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FEMS Microbiology Ecology 53 (1988) 45-52 45 Published by Elsevier FEC 00147 Effects of oxygen on the growth and metabolism of A ctinomyces viscosus M.H. De Jong, J.S. Van der Hoeven, C.W.A. Van den K i e b o o m and P.J.M. Camp Department of Preventive and Community Dentistry, University of Nijmegen, Nijmegen, The Netherlands Received 26 June 1987 Revision received 17 August 1987 Accepted 19 August 1987 Key words: Actinomyces; Oxygen; Cytochrome; Dental plaque 1. SUMMARY Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aerotolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus. To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium (D = 0.2 h -1) with exposure to variable amounts of oxygen. The Y~u¢o~ increased from 62.5 g.mo1-1 under anaerobic conditions to 149 g.mo1-1 under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [14C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b-type absorbtion bands, were found. Correspondence to: J.S. van der Hoeven, Department of Preventive and CommunityDentistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen,The Netherlands. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation. 2. INTRODUCTION Actinomyces viscosus, Actinomyces naeslundii, Streptococcus mitior and Streptococcus sanguis are pioneers in starting dental plaque formation and remain the predominant microorganisms in supragingival plaque at healthy sites [1,2]. These species are carbohydrate fermenters tolerant to oxygen. Recently we have shown that S. mutans and S. sanguis [3] consume considerable amounts of oxygen by the partial oxidation of sugars to acetic acid, carbon dioxide and water. Oxygen consumption proceeds only at low dissolved oxygen concentrations and is apparently not coupled to electron transport phosphorylation, since the growth yield of the streptococci was only slightly affected [3]. Similarly, Lactobacillus 0168-6496/88/$03.50 © 1988 Federationof EuropeanMicrobiologicalSocieties 46 plantarum can grow at the expense of lactate under aerobic conditions, but its metabolism remains coupled to substrate level phosphorylation [4]. Anaerobically A. viscosus and A. naeslundii ferment glucose according to Schemes 1 and 2 [5,6]. Under aerobic conditions, in batch culture, glucose is partially oxidized according to Scheme 3 [5]. (1) glucose ~ 2 lactic acid (2) glucose + carbon dioxide -~ formic acid + acetic acid + succinic acid (3) glucose + 2 oxygen ~ 2 acetic acid + 2 carbon dioxide + 2 water The high growth yields [6] reported for succinate-producing cells (Scheme 2; Ygl.... e = 65 g. mo1-1) strongly suggest that in A. viscosus the production of succinate [7] is coupled to electron transport phosphorylation [6]. The high growth yield (Yglu¢o~= 87 g. mol-1) reported for aerobic batch cultures [5] suggests that electron transport phosphorylation might also occur under aerobic conditions. In Propionibacterium spp. another group of anaerobes tolerant of oxygen, cytochromes are present and electron transport phosphorylation has been shown to occur under aerobic and anaerobic conditions [8,9]. However, the occurrence of cytochromes or oxidative phosphorylation in A. viscosus has never been investigated. We have therefore studied the effects of oxygen on the growth and glucose metabolism of A. viscosus and have studied the presence of cytochromes in aerobically and anaerobically grown cells. 3. MATERIALS AND METHODS 3.1. Growth conditions A. viscosus Ut 2, originally isolated from human dental plaque [10], was grown on a filtersterilized chemically defined medium [11], which contained amino acids, nucleotides, vitamins, inorganic salts, trace elements, a bicarbonate buffer and 10 mM glucose to give carbohydrate limitation. The bacteria were grown at a dilution rate of D = 0.2 h-1 at 37°C in a chemostat (500 ml) as described before [3]. The pH was kept at 7.0 by automatic titration with 2 N KOH. The appropriate gas phase was obtained by mixing N 2 (95%), CO 2 (5%), with N 2 (75%), CO2 (5%) and 02 (20%). The filter-sterilized gas was sparged into the medium (5 1. h -1) below the stirrer (100 rev. rain-l). The purity of the culture was routinely checked on aerobically and anaerobically incubated blood agar plates. The identity of representative isolates was routinely checked with the API 20A system. 3.2. Analytical procedures Dry weights were determined in 40-ml samples taken directly from the culture by syringe. The samples were centrifuged and washed three times in demineralized water. The cells were dried in crucibles at 105°C until they reached constant weight. Dry weight determinations were done at least in duplicate in separate cultures. Formic, acetic, succinic, lactic, citric and glutamic acids were determined with isotachophoresis [12]. Spectra of dithionite-reduced cytochromes in whole bacterial cells were obtained with an Aminco Chance spectrophotometer (American Instruments Co., Washington, U.S.A.) [13]. Aerobically grown cells (1 mg. m1-1 dry weight) were centrifuged, resuspended in chemically defined medium without glucose and incubated with glucose (2 mM), acetate (10 raM), formate (10 raM) and succinate (10 mM). The rates of oxygen consumption and the K m value for oxygen (Gottschal, J.C., personal communication) were determined in these cell suspensions using a polarographic oxygen electrode. 3.3. Metabolism of [ ] 4C]ghlcose 170/~Ci of uniformly labelled [14C]glucose (270 mCi- mmol-1) was added to a chemostat (500 ml) with a culture of A. viscosus in steady state (D = 0.19 h - l ; pH 7.0). The culture was gassed with a mixture of N 2 (80.4%), CO 2 (5%) and 0 2 (14.6%); the dry weight was 1.30 mg- ml-1. At regular time intervals after the addition of [14C]glucose 1.0-rftl samples were taken directly from the culture by syringe and added to 0.1 ml KOH. These samples were centrifuged immediately at 20 °C for 4 min at 18 400 × g. The pellet fraction was washed once and subsequently resuspended in 1.0 ml water. 47 Two 0.1 ml portions were taken from the supernatant fraction. The radioactivity in one portion was counted directly, while 0.1 ml 1.0 M N a H C O 3 and subsequently 0.1 ml 2.0 N HC1 were added to the other portion before counting radioactivity. The latter procedure decreased the p H below 1.0 and removed all 14C-labeled carbon dioxide produced by the bacteria. The radioactivity in 0.1 ml portions of the pellet and the two supernatant fractions was counted in 10 ml Aqualuma plus (Baker). The amount of 14C-labeled carbon dioxide in the culture fluid was calculated by substraction of the amounts of radioactivity in the two supernatant fractions after correction for the dilution. In addition the 14C-labeled carbon dioxide evolving from the culture fluid into the gas phase was captured in three successive washbotties containing 1 N K O H and were counted separately. only parts of RNA and D N A synthesized from glucose were ribose and deoxyribose, and that all amino acids required for protein synthesis were derived from the medium and not synthesized from glucose. The main phospholipid in the cells was assumed to be phosphatidylethanolamine with two C16 fatty acids. Glycerolphosphate and the two C16 fatty acids were assumed to be synthesized from glucose, the latter two via acetyl-CoA [14]. The fraction of the molecular weight of the monomers derived from glucose was calculated as follows: number of C-atoms derived from glucose per monomer ×12, divided by the molecular weight of the monomer. To obtain the amount of glucose-carbon incorporated into 100 g of biomass (dry weight), these fractions were multiplied by the amount of the respective cellular constituents per 100 g of biomass (see Table 1). 3.4. Calculations 4. RESULTS The amount of glucose-carbon incorporated in the biomass was calculated on the basis of data given by Stouthamer [14] for the composition of microbial cells (see Table 1). It was assumed that all polysaccharides (assumed to be polyglucose) were synthesized directly from glucose, that the A . viscosus was grown in a glucose-limited chemostat on a chemically defined medium (D = 0.2 h -1) under exposure to variable amounts of oxygen. The effects of increasing concentrations of oxygen in the gas phase on the growth yield, Fermentation products 1 5- (mot.mo1-1 glucose) 10" / / /-1 x~---~. T [ Q5. o t'o %0 2 Fig. 1. The effect of increasing concentrationsof oxygenin the gas phase on the fermentation products of A. viscosus growing under glucose limitation (D = 0.2 h-l). C), succinic acid; I, acetic acid; zx, formic acid. 48 _ _ % Glucose - C incorporated 100 '° ...... Dry weight (g cells mo1-1 glucose) ~ 150 * . 100 6O o 50 i ~ 0 i I I 10 15 20 %o 2 Fig. 2. Effect of increasing concentrations of oxygen in the gas-phase on the dry weight (g cells m o l - l glucose) and on the partition of glucose carbon (% glucose-C incorporated) over the biomass (calculated from the dry weight with the aid of Table 1), carbon dioxide (calculated) and acidic fermentation products (measured with isotachophoresis) of A. viscosus growing glucose limited at D = 0.2 h -1. * , dry weight; e, % glucose-carbon in the biomass; I , % glucose-carbon in the acidic fermentation products; O, % glucose-carbon in carbon dioxide. fermentation products and carbon recovery are shown in Figs. 1 and 2. Under anaerobic conditions formic, acetic and succinic acid were produced in almost equimolar amounts according to Table 1 The amount of glucose carbon incorporated in microbial cells, growing under glucose limitation on a chemically defined medium containing all amino acids, nucleotides, vitamins, salts and trace elements Macromolecular substance Amount (g/per 100 g cells) Mean molecular weight monomer Number of glucose carbons incorporated Polysaccharide Protein Lipid RNA DNA 16.6 a 52.4 9.4 15.7 3.2 162 690 300 280 6 (0.44) b 0 (0.00) 37 (0.64) 5 (0.20) 5 (0.21) Total 97.3 Amount of glucose carbon (g carbon 100 g cells) 7.38 0.0 6.05 3.14 0.69 17.26 a Data from Stouthamer [14]. b In parenthesis: fraction of the molecular weight of monomer derived from glucose carbon. Scheme 2. At increasing oxygen concentrations first succinic acid disappeared with a concomitant increase in the amounts of formic and acetic acid produced (Fig. 1). Beyond 5% oxygen the amounts of formic and acetic acid also decreased, until at 20% oxygen in the gas phase these acids were no longer produced. No other fermentation products such as ethanol and pyruvic, lactic, citric or glutamic acids were found (results not shown). In washed cell suspensions of aerobically grown cells oxygen was consumed at a considerable rate (101 nmol O 2- mg -1 dry weight, min -1) and with a high affinity ( K m = 1 # m o l . l - 1 ) , when glucose (2.0 m m o l . 1 - 1 ) was added as an external substrate. Addition of acetic, formic and succinic acids to these cell suspensions did not result in oxygen consumption. The Yglucose first increased rapidly from 62.5 g - t o o l -] under anaerobic conditions, to 122.5 g. mo1-1 at 8% oxygen and subsequently more slowly to 149 g. mol-1 at 20% oxygen. The calculations in Table 1 show that a fixed percentage of the biomass was derived from glucose carbon, consequently the amount of glucose carbon incor- 49 Table 2 Log 6 com .. Partition of glucose carbon Incorporated in Biomass Fermentation products Carbon dioxide Percentage glucose carbon [14C]Glucose Calculated a 35.0 32.7 32.3 31.2 32.8 36.0 a Dry weight used for the calculation of the amount of glucose carbon incorporated into the biomass (see Table 1 and Fig. 2) was from a sample taken 1 day before the addition of [14C]glucose; fermentation products were determined in the same sample using isotachophoresis. The remaining glucose carbon, which was not incorporated into the biomass nor into the acidic fermentation products, was assumed to have been converted to carbon dioxide. porated into the biomass increased proportionally with the yield (Fig. 2). The a m o u n t of glucose c a r b o n recovered from the fermentation products decreased, however, f r o m 75% under anaerobic conditions, to only 7% at 20% oxygen. The remaining glucose carbon, neither incorporated into biomass nor into acidic fermentation products, was supposed to be converted to c a r b o n dioxide (Fig. 2). I n order to verify whether the partition of glucose c a r b o n over biomass, acidic fermentation p r o d u c t s and c a r b o n dioxide, as depicted in Fig. 2 was correct, 170 # C i of uniformly labelled [14C]glucose were added to a chemostat culture. D u r i n g the experiment the oxygen level in the gas phase was 14.6% and the dry weight was 130 r a g . ml-1. The results are shown in Fig. 3. Linear regression of the log 14C counts towards t = 0 gave the a m o u n t s of glucose c a r b o n incorporated into the biomass, acidic fermentation products a n d c a r b o n dioxide. The w a s h o u t rates of [14C]carbon in the biomass and acidic fermentation p r o d u c t s were 0.18 h - t and 0.19 h -1, respectively, and in g o o d agreement with the imposed dilution rate of the culture (0.19 h - l ) . The [14C]carbon dioxide dissolved in the culture fluid h a d a m u c h higher washout rate (1.4 h - l ) . This was p r o b a b l y due to exchange of dissolved c a r b o n dioxide with c a r b o n dioxide f r o m the gas sparged t h r o u g h the culture fluid. Accordingly the a m o u n t o f 1 4 C 0 2 captured in the washbottle with K O H ~o 6 -c 0 2 i 6 ,0 2'0 - - - i ~0 hours Fig. 3. Metabolism of [14C]glucose. Log cpm in the biomass (v P), in the acidic fermentation products (e, S) and in carbon dioxide (O, CO2) at different time intervals after the addition of [lac]glucose to a continuous culture of A. viscosus growing glucose limited under 14.6% oxygen (D = 0.19 h-l). was identical to the a m o u n t lost from the culture fluid (results not shown). In Table 2 the data for the partition of 14C over the various fractions are c o m p a r e d with the data derived from chemical analyses of the fermentation products and the calculated a m o u n t s of glucose-carbon in the bioTable 3 b-type cytochromes in aerobically and anaerobically grown cells of A. viscosus Computer analysis of the observed absorbtion spectrum showed that the spectrum consisted of two absorbtion bands at two different wavelengths (I and II) [13]. The two absorbtion bands had different intensities, which were expressed as the % area of the observed absorbtion spectrum. anaerobe Wavelength % Area aerobe I II I II 558.6 72.5 564.3 27.6 558.0 73.7 563.5 26.4 50 mass and carbon dioxide. The data are in good agreement, with the 14C method giving a slightly higher value for the biomass and a somewhat lower one for carbon dioxide. At high percentages of oxygen in the gas phase, a high Ygl..... (149 g. mol-1), combined with a low carbon recovery from fermentation products was observed (Fig. 2). This strongly suggested an oxidative metabolism coupled to electron transport phosphorylation. We have therefore investigated the presence of cytochromes in cells grown under aerobic and anaerobic conditions. In the dithionite-reduced cytochrome spectra only two absorbtion bands of b-type cytochromes could be detected by computer analysis [13]. Their ratios remained the same under aerobic and anaerobic conditions (Table 3). 5. DISCUSSION A. viscosus, an organism generally considered as an anaerobe, grew with a 2.4-times higher yield in aerated cultures than under anaerobic conditions (Fig. 2). The growth yield in aerated cultures (Yglucose= 149 g-mo1-1) cannot be accounted for by a fermentative metabolism and strongly suggested citric acid cycle activity coupled to electron transport phosphorylation, as was observed before in other oxygen consuming anaerobes, such as Propionibacterium pentosaceum [15]. This suggestion was confirmed by the observation that with increasing oxygen concentrations carbon dioxide became almost the sole metabolic product, while acetate, formate and succinate disappeared concomitantly (Figs. 1 and 2, Table 2). Moreover, in the closely related species A. naeslundii, the citric acid cycle enzymes have been demonstrated [5], yet the hnk to oxidative catabolism in Actinomyces species has not been made. Taken together the above strongly suggests that A. viscosus adjusts its metabolism to the amount of oxygen available in the environment. It can grow either completely fermentatively (Schemes 1 and 2), or via partial (Scheme 3) or complete oxidation of carbohydrates (Scheme 4). (4) glucose + 6 oxygen ~ 6 carbon dioxide + 6 water The cytochrome spectra of A. viscosus cells grown under aerobic and anaerobic conditions were identical: only two cytochrome b-type absorbtion bands were present (Table 3). Under anaerobic conditions these cytochromes are presumably part of an electron transport chain terminating with fumarate reductase [16], which enables the production of 1 mol of ATP per mol succinate formed [17]. Fermentation of 1 mol glucose would then yield 4 mol ATP. Since, with glucose as an electron donor oxygen was consumed with a high rate and affinity, it seems reasonable to assume that under aerobic conditions a very similar electron transport chain is active, now with oxygen as the final electron acceptor. If again, as under anaerobic conditions, 1 ATP would be produced per 2 e- passing the electron transport chain, complete oxidation of glucose would yield 14 mol of ATP. Taking into account the fraction of glucose converted into biomass and the partial oxidation of glucose, an YATP value of 18.4-20.8 g. mo1-1 under anaerobic conditions and an YATP of 18.0--21.8 g-mo1-1 under aerobic conditions can be calculated. These values are in good agreement with each other, but somewhat higher than the YATP values observed for propionic acid bacteria (11.5-16.7 g-mo1-1) growing anaerobically with glucose or fructose [9]. The calculations in Table 1 show that, for a growth medium with a specific composition, a fixed percentage of the dry weight of the cells is derived from glucose. As a consequence, with increasing growth yields, an increasing part of the glucose would be used for the production of biomass. This was verified in the experiment in which [14C]glucose was added to the chemostat. The observed incorporation of [14C]glucose into the biomass was in good agreement with the calculated amount (Table 2). In addition the isotope experiment showed that the remaining glucose carbon, which was not incorporated into the biomass or acidic fermentation products, was indeed converted to carbon dioxide. In conclusion the present experiments show that A. viscosus grows as well in aerated cultures as under anaerobic conditions. Oxidation of glucose was presumably coupled to electron transport phosphorylation, which enabled A. viscosus to 51 i n c r e a s e its g r o w t h y i e l d u n d e r a e r o b i c c o n d i t i o n s significantly. Competing microorganisms from the s a m e e c o s y s t e m , s u c h as S. sanguis, s h o w s i m i l a r r a t e s of o x y g e n u p t a k e [18], b u t t h e y c a n n o t significantly increase their growth yield under aerobic c o n d i t i o n s [3]. T h i s m i g h t b e a c o m p e t i t i v e adv a n t a g e f o r A . viscosus in d e n t a l p l a q u e , w h e r e o n l y l i m i t i n g a m o u n t s o f c a r b o h y d r a t e s [19] a n d o x y g e n [20] a r e a v a i l a b l e . ACKNOWLEDGEMENT W e w i s h to t h a n k L . F . O l t m a n a n d W . d e V r i e s f o r t h e i r p r e p a r a t i o n o f the c y t o c h r o m e s p e c t r a , and stimulating discussions. [10] [11] [12] [13] REFERENCES [1] Theilade, E. and Theilade, J. (1985) Formation and ecology of plaque at different locations in the mouth. Scand. J. Dent. Res. 93, 90-95. [21 Theilade, E., Theilade, J. and Mikkeisen, L. (1982) Microbiological studies on early dento-gingival plaque on teeth and mylar strips in humans. J. Periodont. Res. 17, 12-25. [3] Van Beelen, P., Van der Hoeven, J.S., De Jong, M.H. and Hoogendoorn, H. (1986) The effect of oxygen on the growth and acid production of Streptococcus mutans and Streptococcus sanguis. FEMS Microbiol. Ecol. 38, 25-30. [4] Murphy, M.G., O'Connor, L., Walsh, D. and Condon, S. (1985) Oxygen-dependent lactate utilization by Lactobacillus plantarum. Arch. Microbiol. 141, 75-79. [5] Buchanan, B.B. and Pine, L. (1967) Path of glucose breakdown and cell yields of a facultative anaerobe, Actinomyces naeslundii. J. Gen. Microbiol. 46, 225-236. [6] Van der Hoeven, J.S. and De Jong, M.H. (1984) Continuous culture studies and their relation to the in vivo behavior of oral bacteria. In: Continuous Culture 8 (Dean, A.C.R., Ellwood, D.C. and Evans, C.G.T., Eds.), pp. 89-109. Ellis Horwood, Chichester, U.K. [7] Brown, A.T. and Breeding, L.C. (1980) Carbon dioxide metabolism by Actinomyces oiscosus: Pathways for succinate and aspartate production. Infect. Immun. 28, 82-91. [8] De Vries, W,, Van Wijck-Kaptijn, M.C. and Stouthamer, A.H. (1972) Influence of oxygen on growth, cytochrome synthesis and fermentation pattern in propionic acid bacteria. J. Gen. Microbiol. 71, 515-524. [9] De Vries, W., Van Wijck-Kaptijn, M.C. and Stouthamer, [14] [15] [16] [17] [18] [19] [20] A.H. (1973) Generation of ATP during cytochrome-linked anaerobic electron transport in propionic acid bacteria. J. Gen. Microbiol. 76, 31-41. De Jong, M.H., Schaeken, M.J.M., Van den Kieboom, C.W.A. and Van der Hoeven, J.S. (1983) Colonization of the teeth of rats by human and rodent oral strains of the bacterium Actinomyces viscosus. Arch. Oral Biol. 28, 247-252. Van der Hoeven, J.S., De Jong, M.H., Camp, P.J.M. and Van den Kieboom, C.W.A. (1985) Competition between oral Streptococcus species in the chemostat under alternating conditions of glucose hmitation and excess. FEMS Microbiol. Ecol. 31, 373-379. Van der Hoeven, J.S., Franken, P.J.M., Camp, P.J.M. and Dellebarre, C.W. (1978) Analysis of bacterial fermentation products by Isotachophoresis. Appl. Environ. Microbiol. 35, 17-23. Van Wielink, J.E., Oltman, L,F., Leeuwerik, F.J., De Hollander, J.A. and Stouthamer, A.H. 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(1985) Pyruvate oxidase activity dependent on thiamine pyrophosphate, ravin adenine dinucleotide and orthophosphate in Streptococcus sanguis. FEMS Microbiol. Lett, 25, 53-56. De Jong, M.H., Van der Hoeven, J.S. and Lukassen, J.A.M. (1986) Competition for dietary carbohydrates between streptococci in dental plaque. FEMS Microbiol. Ecol. 38, 341-345. Van der Hoeven, J.S., De Jong, M.H. and Kolenbrander, P.E. (1985) In vivo studies of microbial adherence in dental plaque. In: Molecular Basis of Oral Microbial Adherence (Mergenhagen and Rosan, Eds.), pp. 220-227, American Society for Microbiology, Washington.