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1 Department of Microbiology, Cornell University, Ithaca, NY 14853, USA
2 Agricultural Research Service, USDA, Ithaca, NY 14853, USA
Correspondence
James B. Russell
jbr8{at}Cornell.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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p), but C. sporogenes MD1 grows even if the p is very low. Cell suspensions incubated with additional sodium chloride produced ammonia as rapidly at pH 5.0 as at pH 7.0, but cells incubated with additional sodium lactate were sensitive to even small decreases in extracellular pH. Similar results were obtained if the sodium lactate was replaced by sodium acetate or propionate. When extracellular pH declined, pH increased even if sodium lactate was present. The cells accumulated intracellular lactate anion when the pH was acidic, and intracellular glutamate declined. Because amino acid deamination is linked to a transamination reaction involving glutamate dehydrogenase, the decrease in ammonia production could be explained by the decrease in intracellular glutamate. This latter hypothesis was consistent with the observation that extracellular glutamate addition restored amino acid deamination even though glutamate alone did not allow for the generation of ammonia.
p, protonmotive force; pH, transmembrane pH gradient; 
, transmembrane electrical potential; TCS, tetrachlorosalicylanilide; TPP+, tetraphenylphosphonium ionProprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This problem is exacerbated by secondary fermentations that consume acids or produce ammonia. If pH increases, the growth of moulds and other harmful micro-organisms is favoured (Wilkinson, 1999). Silage fermentation is promoted by lactic-acid-producing bacteria that consume sugar, but some silages (e.g. alfalfa haylage) have little sugar and a very high amino acid content. If amino acids are deaminated by clostridia, silage pH can increase (Ohmomo et al., 2002). Amino acid degradation also decreases the nutritive value of silage as animal feed.
Clostridium sporogenes can utilize amino acids as the sole energy source (Nisman, 1954), and it can readily be isolated from silages (Flythe & Russell, 2004). C. sporogenes also ferments carbohydrates, but only if amino acids are present (Lovitt et al., 1987). Protonmotive force (p) has a variety of cellular functions, and bacteria typically have a p of at least –100 mV (Harold, 1986). However, recent work with C. sporogenes MD1 indicated that growing cells had a p smaller than –15 mV, but p values as large as –120 mV could be detected if growth was inhibited by acidic pH, deprivation of an essential amino acid, or inhibitors of protein synthesis (Flythe & Russell, 2005).
The inhibition of bacterial growth by fermentation acids was traditionally explained by the ability of undissociated acids to move across the cell membrane, act as uncouplers, and decrease p (Jay, 1986). The question then arose of how fermentation acids would inhibit a bacterium that has virtually no p when it is growing. Fermentation acid toxicity has also been correlated with intracellular pH regulation (Russell & Diez-Gonzalez, 1998). If a bacterium lets its intracellular pH decrease, the pH gradient across the cell membrane (pH) is lowered, and the accumulation of fermentation acid anions is not as great. This latter mechanism helps to explain why some bacteria are more sensitive to fermentation acids than others.
The experiments described here sought to determine the effect of fermentation acids on the membrane bioenergetics of C. sporogenes MD1. We hypothesized that fermentation acids would prevent p generation at acidic pH. Because preliminary experiments indicated that this simple hypothesis was incorrect, we decided to examine glutamate pools and amino acid transamination.
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METHODS |
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and grown anaerobically (39 °C) in basal medium that contained (per litre) 292 mg KH2PO4, 480 mg Na2SO4, 480 mg NaCl, 100 mg MgSO4.7H2O, 64 mg CaCl2.2H2O, 600 mg cysteine hydrochloride and 6.9 g NaH2PO4.H2O. The basal medium was boiled to remove O2 and allowed to cool under O2-free N2. Enzymic casein hydrolysate (15 mg ml–1, Gibco Laboratories) was added as an energy source. Initial pH was adjusted by adding HCl or NaOH.
Cell suspensions.
Overnight C. sporogenes MD1 cells (16–24 h, 160 µg protein ml–1, pH 6.7) were harvested by centrifugation (2300 g, 5 min) in anaerobic culture tubes with rubber stoppers (Bellco Glass). The supernatants were removed by aspiration and the N2 atmosphere was maintained by continuous gassing. Cell pellets were resuspended in basal medium. The suspensions were transferred to serum bottles and amended with casein hydrolysate and/or glutamic acid. The pH was adjusted with HCl and the cell suspensions incubated for 20 min unless otherwise indicated (39 °C).
Adjustment of culture pH.
C. sporogenes MD1 did not decrease culture pH when casein hydrolysate was the energy source, but pH could be decreased by adding HCl. To reduce pH, C. sporogenes MD1 was grown in an anaerobic culture vessel (250 ml, 100 ml basal medium) equipped with a pH electrode. The vessel was continuously purged with N2 and enough HCl (6 M) was added to achieve a 0.25 unit reduction in pH. HCl was added every 15 min.
Protonmotive force.
The pH and transmembrane electrical potential, , were determined by methods employing silicone oil centrifugation as previously described (Kashket & Barker, 1977). Briefly, pH was estimated from the distribution of [14C]benzoate (1 µM, 0.81 GBq mmol–1; Amersham Pharmacia Biotech) across the cell membrane using the Henderson–Hasselbalch equation. The was calculated from the uptake of [3H]tetraphenylphosphonium bromide ([3H]TPP+) (10 µM, 8.51 GBq mmol–1; Amersham Pharmacia Biotech) using the Nernst equation (–2.3 [RT/F]xlog[(concentration in)/(concentration out)]). Intracellular volume (3.6 µl mg–1 protein) was estimated from the difference between the distribution of 3H2O (50 µl ml–1, 185 kBq µl–1; Amersham Pharmacia Biotech) and the extracellular marker [14C]taurine (0.5 µM, 4.26 GBq mmol–1; Amersham Pharmacia Biotech). [3H]TPP+ and [14C]benzoate uptakes were corrected for extracellular contamination using [14C]taurine, an osmolyte that is not taken up, and 3H2O. Growing cultures or washed cell suspensions were transferred anaerobically to N2-filled tubes that contained [3H]TPP+ or [14C]benzoate, and incubated at 39 °C for 5 min. Corrections for non-specific [3H]TPP+ binding were made by incubating the cells with a combination of nigericin (5 µM) and valinomycin (5 µM) for 10 min to eliminate the .
Intracellular lactate.
Cell suspensions (160 µg protein; 1 ml) in basal medium were incubated for 25 min (39 °C) with [14C]lactic acid (100 mM, 3.1 MBq mmol–1; Amersham Pharmacia Biotech) prior to silicone oil centrifugation (equal parts mixture of Dexter Hysol 550 and 560). The tubes were frozen and the pellets were clipped into scintillation vials with scintillation fluid. The cell pellets were mixed by vortexing and permitted to dissolve overnight at 25 °C before scintillation (Beckman LS56500). Intracellular lactate concentration was calculated from the ratio of supernatant to pellet radioactivity and the intracellular volume (see above). Nigericin- and valinomycin-treated cells were used to correct [14C]lactate uptakes for the extracellular space in the pellet (see above).
Intracellular glutamate.
Growing cells or washed cell suspensions (1 ml; 0.16 mg protein) were placed in microcentrifuge tubes containing silicone oil (400 µl, 7 parts Dexter Hysol 550 to 3 parts Dexter Hysol 560) layered on top of 100 µl 14 % perchloric acid plus 9 mM EDTA. After centrifugation (13 000 g, 1 min), the perchloric acid layer was removed, neutralized with 100 µl 0.3 M K2CO3 and 0.8 M KOH, mixed by vortexing, and frozen at –20 °C. After thawing and centrifugation to remove precipitate (13 000 g, 5 min), samples were analysed for glutamate by a method employing glutamate dehydrogenase (Chen & Russell, 1989). Briefly, glutamate dehydrogenase (EC 1.4.1.4) and NADP+ were mixed in a triethanolamine buffer. The sample was added and the production of NADPH was observed at 340 nm in a spectrophotometer (model 260, Gilford Instruments).
Other analyses.
Cell protein from NaOH-hydrolysed cells (0.2 M NaOH, 100 °C, 15 min) was determined by the Lowry method using bovine serum albumin as a standard. Ammonia was assayed by the colorimetric method of Chaney & Marbach (1962). Optical density (600 nm) was determined using a spectrophotometer (model 260, Gilford Instruments).
Statistics.
All experiments were performed three or more times. In all cases, the coefficient of variation (standard deviation divided by the mean) was less than 10 %.
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RESULTS |
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). Cultures with additional sodium chloride were less susceptible to a decrease in extracellular pH than those that had additional sodium lactate. When the pH was less than 6.25, cultures with sodium lactate had a lower OD600 than those with sodium chloride, and ammonia production was also inhibited (Fig. 1b).
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). However, cells incubated with additional sodium lactate (100 mM) were sensitive to even small decreases in extracellular pH, and little ammonia production was observed at pH 5.0. Similar results were obtained if the sodium lactate was replaced by sodium acetate or propionate. Cells treated with a protonophore (5 µM tetrachlorosalicylanilide, TCS) or a combination of nigericin and valinomycin (5 µM each) were also sensitive to a decrease in extracellular pH.
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p. The p was at least fivefold greater at pH 5.2 than at pH 6.5, but this potential could be eliminated by a combination of nigericin and valinomycin (Fig. 3a). The protonophore TCS also decreased pH, but TCS bound TPP+ and confounded the measurement of . The intracellular pH of C. sporogenes MD1 cells declined as a function of extracellular pH, but the pH was approximately 0.8 pH unit at pH 5.0. Because similar results were observed with cells resuspended in basal medium containing additional sodium chloride or sodium lactate (100 mM each) (Fig. 3b), the fermentation-acid-dependent decline in ammonia production (Fig. 2
) could not be explained by a decrease in intracellular pH or p. If TCS or a combination of nigericin and valinomycin was present, pH could not be detected and the intracellular pH was therefore lower (Fig. 3b).
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pH and the Henderson–Hasselbalch equation (Fig. 4a). Because less than 2 % of the lactic acid (pKa 3.91) was in the undissociated form even if the intracellular pH was 5.75 (Fig. 4a), the lactic acid accumulation was primarily lactate anion. When intracellular lactate anion increased, the specific activity of amino acid deamination declined (Fig. 4b). C. sporogenes MD1 cells resuspended with either sodium lactate or sodium chloride (100 mM) had more than 100 mM intracellular glutamate at pH 6.5 (Fig. 5). Acidic pH had little impact on intracellular glutamate if sodium chloride was present, but glutamate was virtually undetectable if sodium lactate was present and the pH was 5.0. Similar results were obtained if the cells were incubated with 100 mM sodium acetate (Fig. 5), TCS (5 µM) or nigericin and valinomycin (5 µM) and the pH was 5.0 (data not shown).
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). However, large amounts of extracellular glutamate counteracted the lactate-dependent inhibition of ammonia production. When the pH was 5.7 and lactate was 100 mM, the deamination rate was 380 nmol (mg protein)–1 min–1, but the rate increased to 650 nmol (mg protein)–1 min–1 if 60 mM glutamate was added to the cell suspensions. At pH 5.2, lactate was even more inhibitory, and glutamate once again restored ammonia production.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The idea that lactate might act as an uncoupler in C. sporogenes MD1 was supported by the observation that the protonophore TCS, or a combination of nigericin and valinomycin, inhibited ammonia production. However, fermentation acids did not decrease ATP or p at acidic pH. Because p was not affected, it appeared that the ability of fermentation acids to inhibit the ammonia production of C. sporogenes was occurring via a mechanism that did not involve a decline in p.
Many bacteria maintain a relatively constant intracellular pH over a wide range of extracellular pH (Padan et al., 1981), but fermentative bacteria often let their intracellular pH decline when the environment is acidic (Russell & Hino, 1985; Kashket, 1987). The utility of letting intracellular pH decline when fermentation acids are present can be explained by the potential effects of anions. If undissociated fermentation acids move across the cell membrane and ionize in the more alkaline interior, the anions accumulate. When C. sporogenes MD1 was exposed to acidic extracellular pH and lactate was present, pH increased from 0.2 unit at pH 7.0 to 0.8 unit at pH 5.0 and intracellular lactate anion accumulated exponentially. The increase in intracellular lactate was in accordance with the pKa value of lactate, intracellular pH, and the Henderson–Hasselbalch equation.
The strategy of letting intracellular pH decline is facilitated by schemes of energy derivation that remain active at acidic pH (Russell & Diez-Gonzalez, 1998). Because the protonophore (TCS) and the ionophores decreased intracellular pH, the inhibition of ammonia production could have been at least partially explained by a decrease in intracellular pH. However, the same argument could not be made for sodium chloride- and lactate-treated cells. Ammonia production was inhibited by sodium lactate but not sodium chloride when the extracellular pH was acidic, but the declines in intracellular pH values were similar. The ammonia production rate of C. sporogenes MD1 declined when intracellular lactate anion accumulated, and the question then arose how lactate was affecting the deamination of amino acids.
When Roe et al. (1998) treated Escherichia coli cells with acetic acid, intracellular pH decreased transiently, but the cells accumulated acetate anion. Because the accumulation of acetate anion caused a decrease in intracellular glutamate, it appeared that the cells were balancing one anion with another. The restoration of intracellular pH after the removal of acetate was dependent on glutamate biosynthesis, however, ‘the question of how [the] growth inhibition was achieved...remained unresolved’ (Roe et al., 2002). Later work indicated that acetic acid also led to an inhibition of methionine biosynthesis and possibly homocysteine toxicity (Roe et al., 2002).
In C. sporogenes, the first step in oxidative amino acid deamination typically involves a transamination reaction linked to glutamate dehydrogenase (Gottschalk, 1986). In this reaction, amino groups are transferred to 2-oxoglutarate, and glutamate is produced. Glutamate is then deaminated to release the ammonia. The glutamate dehydrogenase of C. symbiosium has a pH optimum of 8.0 (Coughlan et al. (2001). Furthermore, glutamate dehydrogenase has an equilibrium constant (4.5x10–14 M) that strongly favours glutamate formation rather than glutamate deamination (Frieden, 1959). Because relatively high concentrations of glutamate would be necessary for ammonia production, and glutamate is an intracellular osmolyte in a variety of bacteria (Neidhardt et al., 1990), we decided to examine the effect of fermentation acids on the intracellular pool of glutamate. Our results indicated that lactate accumulation caused a marked decline in the intracellular glutamate of MD1. Because this effect was not seen unless the pH was acidic and lactate was present, it appeared that lactate was inhibiting transamination via a decrease in intracellular glutamate. Based on the observation that acetate also caused glutamate efflux, it appeared that the effect was not lactate-specific.
The hypothesis that the decline in glutamate was responsible for the inhibition of ammonia production was consistent with the observation that the addition of extracellular glutamate restored the ammonia production of C. sporogenes MD1. Because glutamate alone was not deaminated and glutamate did not stimulate ammonia production from casein hydrolysate unless lactate was present and pH was acidic, the potential argument that glutamate itself was simply being deaminated could be dismissed. Further work will be needed to define the signal triggering glutamate efflux, but the mechanism is probably related to osmotic homeostasis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Chen, G. & Russell, J. B. (1989). Transport and metabolism of glutamine and glutamate by the ruminal bacterium, Streptococcus bovis. J Bacteriol 171, 2981–2985.
Coughlan, S., Wang, X. G., Britton, K. L., Stillman, T. J., Rice, D. W., Chiaraluce, R., Consalvi, V., Scandurra, R. & Engel, P. C. (2001). Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme's unusual pH dependence. Biochim Biophys Acta 1544, 10–17.
Flythe, M. D. & Russell, J. B. (2004). The effect of pH and a bacteriocin (bovicin HC5) on Clostridium sporogenes MD1, a bacterium that has the ability to degrade amino acids in ensiled plant materials. FEMS Microbiol Ecol 47, 215–222.[CrossRef]
Flythe, M. D. & Russell, J. B. (2005). The ability of acidic pH, growth inhibitors and glucose to increase the protonmotive force and energy spilling of amino acid fermenting Clostridium sporogenes MD1 cultures. Arch Microbiol 183, 236–242.
Frieden, C. (1959). Glutamic dehydrogenase; the order of substrate addition in the enzymatic reaction. J Biol Chem 234, 2891–2896.
Gottschalk, G. (1986). Bacterial Metabolism, pp. 273–275, 2nd edn. New York: Springer.
Harold, F. M. (1986). The Vital Force: a Study of Bioenergetics. New York: W. H. Freeman.
Jay, J. M. (1986). Modern Food Microbiology, 3rd edn. New York: Van Nostrand Reinhold.
Kashket, E. R. (1987). Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance. FEMS Microbiol Rev 46, 233–244.[CrossRef]
Kashket, E. V. & Barker, S. L. (1977). Effects of potassium ions on the electrical and pH gradients across the membrane of Streptococcus lactis cells. J Bacteriol 130, 1017–1023.
Lovitt, R. W., Kell, D. B. & Morris, J. G. (1987). The physiology of Clostridium sporogenes NCIB 8053 growing in defined media. J Appl Bacteriol 62, 81–92.
Neidhardt, F., Ingraham, J. & Schaechter, M. (1990). Physiology of the Bacterial Cell: a Molecular Approach. Sunderland, MA: Sinauer Associates.
Nisman, B. (1954). The Stickland reaction. Bacteriol Rev 18, 16–42.
Ohmomo, S., Tanaka, O., Kitamoto, H. K. & Cai, Y. (2002). Silage and microbial performance, old story but new problems. JARQ 36, 59–71.
Padan, I., Zilberstein, D. & Schuldiner, S. (1981). pH homeostasis in bacteria. Biochim Biophy Acta 650, 151–166.[Medline]
Roe, A. J., Mc Laggan, D., Davidson, I., O'Byrne, C. & Booth, I. R. (1998). Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J Bacteriol 180, 767–772.
Roe, A. J. O'Byrne, C., Mc Laggan, D. & Booth, I. R. (2002). Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity. Microbiology 148, 2215–2222.
Russell, J. B. & Diez-Gonzalez, F. (1998). The effects of fermentation acids on bacterial growth. Adv Microb Physiol 39, 205–234.[Medline]
Russell, J. B. & Hino, T. (1985). Regulation of lactate production in Streptococcus bovis: a spiraling effect that leads to rumen acidosis. J Dairy Sci 68, 1712–1721.
Wilkinson, J. M. (1999). Silage and animal health. Natural Toxins 7, 221–232.[CrossRef][Medline]
Received 17 March 2006;
revised 5 May 2006;
accepted 12 May 2006.
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), sodium lactate (
), sodium acetate (
), sodium propionate (
), TCS (5 µM) (
), or a combination of nigericin and valinomycin (5 µM each) (
). The concentration of all the sodium salts was 100 mM. In each case, cell suspensions were acidified with HCl, and the initial cell protein concentration was 160 µg ml–1.
