Arch Microbiol (1992) 157:471-474
Archives of
Hicrobiolegy ,9 Springer-Verlag 1992
Anaerobic malonate decarboxylation by Citrobacter diversus. Growth and metabolic studies, and evidence of ATP formation Peter H. Janssen* and C. G. Harfoot Department of Biological Sciences, University of Walkato, Private Bag 3105, Hamilton, New Zealand Received November 13, 1991/Accepted January 30, 1992
Abstract. Citrobacter diversus A T C C 27156 was able to grow by decarboxylation of malonate to acetate under strictly anaerobic conditions, in the presence of yeast extract. The growth yield, corrected for growth on yeast extract, was 2.03 g cell dry mass per tool malonate. The addition of malonate to ATP-depleted cell suspensions (less than 0.2 nmol A T P / m g cell protein) resulted in a rapid increase in cellular ATP levels to between 4.5 and 6.0 nmol/mg cell protein. Intact cells decarboxylated malonate at rates of up to 1.5 gmol/min, mg protein. Enzyme assays on malonate-grown cells indicated activation of malonate by an ATP-dependent ligase reaction and by CoA transfer from acetyl-CoA, followed by decarboxylation of malonyl-CoA to acetyl-CoA with subsequent recovery of the invested ATP by substrate level phosphorylation through the activity of acetate kinase. Net A T P synthesis is postulated to be mediated by gradient formation coupled to the decarboxylation of malonyl-CoA. The protonophore CCCP and H +ATPase inhibitor D C C D significantly reduced cellular A T P levels, suggesting a role for proton gradients in the energy metabolism of this strain when growing an malonate. Inhibitors of sodium metabolism or ommission of sodium had no effect on ATP levels or malonate decarboxylation. Key words: Citrobacter diversus - Anaerobic fermentation - Energy metabolism - Malonate - Decarboxylation - P r o t o n gradient
strain 16mall and other Enterobacteriaceae, and Sporomusa malonica grow well on malonate in the presence of yeast extract (Dehning et al. 1989; Janssen and H a r f o o t 1990; Janssen 1991a) and Malonomonas rubra grows on malonate in mineral medium (Dehning and Schink 1989). Malonomonas rubra displays a requirement for sodium (Dehning and Schink 1989), which suggests that the decarboxylation of malonate is coupled to the generation of a transmembrane sodium ion gradient for ATP production, analogous to the A T P generating system of the succinate-decarboxylating Propionigenium rnodestum (Hilpert et al. 1984). The malonate-decarboxylating activity of suspensions of Citrobacter sp. strain 16mall was sensitive to the protonophore 2,4-dinitrophenol, but resistant to the sodium ionophore monensin (Janssen and H a r f o o t 1990). This seems to preclude the involvement of a sodium ion gradient in the energy metabolism of Enterobacteriaceae growing anaerobically on malonate. Since the Enterobacteriaceae have a requirement for yeast extract when growing on malonate, there remain questions as to the exact role of malonate in the metabolism of these organisms, and whether ATP is actually formed coupled to the decarboxylation of malonate. Investigations to answer these questions and to measure enzymes involved in malonate catabolism in one strain of the malonate-fermenting Enterobacteriaceae are described in this communication. Materials and Methods Organism and growth conditions
Under dark anaerobic conditions only a few bacteria have been demonstrated to be able to grow on malonate, decarboxylating it to acetate and CO2. Citrobacter sp. * Present address and address for correspondence: P. H. Janssen, Fakult/it ffir Biologie,Universit/it Konstanz, Postfach 5560, W-7750 Konstanz Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone;
DCCD, dicyclohexylcarbodiimide; MOPS, 3-(N-morpholino) propanesulfonic acid
Citrobacter diversus ATCC 27156 was obtained from the National
Health Institute (now New Zealand Communicable Diseases Centre), Porirua, New Zealand (collection number NHI 868) and maintained in the freshwater medium of Janssen and Harfoot (1990) with 1 g yeast extract/l, 50 mM Na2 malonate and sulfide reductant at 34 ~ Growth experiments were carried out in the same medium. Optical density readings, HPLC analysis of samples, dry weight yield determinations and culture manipulations were as previously described (Janssen and Harfoot 1990). For experiments with cell suspensions, bulk cultures were grown in 100 ml volumes in 160-ml serum vials with 100 mM malonate and 50 mM MOPS (pH adjusted to 7.0 with NaOH).
472
Cell suspension experiments Cell suspensions were prepared as described elsewhere (Janssen 1992) without the addition of thioglycolate. ATP formation and malonate decarboxylation were followed as described by Janssen (1992). Malonic or acetic acid (neutralized to pH 7.0 with KOH). NaC1 or KC1 were added to 20 mM final concentration from anoxic stock solutions as required. When testing the effects of potential metabolic inhiNtors on cellular ATP levels, 20 mM KOH-neutralized malonic acid was added at the beginning of the experiment, and inhibitors added after 18 rain from ethanolic stock solutions at 0.1 ml per 20 ml buffer. As a control, 0.1 ml ethanol was added. Arsenate was added from an HCl-neutralized stock solution in buffer. Oubain and DCCD, at the levels present in the samples for ATP analysis, did not interfer with the luciferin/luciferase assay.
Enzymes assays Cell-free extracts were prepared by ultrasonication as described by Janssen and Harfoot (1990). Protein was estimated by a micromethod adaptation of the Lowry method (Scopes 1982). Enzymes and biochemicals were purchased from Sigma Chemicals (St. Louis, Mo., USA). Measurements of malonate decarboxylating actwity by crude cell-free extracts was carried out in Nz-flushed stoppered tubes at 34 ~ containing 1.5 ml 100mM MOPS/KOH pH 7.0, 33 mM malonate and crude cell-free extract (0.5 mg protein). Malonate decarboxylation and acetate formation was monitored by HPLC analysis of perchloric acid treated samples (Janssen 1992). End-point enzyme assays were carried out over 15 min at 34 ~ in stoppered N2-flushed test-tubes. At each 5 rain time point three assays (and appropriate controls) were halted and put on ice. Acetate kinase (EC 2.7.2.1) was assayed basically by method 1 of Nishimura and Griffith (1981), omitting succinate from the assay mixture. Malonate-CoA ligase (EC 6.2.1.-) was assayed using the following reaction mixture (modified from Wofford et al. 1986): 100 mM Tris, 15 mM NaF, 0.3 mM Triton X-100, 300 mM hydroxylamine 9HC1, 5 mM MgC12, 2 mM Na2 malonate, 5 mM ATP, 15 mM mercaptoethanol, 0.4 mM CoA, 2 mM GSH, pH adjusted to 8.3 with KOH. The formation of the CoA ester of malonate was measured by following its reaction with hydroxylamine using FeC13 reagent as for acetate kinase (Nishimura and Griffith 1981). Continuous enzyme assays were carried out a 34 ~ as described earlier (Janssen 1991b). Phosphotransacetylase (EC 2.3.1.8) was assayed as described by Bergmeyer et al. (1974). Malonate CoA transferase (EC 2.8.3.3) was assayed by following acetyl-CoA formation using the following reaction mixture: 100raM Tris, 20 mM (NH4)2SO4, 20 mM Na acetate, 0.4 mM malonyl-CoA, 1 mM dithiothreitol, 2 mM NAD, 5 mM malate, 1 U citrate synthase, 2.5 U malate dehydrogenase, pH adjusted to 7.4 with HC1. The activity measured was corrected for acetyl-CoA formation due to malonyl-CoA decarboxylase activity. Malonyl-CoA decarboxylase (EC 4.1.1.9) was assayed using the following mixture: 100 mM imidazole, 5 mM MgC12, 0.4 mM malonyl-CoA, 2 mM NAD, 5 mM malate, 1 mM dithiothreitol, 1 U citrate synthase, 2.5 U malate dehydrogenase, pH adjusted to 6.5 with HC1. Oxidatmn and reduction of pyridine nucleotides was followed at 340 nm (Bergmeyer 1975).
y
150
loo
,ti
50 o
0
1'0 2'0 30 ' 40' Malonate used (mmol)
50
Fig. 1. Growth yield of Citrobacter diversusATCC 27156 on a range of malonate in the presence of 1 g yeast extract/1 under strictly anaerobic conditions. The malonate concentrations are the amounts metabolized
acetate in s t o i c h i o m e t r i c a m o u n t s . T h e g r o w t h yield was p r o p o r t i o n a l to the a m o u n t of m a l o n a t e d e c a r b o x y l a t e d , u p to at least 45 retool/1 (Fig. 1), a n d was 2.03 g cell d r y m a s s p e r m o l m a l o n a t e (corrected for g r o w t h on yeast extract). T h e yield is the r a n g e (1.3 to 2.1 g cell d r y m a s s p e r m o l m a l o n a t e ) r e p o r t e d for o t h e r m a l o n a t e - d e g r a d i n g b a c t e r i a ( D e h n i n g et al. 1989; D e h n i n g a n d Schink 1989; J a n s s e n a n d H a r f o o t 1990; J a n s s e n 1991a). Yeast e x t r a c t was n o t r e q u i r e d for a n a e r o b i c g r o w t h o n c i t r a t e o r glucose.
A T P formation by cell suspensions A d d i t i o n o f m a l o n a t e to cell s u s p e n s i o n s o f m a l o n a t e g r o w n Citrobacter diversus resulted in a n i m m e d i a t e increase in cellular A T P levels (to b e t w e e n 4.5 a n d 6.0 n m o l / m g p r o t e i n ) with c o n c o m i t a n t d e g r a d a t i o n o f m a l o n a t e (Fig. 2). M a l o n a t e was d e c a r b o x y l a t e d at rates o f u p to 1.5 g m o l / m i n 9 protein. Cell s u s p e n s i o n s to
5
20
4
15.
[
10
5 Results and discussion
Growth studies
-~0
0
Citrobacter diversus A T C C 27156 was m a i n t a i n e d b y a p p r o x i m a t e l y m o n t h l y s u b c u l t u r e using m a l o n a t e as the g r o w t h s u b s t r a t e u n d e r strictly a n a e r o b i c (sulfide-reduced) c o n d i t i o n s for m o r e t h a n four years. This strain grew o n m a l o n a t e , in the p r e s e n c e of 1.0 g extract/1 (~t = 0.40 h - 1 at 34 ~ with c o n c o m i t a n t f o r m a t i o n of
Fig. 2. ATP formation([]) by cell suspensions of malonate-grown Citrobacter diversus ATCC 27156 concomitant with decarboxylation ofmalonate (9 The ATP content of cells to which no addition was made or to which 20 mM KC1 or acetate was added is also shown ( I )
0
10
20 30 Time (min)
40
50
473 Table 1. Effect of potential metabolic inhibitors on cellular ATP levels and the rate of malonate decarboxylation by cell suspensions of malonate-grown Citrobacter diversus ATCC 27156. The results are expressed relative to appropriate controls (nominally 100%)
Cellular ATP levels after addition
Inhibitor and concentration
CCCP Monensina DCCD Ouabain a Ethanol only control Arsenate Buffer only control
150 gM 75 gM 150 I-tM 75 gM 5 mM
Malonate decarboxylating activity
(nmol/mg)
(%)
(gmol/min - mg)
(%)
0.05 4.80 1.94 5.69 5.71 0.05 4.90
1 84 34 100 100 1 100
0.30 0.49 0.56 0.55 0.52 0.25 0.59
58 94 108 106 100 42 100
a Tested in the presence of 20 mM NaC1 which 20 m M KC1 or acetate were added, or to which no additions were made, showed no changes in cellular ATP levels. This basal level, regarded as ATP depleted, was less than 0.2 nmol/mg protein. Addition of 20 m M NaC1 to the anoxic buffer before malonate addition resulted in identical rates of malonate degradation and ATP production. These results indicate that malonate was actually functioning as an energy source for Citrobacter diversus, and not merely as a supplement improving the growth yield on yeast extract. Studies using potential metabolic inhibitors (Table 1) suggested no role for sodium gradients in the malonate metabolism of Citrobacter diversus, supporting the findings of an earlier study on Citrobacter sp. 16mall (Janssen and H a r f o o t 1990). The sodium ionophore monensin and the putative Na+-ATPase inhibitor ouabain (Zollner 1989) both had little effect on the levels of cellular A T P or on the rate of malonate decarboxylation by cell suspensions in the presence of sodium. In contrast, the protonophore C C C P and the H+-ATPase inhibitor D C C D did have an effect on cellular ATP levels, indicating a role for proton gradients. Inhibition of the membrane-bound H+-ATPase complex by D C C D had no effect on the rate of malonate decarboxylation, but did reduce cellular A T P levels. This suggests that inhibiting the flow of protons from a postulated proton gradient via the H +-ATPase complex back into the bacterial cell does not affect the independent process generating the gradient. C C C P decreased the rate of malonate decarboxylation, suggesting that the gradient could also be involved in substrate transport. The data obtained indicates the A T P is generated by the gradient, rather than the gradient being generated at the expense of ATP. Measurement of enzyme activities suggest that malonate is activated by a ligase (EC 6.2.1.-, 0.24 gmol/ m i n . m g protein), decarboxylated to acetyl-CoA, then further metabolised by phosphotransacetylase (EC 2.3.1.8, 2.22 gmol/min 9mg protein) to acetyl-P, then to acetate with concomitant ATP formation by acetate kinase (EC 2.7.2.1, 1.12 g m o l / m i n . m g protein). There was also evidence for the activation for malonate by CoA transfer from acetyl-CoA (EC 2.8.3.3, 0.08 gmol/min - mg protein). Simultaneous activity of these two malonate activating systems was also found in a malonate-degrading Pseudomonas sp.-(Hayaishi 1955), and is also postulated to be present in the malonate-degrading
species Klebsiella oxytoca, Rhodobacter capsulatus and Sporomusa malonica (Dehning 1990). Both of these activating mechanisms result in pathways producing no net ATP gain. In the present study malonyl-CoA decarboxylase (EC 4.1.1.9) could be measured only at low activities (0.02 rtmol/min 9mg protein). The use of different buffers at various pH values did not result in higher activities. Crude cell-free extracts were able to decarboxylate free malonate. This activity was stimulated 3-fold by the addition of 1.0raM acetyl-phosphate and 12.5-fold (1.65 gmol/min 9mg protein) by the addition of 0.2 m M acetyl-CoA. Addition of CoA had no stimulatory effect. This supports the idea of a pathway with acetyl-CoA as an intermediate and reactant. Net ATP formation during growth on malonate appears to be mediated by the generation of a proton gradient coupled to the decarboxylation reaction and the activity of an H+-ATPase. This was also postulated in a study on a similar malonate-degrading bacterium (Janssen and Harfoot 1990). This may be directly generated by a membrane-bound decarboxylase (Hilpert et al. 1984), by an antiport exchange system (Anantharam et al. 1989; Poolman et al. 1991), or coupled to endproduct excretion (Michels et al. 1979; Michel and Macy 1990).
Acknowledgements: The authors thank the directors of the Thermophile Research Unit, University of Waikato, for access to facilities, and the Water Quality Centre, DSIR, Hamilton, for use of the integrating photometer. References Anantharam V, Allison MJ, Maloney PC (1989) Oxalate: formate exchange. The basis for energy coupling in Oxalobacter. J Biol Chem 264:7244-7250 Bergmeyer HU (1975) Neue Werte f/ir &e molaren ExtinktionsKoeffizienten von NADH und NADPH zum Gebrauch im Routine-Laboratorium. J Clin Chem Clin Biochem 13 : 507- 508 BergmeyerHU, GawehnK, Grassl M (1974) Enzymes as biochemical reagents. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 1, 2nd English edn. Verlag Chemic, Weinheim, pp 425-522 Dehning I (1990) Abbau von Oxalat und Malonat durch anaerobe Bakterien. Dissertation, Mikrobiologie I, Universit/it Tfibingen, FRG
474 Dehning 1, Schink B (1989) Malonomonas rubra gen. nov., sp. nov., a microaerotolerant anaerobic bacterium growing by decarboxylation of malonate. Arch Microbiol 151:427-433 Dehning I, Stieb M, Schink B (1989) Sporomusa malonica sp. nov., a homoacetogenic bacterium growing by decarboxylation of malonate or succinate. Arch Microbiol 151:421-425 Hayaishi O (1955) Enzymatic decarboxylation of malonic acid. J Biol Chem 215:125-136 Hilpert W, Schink B, Dimroth P (1984) Life by a new deearboxylation-dependent mechanism with Na + as coupling ion. EMBO J 3:1665-1670 Janssen PH (1991a) Growth of enterobacteria on malonate under strictly anaerobic conditions. Syst Appl Microbiol 14:93-97 Janssen PH (1991 b) Fermentation of L-tartrate by a newly isolated Gram-negative glycolytic bacterium. Antonie van Leeuwenhoek 59:191-198 Janssen PH (I 992) Growth yield increase and ATP formation linked to succinate decarboxylation in Veillonella parvula. Arch Microbiol 157:442-445 Janssen PH, Harfoot CG (1990) Isolation of a Citrobaeter species able to grow on malonate under strictly anaerobic conditions. J Gen Microbiol 136:1037-1042
Michel TA, Macy JM (1990) Generation of a membrane potential by sodium-dependent succinate efflux in Selenomonas rum# nantium. J Bacteriol 172:1430-1435 Michels PAM, Michels JPJ, Boonstra J, Konings WN (1979) Generation of an electrochemical proton gradient in bacteria by the excretion of metabolic and products. FEMS Microbiology Letters 5:357-364 Nishimura JS, Griffith MJ (1981) Acetate kinase from Veillonella alcalescens. In: Lowenstein JM (ed) Methods in enzymology. vol 71: lipids part C. Academic Press, New York London, pp 311-316 Poolman B, Molenaar D, Smid EJ, Ubbink T, Abee T, Renault PP, Konings WN (1991) Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy. J Bacteriol 173:6030-6037 Scopes R (1982) Protein purification: principles and practice. Springer, New York Berlin Heidelberg Wofford NQ, Beaty PS, McInerny MJ (1986) Preparation of cellfree extracts and the enzymes involved in fatty acid metabolism in Syntrophomonas wolfei. J Bacteriol 167:179-185 Zollner H (1989) Handbook of enzyme inhibitors. Verlag Chemic, Weinheim, FRG
Archives of
Hicrobiolegy ,9 Springer-Verlag 1992
Anaerobic malonate decarboxylation by Citrobacter diversus. Growth and metabolic studies, and evidence of ATP formation Peter H. Janssen* and C. G. Harfoot Department of Biological Sciences, University of Walkato, Private Bag 3105, Hamilton, New Zealand Received November 13, 1991/Accepted January 30, 1992
Abstract. Citrobacter diversus A T C C 27156 was able to grow by decarboxylation of malonate to acetate under strictly anaerobic conditions, in the presence of yeast extract. The growth yield, corrected for growth on yeast extract, was 2.03 g cell dry mass per tool malonate. The addition of malonate to ATP-depleted cell suspensions (less than 0.2 nmol A T P / m g cell protein) resulted in a rapid increase in cellular ATP levels to between 4.5 and 6.0 nmol/mg cell protein. Intact cells decarboxylated malonate at rates of up to 1.5 gmol/min, mg protein. Enzyme assays on malonate-grown cells indicated activation of malonate by an ATP-dependent ligase reaction and by CoA transfer from acetyl-CoA, followed by decarboxylation of malonyl-CoA to acetyl-CoA with subsequent recovery of the invested ATP by substrate level phosphorylation through the activity of acetate kinase. Net A T P synthesis is postulated to be mediated by gradient formation coupled to the decarboxylation of malonyl-CoA. The protonophore CCCP and H +ATPase inhibitor D C C D significantly reduced cellular A T P levels, suggesting a role for proton gradients in the energy metabolism of this strain when growing an malonate. Inhibitors of sodium metabolism or ommission of sodium had no effect on ATP levels or malonate decarboxylation. Key words: Citrobacter diversus - Anaerobic fermentation - Energy metabolism - Malonate - Decarboxylation - P r o t o n gradient
strain 16mall and other Enterobacteriaceae, and Sporomusa malonica grow well on malonate in the presence of yeast extract (Dehning et al. 1989; Janssen and H a r f o o t 1990; Janssen 1991a) and Malonomonas rubra grows on malonate in mineral medium (Dehning and Schink 1989). Malonomonas rubra displays a requirement for sodium (Dehning and Schink 1989), which suggests that the decarboxylation of malonate is coupled to the generation of a transmembrane sodium ion gradient for ATP production, analogous to the A T P generating system of the succinate-decarboxylating Propionigenium rnodestum (Hilpert et al. 1984). The malonate-decarboxylating activity of suspensions of Citrobacter sp. strain 16mall was sensitive to the protonophore 2,4-dinitrophenol, but resistant to the sodium ionophore monensin (Janssen and H a r f o o t 1990). This seems to preclude the involvement of a sodium ion gradient in the energy metabolism of Enterobacteriaceae growing anaerobically on malonate. Since the Enterobacteriaceae have a requirement for yeast extract when growing on malonate, there remain questions as to the exact role of malonate in the metabolism of these organisms, and whether ATP is actually formed coupled to the decarboxylation of malonate. Investigations to answer these questions and to measure enzymes involved in malonate catabolism in one strain of the malonate-fermenting Enterobacteriaceae are described in this communication. Materials and Methods Organism and growth conditions
Under dark anaerobic conditions only a few bacteria have been demonstrated to be able to grow on malonate, decarboxylating it to acetate and CO2. Citrobacter sp. * Present address and address for correspondence: P. H. Janssen, Fakult/it ffir Biologie,Universit/it Konstanz, Postfach 5560, W-7750 Konstanz Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone;
DCCD, dicyclohexylcarbodiimide; MOPS, 3-(N-morpholino) propanesulfonic acid
Citrobacter diversus ATCC 27156 was obtained from the National
Health Institute (now New Zealand Communicable Diseases Centre), Porirua, New Zealand (collection number NHI 868) and maintained in the freshwater medium of Janssen and Harfoot (1990) with 1 g yeast extract/l, 50 mM Na2 malonate and sulfide reductant at 34 ~ Growth experiments were carried out in the same medium. Optical density readings, HPLC analysis of samples, dry weight yield determinations and culture manipulations were as previously described (Janssen and Harfoot 1990). For experiments with cell suspensions, bulk cultures were grown in 100 ml volumes in 160-ml serum vials with 100 mM malonate and 50 mM MOPS (pH adjusted to 7.0 with NaOH).
472
Cell suspension experiments Cell suspensions were prepared as described elsewhere (Janssen 1992) without the addition of thioglycolate. ATP formation and malonate decarboxylation were followed as described by Janssen (1992). Malonic or acetic acid (neutralized to pH 7.0 with KOH). NaC1 or KC1 were added to 20 mM final concentration from anoxic stock solutions as required. When testing the effects of potential metabolic inhiNtors on cellular ATP levels, 20 mM KOH-neutralized malonic acid was added at the beginning of the experiment, and inhibitors added after 18 rain from ethanolic stock solutions at 0.1 ml per 20 ml buffer. As a control, 0.1 ml ethanol was added. Arsenate was added from an HCl-neutralized stock solution in buffer. Oubain and DCCD, at the levels present in the samples for ATP analysis, did not interfer with the luciferin/luciferase assay.
Enzymes assays Cell-free extracts were prepared by ultrasonication as described by Janssen and Harfoot (1990). Protein was estimated by a micromethod adaptation of the Lowry method (Scopes 1982). Enzymes and biochemicals were purchased from Sigma Chemicals (St. Louis, Mo., USA). Measurements of malonate decarboxylating actwity by crude cell-free extracts was carried out in Nz-flushed stoppered tubes at 34 ~ containing 1.5 ml 100mM MOPS/KOH pH 7.0, 33 mM malonate and crude cell-free extract (0.5 mg protein). Malonate decarboxylation and acetate formation was monitored by HPLC analysis of perchloric acid treated samples (Janssen 1992). End-point enzyme assays were carried out over 15 min at 34 ~ in stoppered N2-flushed test-tubes. At each 5 rain time point three assays (and appropriate controls) were halted and put on ice. Acetate kinase (EC 2.7.2.1) was assayed basically by method 1 of Nishimura and Griffith (1981), omitting succinate from the assay mixture. Malonate-CoA ligase (EC 6.2.1.-) was assayed using the following reaction mixture (modified from Wofford et al. 1986): 100 mM Tris, 15 mM NaF, 0.3 mM Triton X-100, 300 mM hydroxylamine 9HC1, 5 mM MgC12, 2 mM Na2 malonate, 5 mM ATP, 15 mM mercaptoethanol, 0.4 mM CoA, 2 mM GSH, pH adjusted to 8.3 with KOH. The formation of the CoA ester of malonate was measured by following its reaction with hydroxylamine using FeC13 reagent as for acetate kinase (Nishimura and Griffith 1981). Continuous enzyme assays were carried out a 34 ~ as described earlier (Janssen 1991b). Phosphotransacetylase (EC 2.3.1.8) was assayed as described by Bergmeyer et al. (1974). Malonate CoA transferase (EC 2.8.3.3) was assayed by following acetyl-CoA formation using the following reaction mixture: 100raM Tris, 20 mM (NH4)2SO4, 20 mM Na acetate, 0.4 mM malonyl-CoA, 1 mM dithiothreitol, 2 mM NAD, 5 mM malate, 1 U citrate synthase, 2.5 U malate dehydrogenase, pH adjusted to 7.4 with HC1. The activity measured was corrected for acetyl-CoA formation due to malonyl-CoA decarboxylase activity. Malonyl-CoA decarboxylase (EC 4.1.1.9) was assayed using the following mixture: 100 mM imidazole, 5 mM MgC12, 0.4 mM malonyl-CoA, 2 mM NAD, 5 mM malate, 1 mM dithiothreitol, 1 U citrate synthase, 2.5 U malate dehydrogenase, pH adjusted to 6.5 with HC1. Oxidatmn and reduction of pyridine nucleotides was followed at 340 nm (Bergmeyer 1975).
y
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50 o
0
1'0 2'0 30 ' 40' Malonate used (mmol)
50
Fig. 1. Growth yield of Citrobacter diversusATCC 27156 on a range of malonate in the presence of 1 g yeast extract/1 under strictly anaerobic conditions. The malonate concentrations are the amounts metabolized
acetate in s t o i c h i o m e t r i c a m o u n t s . T h e g r o w t h yield was p r o p o r t i o n a l to the a m o u n t of m a l o n a t e d e c a r b o x y l a t e d , u p to at least 45 retool/1 (Fig. 1), a n d was 2.03 g cell d r y m a s s p e r m o l m a l o n a t e (corrected for g r o w t h on yeast extract). T h e yield is the r a n g e (1.3 to 2.1 g cell d r y m a s s p e r m o l m a l o n a t e ) r e p o r t e d for o t h e r m a l o n a t e - d e g r a d i n g b a c t e r i a ( D e h n i n g et al. 1989; D e h n i n g a n d Schink 1989; J a n s s e n a n d H a r f o o t 1990; J a n s s e n 1991a). Yeast e x t r a c t was n o t r e q u i r e d for a n a e r o b i c g r o w t h o n c i t r a t e o r glucose.
A T P formation by cell suspensions A d d i t i o n o f m a l o n a t e to cell s u s p e n s i o n s o f m a l o n a t e g r o w n Citrobacter diversus resulted in a n i m m e d i a t e increase in cellular A T P levels (to b e t w e e n 4.5 a n d 6.0 n m o l / m g p r o t e i n ) with c o n c o m i t a n t d e g r a d a t i o n o f m a l o n a t e (Fig. 2). M a l o n a t e was d e c a r b o x y l a t e d at rates o f u p to 1.5 g m o l / m i n 9 protein. Cell s u s p e n s i o n s to
5
20
4
15.
[
10
5 Results and discussion
Growth studies
-~0
0
Citrobacter diversus A T C C 27156 was m a i n t a i n e d b y a p p r o x i m a t e l y m o n t h l y s u b c u l t u r e using m a l o n a t e as the g r o w t h s u b s t r a t e u n d e r strictly a n a e r o b i c (sulfide-reduced) c o n d i t i o n s for m o r e t h a n four years. This strain grew o n m a l o n a t e , in the p r e s e n c e of 1.0 g extract/1 (~t = 0.40 h - 1 at 34 ~ with c o n c o m i t a n t f o r m a t i o n of
Fig. 2. ATP formation([]) by cell suspensions of malonate-grown Citrobacter diversus ATCC 27156 concomitant with decarboxylation ofmalonate (9 The ATP content of cells to which no addition was made or to which 20 mM KC1 or acetate was added is also shown ( I )
0
10
20 30 Time (min)
40
50
473 Table 1. Effect of potential metabolic inhibitors on cellular ATP levels and the rate of malonate decarboxylation by cell suspensions of malonate-grown Citrobacter diversus ATCC 27156. The results are expressed relative to appropriate controls (nominally 100%)
Cellular ATP levels after addition
Inhibitor and concentration
CCCP Monensina DCCD Ouabain a Ethanol only control Arsenate Buffer only control
150 gM 75 gM 150 I-tM 75 gM 5 mM
Malonate decarboxylating activity
(nmol/mg)
(%)
(gmol/min - mg)
(%)
0.05 4.80 1.94 5.69 5.71 0.05 4.90
1 84 34 100 100 1 100
0.30 0.49 0.56 0.55 0.52 0.25 0.59
58 94 108 106 100 42 100
a Tested in the presence of 20 mM NaC1 which 20 m M KC1 or acetate were added, or to which no additions were made, showed no changes in cellular ATP levels. This basal level, regarded as ATP depleted, was less than 0.2 nmol/mg protein. Addition of 20 m M NaC1 to the anoxic buffer before malonate addition resulted in identical rates of malonate degradation and ATP production. These results indicate that malonate was actually functioning as an energy source for Citrobacter diversus, and not merely as a supplement improving the growth yield on yeast extract. Studies using potential metabolic inhibitors (Table 1) suggested no role for sodium gradients in the malonate metabolism of Citrobacter diversus, supporting the findings of an earlier study on Citrobacter sp. 16mall (Janssen and H a r f o o t 1990). The sodium ionophore monensin and the putative Na+-ATPase inhibitor ouabain (Zollner 1989) both had little effect on the levels of cellular A T P or on the rate of malonate decarboxylation by cell suspensions in the presence of sodium. In contrast, the protonophore C C C P and the H+-ATPase inhibitor D C C D did have an effect on cellular ATP levels, indicating a role for proton gradients. Inhibition of the membrane-bound H+-ATPase complex by D C C D had no effect on the rate of malonate decarboxylation, but did reduce cellular A T P levels. This suggests that inhibiting the flow of protons from a postulated proton gradient via the H +-ATPase complex back into the bacterial cell does not affect the independent process generating the gradient. C C C P decreased the rate of malonate decarboxylation, suggesting that the gradient could also be involved in substrate transport. The data obtained indicates the A T P is generated by the gradient, rather than the gradient being generated at the expense of ATP. Measurement of enzyme activities suggest that malonate is activated by a ligase (EC 6.2.1.-, 0.24 gmol/ m i n . m g protein), decarboxylated to acetyl-CoA, then further metabolised by phosphotransacetylase (EC 2.3.1.8, 2.22 gmol/min 9mg protein) to acetyl-P, then to acetate with concomitant ATP formation by acetate kinase (EC 2.7.2.1, 1.12 g m o l / m i n . m g protein). There was also evidence for the activation for malonate by CoA transfer from acetyl-CoA (EC 2.8.3.3, 0.08 gmol/min - mg protein). Simultaneous activity of these two malonate activating systems was also found in a malonate-degrading Pseudomonas sp.-(Hayaishi 1955), and is also postulated to be present in the malonate-degrading
species Klebsiella oxytoca, Rhodobacter capsulatus and Sporomusa malonica (Dehning 1990). Both of these activating mechanisms result in pathways producing no net ATP gain. In the present study malonyl-CoA decarboxylase (EC 4.1.1.9) could be measured only at low activities (0.02 rtmol/min 9mg protein). The use of different buffers at various pH values did not result in higher activities. Crude cell-free extracts were able to decarboxylate free malonate. This activity was stimulated 3-fold by the addition of 1.0raM acetyl-phosphate and 12.5-fold (1.65 gmol/min 9mg protein) by the addition of 0.2 m M acetyl-CoA. Addition of CoA had no stimulatory effect. This supports the idea of a pathway with acetyl-CoA as an intermediate and reactant. Net ATP formation during growth on malonate appears to be mediated by the generation of a proton gradient coupled to the decarboxylation reaction and the activity of an H+-ATPase. This was also postulated in a study on a similar malonate-degrading bacterium (Janssen and Harfoot 1990). This may be directly generated by a membrane-bound decarboxylase (Hilpert et al. 1984), by an antiport exchange system (Anantharam et al. 1989; Poolman et al. 1991), or coupled to endproduct excretion (Michels et al. 1979; Michel and Macy 1990).
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474 Dehning 1, Schink B (1989) Malonomonas rubra gen. nov., sp. nov., a microaerotolerant anaerobic bacterium growing by decarboxylation of malonate. Arch Microbiol 151:427-433 Dehning I, Stieb M, Schink B (1989) Sporomusa malonica sp. nov., a homoacetogenic bacterium growing by decarboxylation of malonate or succinate. Arch Microbiol 151:421-425 Hayaishi O (1955) Enzymatic decarboxylation of malonic acid. J Biol Chem 215:125-136 Hilpert W, Schink B, Dimroth P (1984) Life by a new deearboxylation-dependent mechanism with Na + as coupling ion. EMBO J 3:1665-1670 Janssen PH (1991a) Growth of enterobacteria on malonate under strictly anaerobic conditions. Syst Appl Microbiol 14:93-97 Janssen PH (1991 b) Fermentation of L-tartrate by a newly isolated Gram-negative glycolytic bacterium. Antonie van Leeuwenhoek 59:191-198 Janssen PH (I 992) Growth yield increase and ATP formation linked to succinate decarboxylation in Veillonella parvula. Arch Microbiol 157:442-445 Janssen PH, Harfoot CG (1990) Isolation of a Citrobaeter species able to grow on malonate under strictly anaerobic conditions. J Gen Microbiol 136:1037-1042
Michel TA, Macy JM (1990) Generation of a membrane potential by sodium-dependent succinate efflux in Selenomonas rum# nantium. J Bacteriol 172:1430-1435 Michels PAM, Michels JPJ, Boonstra J, Konings WN (1979) Generation of an electrochemical proton gradient in bacteria by the excretion of metabolic and products. FEMS Microbiology Letters 5:357-364 Nishimura JS, Griffith MJ (1981) Acetate kinase from Veillonella alcalescens. In: Lowenstein JM (ed) Methods in enzymology. vol 71: lipids part C. Academic Press, New York London, pp 311-316 Poolman B, Molenaar D, Smid EJ, Ubbink T, Abee T, Renault PP, Konings WN (1991) Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy. J Bacteriol 173:6030-6037 Scopes R (1982) Protein purification: principles and practice. Springer, New York Berlin Heidelberg Wofford NQ, Beaty PS, McInerny MJ (1986) Preparation of cellfree extracts and the enzymes involved in fatty acid metabolism in Syntrophomonas wolfei. J Bacteriol 167:179-185 Zollner H (1989) Handbook of enzyme inhibitors. Verlag Chemic, Weinheim, FRG
