Department of Bioresource Science, Ibaraki University College of Agriculture, Ami-machi, Ibaraki 300-0393, Japan1
Department of Microbiology, Okayama University Dental School, Shikata-cho 2-chome, Okayama 700-8525, Japan2
Author for correspondence: Hiroyuki Ohta. Tel: +81 298 88 8684. Fax: +81 298 88 8525. e-mail: hohta{at}acs.ibaraki.ac.jp
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ABSTRACT |
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Keywords: fructose metabolism, microaerophile, chemostat culture, potassium ion, periodontopathogen
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INTRODUCTION |
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In this study, we examined whether or not respiratory-chain phosphorylation occurs in A. actinomycetemcomitans. To this end, several growth parameters, including the specific rate of ATP production from fructose catabolism, were estimated for steady-state cultures at different dilution rates. Our study also focused on the effect of potassium on the ATP production rate. Potassium is influential in bacterial energy metabolism (Hueting et al., 1979 ) and a predominant cation in oral environments such as the dental plaque and gingival fluid (Tatevossian & Gould, 1976
).
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METHODS |
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Chemostat culture.
The bacterium was grown in a chemostat system as described before (Ohta et al., 1989 ). For anaerobic cultures, the system was kept under a stream (200300 ml h-1) of N2, which was freed of traces of oxygen by passage over a gas-purifying column (Gas Clean GC-RX; Nikka Seiko). In the chemostat system the redox potential was measured continuously using a platinum electrode with an Ag/AgCl reference cell and used to control the stirring speed in the culture. The electrode signal was measured with a redox regulator (model F011; Tokyo Rikakikai) calibrated against a quinhydrone reference solution at pH 4·0. In this system, manipulation of the air flow in combination with feedback control on the stirring speed made it possible to maintain a constant redox potential (±510 mV) between approximately -400 mV (anaerobic) and +300 mV (aerobic; near air saturation), depending on the exact medium composition and the air supply (Ohta & Gottschal, 1988
; Ohta et al., 1996b
). In this study the redox potential of microaerobic culture was maintained at -200 mV. The temperature of the culture was maintained at 37 °C, and the pH was maintained at 7·0 with automatic addition of 2 M NaOH or 2 M HCl. The OD660 of cultures was measured in a 1 cm light path cuvette to determine the cell densities. The averaged coefficient of the dry cell weight at OD660 was 0·852±0·055 mg dry wt ml-1 per OD660 unit (mean±SD of 12 runs at dilution rates between 0·04 and 0·25 h-1) (Ohta et al., 1989
). The relationship is linear up to 0·6 units of OD660 (Ohta et al., 1989
). The purity of the cultures was routinely checked on tryptic soy agar (BBL Microbiology Systems) plates.
Measurement of oxygen uptake by washed cells.
Bacterial cells were sampled from chemostat cultures, washed twice with 100 mM potassium MOPS buffer (pH 7·0) containing 66 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 0·5 mM MgCl2, and suspended at 3450 mg dry wt ml-1 in the MOPS buffer. Oxygen consumption by cell suspensions was measured at 37 °C in a Hansatech oxygen electrode unit (type DW1). It consisted of a water-jacketed reaction chamber (volume, 1 ml), with a Clark-type oxygen electrode disc set in the floor. The chamber was closed with a stopper. In this unit, the magnetic follower that stirred the cell suspension spun directly above the membrane-covered electrode. The chamber contained the air-saturated MOPS buffer and an appropriate amount of the cell suspension was injected through a hole in the stopper. After the output signal had become constant, the reaction was started by the addition of fructose (final concentration 10 mM). The calibration of the electrode was made on the assumption that the dissolved oxygen concentration in air-saturated water was 217 µM at 37 °C (Kielley, 1963 ).
Chemical analysis.
Formate, succinate and ethanol were determined by GLC, and fructose by the enzymic method described before (Ohta et al., 1989 ). Acetate was determined using an enzyme system consisting of acetyl-CoA synthetase, citrate synthase and malate dehydrogenase (Boehringer Mannheim).
Calculations.
The specific rate of ATP production during fructose fermentation (qATP(fermen)) was calculated by the following equation (Ohta et al., 1989 ):
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where qfructose and qacetate are the specific rates of fructose consumption and acetate formation, respectively, and q is expressed in mmol substrate consumed or formed (g dry wt)-1 h-1. The ATP yield from fructose fermentation (mol ATP formed per mol fructose metabolized) was calculated as the ratio of qATP(fermen) to qfructose. For the anaerobic cultures, the YATP (g cells produced per mol ATP consumed) was estimated by the ratio of the growth yield on fructose (Yfructose) to the ATP yield from fructose fermentation. Based on the discussion by Stouthamer & Bettenhaussen (1975) , we assumed that the YATP was dependent on the growth rate and did not change significantly with a change from anaerobic to microaerobic conditions when medium composition and culture pH were kept constant. Thus, for microaerobic cultures, the specific rates of total ATP production (qATP(total)) and ATP production through respiratory chain phosphorylation (qATP(respi)) were estimated by the following equation:
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RESULTS AND DISCUSSION |
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Assuming the following relationship (Tempest & Neijssel, 1984 )
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the maximum growth yield (Ymax) and the maintenance coefficient (m) were estimated: 50·3 g dry wt (mol fructose)-1 and mfructose 0·359 mmol (g dry wt)-1 h-1, respectively.
The main fermentation products from anaerobic fructose-limited chemostat cultures of A. actinomycetemcomitans were acetate, formate, ethanol and succinate. Lactate was not detected at any of the dilution rates tested. Carbon recoveries, which were estimated from the ratios of amounts of fermentation-product-carbons to amounts of fructose-carbon consumed, were between 88 and 91%. The specific formation rates of fermentation products as functions of the dilution rate are shown in Fig. 2(a). Acetate was the most abundant product and its formation was proportional to dilution rate. Succinate was the second most abundant product at dilution rates below 0·10 h-1, but its formation did not increase with increasing dilution rate. Increases in the formation of formate and ethanol showed biphasic patterns and the slopes were higher at dilution rates above 0·10 h-1.
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To examine whether or not an induction of respiratory capacity with fructose occurs upon microaerobic culturing, washed cells were prepared from the anaerobic (Eh=-410 mV) and microaerobic (Eh=-270 and -200 mV) fructose-limited chemostat cultures run at D=0·10 h-1 then oxygen uptake rates by the two sets of washed cells were compared. Fructose-dependent oxygen uptake activity of the microaerobically grown cells was slightly lower [1·261·36 mmol O2 h-1 (g dry wt)-1(mean of duplicate determinations)] than that of anaerobically grown cells [1·54 mmol O2 h-1 (g dry wt)-1], indicating that no induction but a small suppression of respiratory capacity occurred in the microaerobic culture. It was also noted that the measured oxygen uptake rates were significantly low compared with those of washed cells of batch-grown Escherichia coli K-12 [about 8 mmol O2 h-1 (g dry wt)-1] (Ohta & Taniguchi, 1988 ).
Effect of K+ on growth
Our growth medium (AA medium) contained 5·2 mM K+, which was higher than the amount (1 mM) just sufficient to meet the cellular requirement of a rapidly growing culture of the facultatively anaerobic, sugar-fermenting Klebsiella aerogenes (K. pneumoniae) (Tempest & Neijssel, 1984 ). However, considering that oral environments such as dental plaque and gingival fluid contain relatively high amounts of K+ (61·5±13·5 mM for plaque fluid and 17·4±9·0 mM for gingival fluid) (Tatevossian & Gould, 1976
), higher extracellular K+ concentrations might be required for rapid growth of A. actinomycetemcomitans. Therefore, fructose-limited growth of strain 301-b was examined using media containing high K+ concentrations. In these media, the Na+ concentration was fixed at 24 mM. The Yfructose of the steady-state microaerobic cultures (Eh=-200 mV) with 81 and 162 mM K+ were 1·3 times higher than the values of the low (5·2 mM) K+ culture (Table 1
). In contrast, for the anaerobic cultures, increases in extracellular K+ concentrations did not result in an increase in the Yfructose. As a control, the effect of Na+ on the anaerobic and microaerobic fructose-limited growth (at D=0·15 h-1) was also examined; the medium K+ concentration was fixed at 5·2 mM. No significant changes in the Yfructose were found for anaerobic or microaerobic cultures with increasing Na+ concentration from 24 to 55 then to 106 mM (Table 1
).
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This indicates that under the anaerobic condition, the specific rate of fermentation-derived ATP production was not affected by the increases in extracellular K+ and Na+ concentrations, and the ATP yield was constant with the value of 3·13 mol ATP per mol fructose.
Estimation of ATP production during microaerobic growth
In the microaerobic control cultures, the specific rate of ATP production from fermentation (qATP(fermen)) was also a linear function of the fructose consumption rate (qfructose) (Fig. 6a). However, the estimated ATP yield value [2·49 mol ATP (mol fructose)-1] was lower than the anaerobic value [3·13 mol ATP (mol fructose)-1]. Assuming that the YATP was similar between anaerobic and microaerobic cultures, this result did not account for the increased Yfructose values in the microaerobic cultures at lower dilution rates. Hence, it was very likely that that the additional ATP was derived from respiration. The YATP calculated from the anaerobic cultures increased linearly from 11·7 g dry wt (mol ATP)-1 at D=0·04 h-1 to 14·9 g dry wt (mol ATP)-1 at D=0·20 h-1 (Fig. 1a
). Based on the assumption that the YATP was dependent of the growth rate and did not change significantly with a change from anaerobic to microaerobic conditions at a fixed growth rate, the specific rates of total ATP production (qATP(total)) and ATP production via respiration (qATP(respi)) were estimated for each steady state culture. As shown in Fig. 6(a)
, the qATP(respi) was expected to parallel the qATP(fermen) until it reached a maximum value at a qfructose of 1·58 mmol (g dry wt)-1 h-1, and to decrease above this qfructose value. From the ratio of qATP(respi) to qfructose, the respiration-derived ATP yield was estimated to be between 1·2 and 2·0 mol ATP (mol fructose)-1 below qfructose=1·58 mmol (g dry wt)-1 h-1.
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Satisfactory explanations are not yet available for the requirement of K+ in cells growing rapidly under microaerobic conditions. One possibility is that mechanisms leading to a significant energy loss occur when cells are grown at higher rates in low-K+ conditions. The most probable mechanism is a K+ efflux system because a K+ efflux system (kefC) was reported in not only E. coli (Bakker et al., 1987 ) but also Haemophilus influenzae: the latter is phylogenetically related to A. actinomycetemcomitans (Fleischmann et al., 1995
). If this is the case for A. actinomycetemcomitans, it will be of interest to examine whether the leakage of K+ from the cytoplasm is enhanced by exposure of cells to oxygen. The occurrence of a significant energy loss is also very likely for the high-Na+ culture at higher growth rates because a negative effect of increasing Na+ on the Yfructose was observed (Fig. 3b
). Based on the database of the on-going genome analysis of A. actinomycetemcomitans strain HK1651 (B. A. Roe, F. Z. Najar, S. Clifton, T. Ducey, L. Lewis and D. W. Dyer, University of Oklahoma, Department of Chemistry and Biochemistry, and the University of Oklahoma Health Science Centre, Department of Microbiology and Immunology; http://www.genome.ou.edu/act.html), Na+/H+ antiporter proteins are expected to be present in this organism, and thus in high-Na+ conditions, extra energy will be needed to drive the Na+/H+ antiporters for keeping intracellular Na+ concentration low.
K+ is the major cytoplasmic cation of growing bacterial cells (Silver, 1978 ). In Gram-negative bacteria such as E. coli and K. aerogenes (K. pneumoniae), intracellular K+ concentrations are as high as 0·10·5 M (Tempest et al., 1966
; Kakinuma, 1998
). In E. coli, K+ can be taken up via a number of systems that differ in their affinity for K+; there is a constitutive low-affinity system (Trk) and an inducible high-affinity system (Kdp) (Bakker, 1993
; Epstein et al., 1993
). In H. influenzae, the gene encoding the Trk K+ uptake protein is present but the gene of the Kdp system is absent (Fleischmann et al., 1995
). This seems also to be true for A. actinomycetemcomitans, based on the database of its on-going genome analysis. The absence of the high-affinity system for K+ uptake might be disadvantageous for A. actinomycetemcomitans in low-K+ environments. This would be consistent with the fact that the primary ecological niche of A. actinomycetemcomitans is a K+-rich environment such as dental plaque (61·5±13·5 mM) and gingival fluid (17·4±9·0 mM) (Tatevossian & Gould, 1976
). Interestingly, it was reported that in patients suffering a more severe periodontitis, the gingival fluid Na+ concentrations tended to be lower, and those for K+ to be significantly higher (Bang et al., 1973
). As discussed by Bang et al. (1973)
, in more severe cases of periodontitis the higher number of degenerating epithelial, connective tissue and blood cells contributes to increasing the K+ concentration of the exudates by the liberation of their intracellular content. Hence, it may be speculated that both moderate levels of oxygen tension and increased K+ contents prevail in periodontal lesion sites and these constitute conditions favourable for the growth of A. actinomycetemcomitans.
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REFERENCES |
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Received 23 January 2001;
revised 22 May 2001;
accepted 5 June 2001.
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