the enzymes isocitrate lyase, malate synthase and pyruvate carboxylase, which excludes the glyoxalate bypass or direct conversion of pyruvate to oxalacetate in these organisms. Since fumarate reductase was not detected, fumarate is presumably reduced by succinic dehydrogenase. Warringa et al. (1958) demonstrated the reversibility of succinic dehydrogenase and its physiological importance for an oral anaerobic coccus. All the strains of haemophili tested produced succinate in broth containing pyruvate, malate or fumarate instead of glucose (Table 3). In contrast, only strain NCTC 4101 formed succinate in the presence of 2-oxoglutarate. It could be that the other strains were impermeable to 2oxoglutarate but the enzyme assays and the results of the experiment with radioactively labelled substrates suggest this is due to an incomplete tricarboxylic acid cycle. The probable pathway in haemophili is carboxylation of phosphoenolpyruvate to oxalacetate by phosphoenolpyruvate-carboxylase,then via malate and fumarate to succinate catalysed by malate dehydrogenase, fumarase and succinic dehydrogenase, respectively. Acetate would presumably be formed from pyruvate via acetyl-CoA. The pattern of incorporation into cell constituents of [14C]glucose, amino acids and carboxylic acids by growing cells of H . parainflzienzae S15 (Table 4) is revealing, particularly with respect to the relative labelling of aspartate and glutamate. Glucose carbon was widely distributed, about 34% entering the lipid fraction via acetate and fatty acid synthesis. [ 14C]Acetate itself was 89% incorporated into lipids, with negligible labelling of aspartate and glutamate. The 3C substrates alanine and lactate, each convertible to acetate via pyruvate, gave similar fractions of their incorporation to the lipids although the extent of incorporation was quite different. With alanine much of the incorporation was into proteins, like the other amino acids aspartate and glutamate. Negligible label from alanine flowed into aspartate or glutamate and the largest fraction of radioactivity from either aspartate or glutamate was directly incorporated into protein. Those substrates which labelled the dicarboxylic amino acids significantly all formed aspartate more than glutamate in the ratios 4.5 : 1 for glucose, 5 : 1 for lactate and 7 : 1 for malate. It is clear that glutamate can be metabolized by haemophili but these results support the enzymological findings that such metabolism is not via the tricarboxylic acid cycle. t 20 40 60 80 100 120 140 Time (min) Fig. 1. Time-course for the conversion of radioactively labelled [U-14C]glucose into succinate and acetate/lactate by an oral strain (S 15) of Haemophilus paruinfluenzue. A,Succinate formed ; 0 ,acetate or lactate formed; I, glucose used. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 17:37:19 Acid end-products of haemophili 1793 Figure 1 summarizes the results of a time-course conversion of 0.33 mM-glucose into succinate, acetate and lactate by a washed cell suspension of H . parainjluenzae S15. Acetate and lactate could not be separated by the TLC system used which was chosen for its clear separation of succinate from other metabolites. The metabolism over 2 h led to 75% of glucose being converted to these three acids with 25 % incorporation. This shows that these metabolites, which were measured in spent broth, can be formed from glucose in large quantities and are unlikely to have been produced from other media constituents. Although these investigations of Haemophilus metabolism are only preliminary and exploratory, it seems reasonable to suggest that haemophili produce succinic acid as a major end-product of metabolism because many strains lack some of the enzymes of the tricarboxylic acid cycle. REFERENCES HOFSTAD, T. (1974). The distribution of heptose and 2keto-3-deoxyoctonate in Bacteriodaciae. Journal of General Microbiology 85, 3 14-320. HOLDEMAN, L. V., CATO,E. P. & MOORE,W. E. C. (1977). Anaerobe Laboratory Manual, 4th edn, pp. 30-38,134-140. Blacksburg : VPI Anaerobe Laboratory, Virginia Polytechnic Institute and State University. HOLLANDER, R. (1976). Energy metabolism of some representatives of the Haemophilus group. Antonie van Leeuwenhoek 42,429-444. JURTSHUK, P., MAY,A. K., POPE,L. M. & ASTON,P. R. (1969). Comparative studies on succinate and terminal oxidase activity in microbial electron transport system. Canadian Journal of Microbiology 15, 797807. KILIAN,M. (1976). 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British Journal of Oral Surgery 12,112. TUYAU,J. E. & SIMS, W. (1983). Aspects of the pathogenicity of some oral and other haemophili. Journal of Medical Microbiology 16, 467475. WARRINGA, M.G. P. J., SMITH,0.H., GIUDIITA,A. & SINGER,T. P. (1958). Studies on succinic dehydrogenase VIII. Isolation of a succinic dehydrogenasefumaric reductase from an obligate anaerobe. Journal of Biological Chemistry 230, 97-109. WHITE,D. C. (1966). The obligatory involvement of the electron transport system in the catabolic metabolism of Haemophilus parainjuenzae. Antonie van Leeuwenhoek 32, 139-158. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 17:37:19