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Regulatory and molecular properties of citrate synthases from methylotrophs
FEMS Microbiology Letters 34 (1986) 191-194 Published by Elsevier
191
FEM 02393
Regulatory and molecular properties of citrate synthases from methylotrophs ( Hyphomicrobium spp.; Bacillus sp $2A1; Arthrobacter 2B2; Methylophilus methylotrophus; NADH-inhibition; metabolic regulation)
R. O t t o * University of Bath, Clacerton Down, Bath, BA2 7.4 Y, U.K. Received 20 November 1985 Revision received and accepted 23 December 1985
1. SUMMARY Gram-negative methylotro~hs contain a high-Mr 'large' citrate synthase. Gram'positive methylotrophs, on the other hand, containa' small' citrate synthase. These differences in M r coincided partly with differences in NADH sensitivity. Citrate synthases from obligate Gram-negative and Grampositive facultative methylotrophs were insensitive to feedback inhibition by NADH; only the enzymes from Gram-negative facultative methylotrophs were inhibited by NADH.
2. INTRODUCTION The citric acid cycle (TCA,cycle) of aerobic chemoheterotrophic bacteria serves a dual purpose: (i) it furnishes the organism with essential precursors for biosynthesis; and (ii) it generates reducing equivalents. The correct execution of this complex task requires elaborate control mechanisms, and indeed, it has been shown that the TCA-cycle contains several control points [1]. One * Present address: Molukkenstraat 36, 9715 NV Groningen, The Netherlands.
of the most prominent of these control points is the reaction catalysed by citrate synthase (EC 4.1.3.7). Bacterial citrate synthases display a marked but still partly unexplained diversity in their molecular and regulatory properties [1]. Two classes of bacterial citrate synthases can be distinguished: (i) the citrate synthases from gramnegative bacteria with an M r of approx. 250 000, and usually sensitive to feedback inhibition by the end-product of the TCA-cycle, NADH; and (ii) the much smaller NADH-insensitive citrate synthases from gram-positive bacteria, with an Mr of about 100000 [1]. In contrast to most heterotrophs, Gram-negative methylotrophs use the TCA cycle only in its capacity as an anabolic pathway. As a result, the cycle has undergone some modifications: oxoglutarate dehydrogenase activity is either absent or severely repressed [2]; and citrate synthase is in many cases insensitive to feedback inhibition by N A D H [3]. In some methylotrophs, however, notably those equipped with the serine pathway of formaldehyde fixation, the citrate synthase is still sensitive to feedback inhibition by N A D H [3-6]: these organisms also retain low levels of oxoglutarate dehydrogenase during methylotrophic growth [2]. It has been speculated that the presence of a NADH-sensitive citrate synthase
0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies
192 in these organisms prevents unwanted dissimilation by the TCA cycle of newly formed carbon-carbon bonds derived from Cl-substrates [21. In this paper, the molecular and regulatory properties of the citrate synthases of some obligate, facultative, Gram-negative and Gram-positive methylotrophs have been examined more closely.
3. MATERIALS A N D M E T H O D S
3.1. Growth medium and culture conditions Methylophilus methylotrophus NCIB10515, strain AS1, Arthrobacter sp. 2B2 [7], Hyphomicrobium sp. strain X [8], Hyphomicrobium sp. strain Hanham (obtained from Dr. M.M. Attwood, University of Sheffield, U.K.) and Bacillus sp. S2A1 (NCIBl1343) were grown in a mineral medium [9] fortified with 1 mg D-biotin. 1-1 and with either 40 mM methylamine, methanol, ethanol or glucose as carbon source. Bacteria were grown at 30°C in shake flasks on a rotary shaker.
3.2. Preparation of cell extracts Cells were harvested by centrifugation for 10 min at 5000 x g and 4°C, washed with 50 mM Tris-HCl, pH 7.5, and disrupted by sonication (ten 30-s bursts allowing 1 min intervals for cooling on ice) using an Ultrasonics sonicator (Ultrasonics, Shipley, U.K.) equipped with a 4.5 mm probe, set at 50 W. Cell debris was removed by centrifugation for 30 min at 48 000 x g.
3.3. Sizing of bacterial citrate synthases Bacterial citrate synthases were sized relative to rabbit muscle lactate dehydrogenase (EC 1.1.1.27). Crude cell extract (1 ml) was mixed with 2300 nkat lactate dehydrogenase and subsequently applied to a column packed with Sephadex G-200 (20 x 1.5 cm). The column was eluted with 50 mM Tris-HC1, p H 7.5. Fractions (1 ml) were screened for citrate synthase and lactate dehydrogenase.
3.4. Enzyme assays Enzyme activities are expressed as n k a t - ( m g protein) -a. Protein was determined according to
Lowry et al. [10]. The following enzymes were assayed by published procedures: citrate synthase [11], lactate dehydrogenase (EC 1.1.1.27) [12], and oxoglutarate dehydrogenase with the N A D reduction method [13]. Kinetic constants were determined from direct linear plots [14].
4. RESULTS A N D DISCUSSION The results of the relative size determination of the citrate synthases of several methylotrophs are summarised in Table 1. Arthrobacter 2B2 and Bacillus $2A1 contain a 'small' citrate synthase ( M r < 150000); all the Gram-negative methylotrophs, on the other hand, contain the 'large' type ( M r > 150000). The division between Gram-negative and Gram-positive methylotrophs based on the size of citrate synthase is evident. Furthermore, the relative size of the citrate synthases of facultative methylotrophs was independent of the nature of the growth substrate (Table 1). It is known that this difference in M r coincides with a difference in sensitivity to NADH. Contrary to the enzymes from Gram-positive bacteria, the citrate synthases from Gram-negative bacteria are sensitive to feedback inhibition by N A D H [1]. This difference was also observed in Bacillus sp. S2A1 and Arthrobacter sp. 2B2; the citrate synthases of these 2 Gram-positive methylotrophs were unaffected by 5 mM N A D H , but of all the Gram-negatives tested, only the citrate synthase of the facultative methylotroph Hyphomicrobium sp. strain X showed appreciable inhibition by N A D H . The citrate synthases from M. methylotrophus and Hyphomicrobium sp. strain Hanham were insensitive, and are therefore clear exceptions, this probably reflects the highly specialised life style of these organisms. Despite this, the results together with literature data [3-6] indicate that the regulatory and molecular properties of the citrate synthases of methylotrophs follow the same pattern and division, as in so many other non-methylotrophs [1]. However, the different responses of the citrate synthases from the 2 Hyphomicrobium strains to N A D H presents an interesting example of enzyme diversity within a single genus and the
193 Table 1 Molecular sizes of citrate synthases from methylotrophic organisms Organism
C.S. (C1)
C.S. (H)
Gram
Ass.
Type
M. methylotrophus Hyphomicrobium sp. strain Hanham Hyphomicrobium sp. strain X Arthrobacter sp. strain 2B2 Bacillus sp. strain S2A1
L
N.A. N.A. L S S
+ +
RuMP serine serine RuMP RuMP
obl obl fac fac fac
L L S S
Abbreviations: First column: C.S. (C1), citrate synthase expressed during methylotrophic growth (Methylophilus methylotrophus was grown on methanol, all other strains were grown on methylamine as carbon and energy source); L, "large" citrate synthase; S, "small" citrate synthase. Second column; C.S. (H), citrate synthase expressed during growth on multicarbon substrates (Bacillus S2A1 and Arthrobacter 2B2 were grown on glucose, Hyphomicrobium X was grown on ethanol); N.A., not applicable. Third column; Gram character. Fourth column; Pathway of formaldehyde fixation; ribulose monophosphate cycle (RUMP), serine pathway (serine). Fifth column; Nutritional type; obligate (obl), facultative methyiotroph (fac).
kinetic properties of these enzymes were studied in more detail. The citrate synthase of Hyphomicrobium sp. strain X was almost completely inhibited even by low NADH concentrations, whilst the enzyme of the obligate methylotroph Hyphomicrobiurn sp. strain Hanham was unaffected (Fig. 1). The inhibition of the citrate synthase of Hyphomicrobium sp. strain X by NADH was non-competitive with respect to acetyl-CoA (Table 2). The inhibitor constant (Ki) of the citrate
loc z 0 t.-
z
o
o
o
o
Do mM NADH Fig. 1. Relation between degree of inhibition of citrate synthase and NADH concentration. O, Hyphomicrobium sp..strain X; O, Hyphomicrobium sp. strain Hanham. The solid line representing the theoretical relationship was calculated by assuming that K i (NADH) is 0.4 mM.
synthase of Hyphomicrobiumsp. strain X (0.4 mM) was considerably lower than that of Hyphornicrobium sp. strain Hanham (12 mM) (Table 2). This type of inhibition suggests the presence of a binding site for NADH separate from the active site; presumably this site has been modified in the obligate methylotroph Hyphomicrobium sp. strain Hanham. The inhibition of the citrate synthase by 0.75 mM NADH could be overcome by the addition of 1 mM AMP (Table 2). In this respect the citrate synthase of Hyphomicrobiumsp. strain X is very similar to the citrate synthases of other aerobic Gram-negative bacteria [1]. The insensitivity of the citrate synthase of Hyphomicrobium sp. strain Hanham to NADH is also interesting for another reason. This organism contains low levels of oxoglutarate dehydrogenase activit2( (0.03 nkat. mg-1), and hence, TCA-cycle activity. Zatman's hypothesis [2] concerning the regulation of citrate synthase in methylotrophs stipulates that under these circumstances an organism should have a NADH-sensitive citrate synthase. This is apparently not always the case, and in this respect Hyphomicrobium sp. strain Hanham is similar to another serine-pathway obligate methylotroph Methylococcus trichosporum OB3b, which was also shown to possess a NADHinsensitive citrate synthase [3]. This makes it unlikely that the reason for the presence of a NADH-sensitive citrate synthase in other serinepathway Gram-negative facultative methylotrophs is to prevent unwanted dissimilation of metabo-
194 Table 2 Kinetic parameters of the citrate synthases of Hyphomicrobium sp. strain X and Hyphomicrobium sp. strain Hanham. Kinetic parameters were determined using a single batch of enzyme; standard errors were calculated according to Cornish-Bowden and Eisenthal [15] Strain
X
Hanham
Km ( # M acetyl-CoA) Vmax (nkat- mg protein- l) in the presence of 0.75 mM N A D H K m (/~M acetyl-CoA) Vma~ (nkat- mg protein- 1) in the presence of 5 mM N A D H K m ( ~ M acetyl-CoA) Vm~X (nkat. mg protein- 1 ) in the presence of 0.75 mM N A D H and 1 mM AMP K m (/~M acetyl-CoA) Vm~x (nkat. mg protein- 1 ) K i (mM NADH)
161 _+22 0.68-+ 0.04
143 +11 0.3 + 0.01
159 -+ 34 0.23 -+ 0.03
N.T. N.T.
N.T. N.T.
156 _+41 0.23 _+ 0.05
214 _+40 0.73 -4- 0.10 0.4
N.T. N.T. 12
N.T. = not tested.
lites derived from the serine pathway. It is more likely that the presence of a NADH-sensitive citrate synthase is inherent to the more nutritionally versatile life style of these organisms. In their natural habitat, which is usually very poor in nutrients, facultative methylotrophs probably grow on a mixture of different substrates rather than a single substrate, and this involves both one-carbon as well as multicarbon substrates. Under these circumstances, regulation of the citrate synthase and hence the TCA-cycle is essential. Moreover, multicarbon and one-carbon substrates do not occur sequentially in natural milieux but are released simultaneously as a result of the slow decomposition of organic matter. Therefore, a facultative methylotroph has little to gain by expressing 2 different citrate synthases, one during methylotrophic growth and one (NADH-sensitive) during growth on multicarbon substrates [5].
ACKNOWLEDGEMENTS I gratefully acknowledge the help of Dr. M.M. Attwood, Prof. J.R. Quayle, Prof. P.D.J. Weitzman and Dr. L.J. Zatman in providing both materials and experimental facilities, and the Science and Engineering Research Council for financial support under grant number GR/C/71644.
REFERENCES [1] Weitzman, P.D.J. (1981) Adv. Microb. Physiol. 22, 185-244. [2] Zatman, L.J. (1981) in Microbial growth on C-1 Compounds, (Dalton, H., Ed.) pp. 42-54. Heyden, London. [3] Colby, J. and Zatman, L.J. (1975) Biochem. J. 150, 141-144. [4] Cox, R.B. and Quayle, J.R. (1976) J. Gen. Microbiol. 95, 121-133. [5] Babel, W. and Muller-Kraft, G. (1979) Z. Allg. Mikrobiol. 19, 687-693. [6] Anthony, C. and Taylor, I.J. (1975) Proc. Soc. Gen. Microbiol. 4, 67. [7] Colby, J. and Zatman, L.J. (1973) Biochem. J. 132, 101-112. [8] Attwood, M.M. and Harder, W. (1972) Antonie van Leeuwenhoek 39, 369-378. [9] Large, P.J. and Haywood, G.W. (1981) FEMS Microbiol. Lett. 11,207-209. [10] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [11] Srere, P. (1969) in Methods in Enzymology, Vol. 13 (Lowenstein, J.M., Ed.), pp. 3-11. Academic Press, New York and London. [12] Bergmeyer, H.U. (1974) Methods of Enzymatic Analysis, Vol. 1, pp. 481-482. Academic Press, New York and London. [13] Reed, L.J. and Mukherjee, B.B. (1969) Methods in Enzymology, Vol. 13 (Lowenstein, J.M., Ed.), pp. 55-61. Academic Press, New York and London. [14] Eisenthal, R. and Cornish-Bowden, A. (1974) Biochem. J. 139, 715-720. [15] Cornish-Bowden, A. and Eisenthal, R. (1974) Biochem. J. 139, 721-730.
191
FEM 02393
Regulatory and molecular properties of citrate synthases from methylotrophs ( Hyphomicrobium spp.; Bacillus sp $2A1; Arthrobacter 2B2; Methylophilus methylotrophus; NADH-inhibition; metabolic regulation)
R. O t t o * University of Bath, Clacerton Down, Bath, BA2 7.4 Y, U.K. Received 20 November 1985 Revision received and accepted 23 December 1985
1. SUMMARY Gram-negative methylotro~hs contain a high-Mr 'large' citrate synthase. Gram'positive methylotrophs, on the other hand, containa' small' citrate synthase. These differences in M r coincided partly with differences in NADH sensitivity. Citrate synthases from obligate Gram-negative and Grampositive facultative methylotrophs were insensitive to feedback inhibition by NADH; only the enzymes from Gram-negative facultative methylotrophs were inhibited by NADH.
2. INTRODUCTION The citric acid cycle (TCA,cycle) of aerobic chemoheterotrophic bacteria serves a dual purpose: (i) it furnishes the organism with essential precursors for biosynthesis; and (ii) it generates reducing equivalents. The correct execution of this complex task requires elaborate control mechanisms, and indeed, it has been shown that the TCA-cycle contains several control points [1]. One * Present address: Molukkenstraat 36, 9715 NV Groningen, The Netherlands.
of the most prominent of these control points is the reaction catalysed by citrate synthase (EC 4.1.3.7). Bacterial citrate synthases display a marked but still partly unexplained diversity in their molecular and regulatory properties [1]. Two classes of bacterial citrate synthases can be distinguished: (i) the citrate synthases from gramnegative bacteria with an M r of approx. 250 000, and usually sensitive to feedback inhibition by the end-product of the TCA-cycle, NADH; and (ii) the much smaller NADH-insensitive citrate synthases from gram-positive bacteria, with an Mr of about 100000 [1]. In contrast to most heterotrophs, Gram-negative methylotrophs use the TCA cycle only in its capacity as an anabolic pathway. As a result, the cycle has undergone some modifications: oxoglutarate dehydrogenase activity is either absent or severely repressed [2]; and citrate synthase is in many cases insensitive to feedback inhibition by N A D H [3]. In some methylotrophs, however, notably those equipped with the serine pathway of formaldehyde fixation, the citrate synthase is still sensitive to feedback inhibition by N A D H [3-6]: these organisms also retain low levels of oxoglutarate dehydrogenase during methylotrophic growth [2]. It has been speculated that the presence of a NADH-sensitive citrate synthase
0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies
192 in these organisms prevents unwanted dissimilation by the TCA cycle of newly formed carbon-carbon bonds derived from Cl-substrates [21. In this paper, the molecular and regulatory properties of the citrate synthases of some obligate, facultative, Gram-negative and Gram-positive methylotrophs have been examined more closely.
3. MATERIALS A N D M E T H O D S
3.1. Growth medium and culture conditions Methylophilus methylotrophus NCIB10515, strain AS1, Arthrobacter sp. 2B2 [7], Hyphomicrobium sp. strain X [8], Hyphomicrobium sp. strain Hanham (obtained from Dr. M.M. Attwood, University of Sheffield, U.K.) and Bacillus sp. S2A1 (NCIBl1343) were grown in a mineral medium [9] fortified with 1 mg D-biotin. 1-1 and with either 40 mM methylamine, methanol, ethanol or glucose as carbon source. Bacteria were grown at 30°C in shake flasks on a rotary shaker.
3.2. Preparation of cell extracts Cells were harvested by centrifugation for 10 min at 5000 x g and 4°C, washed with 50 mM Tris-HCl, pH 7.5, and disrupted by sonication (ten 30-s bursts allowing 1 min intervals for cooling on ice) using an Ultrasonics sonicator (Ultrasonics, Shipley, U.K.) equipped with a 4.5 mm probe, set at 50 W. Cell debris was removed by centrifugation for 30 min at 48 000 x g.
3.3. Sizing of bacterial citrate synthases Bacterial citrate synthases were sized relative to rabbit muscle lactate dehydrogenase (EC 1.1.1.27). Crude cell extract (1 ml) was mixed with 2300 nkat lactate dehydrogenase and subsequently applied to a column packed with Sephadex G-200 (20 x 1.5 cm). The column was eluted with 50 mM Tris-HC1, p H 7.5. Fractions (1 ml) were screened for citrate synthase and lactate dehydrogenase.
3.4. Enzyme assays Enzyme activities are expressed as n k a t - ( m g protein) -a. Protein was determined according to
Lowry et al. [10]. The following enzymes were assayed by published procedures: citrate synthase [11], lactate dehydrogenase (EC 1.1.1.27) [12], and oxoglutarate dehydrogenase with the N A D reduction method [13]. Kinetic constants were determined from direct linear plots [14].
4. RESULTS A N D DISCUSSION The results of the relative size determination of the citrate synthases of several methylotrophs are summarised in Table 1. Arthrobacter 2B2 and Bacillus $2A1 contain a 'small' citrate synthase ( M r < 150000); all the Gram-negative methylotrophs, on the other hand, contain the 'large' type ( M r > 150000). The division between Gram-negative and Gram-positive methylotrophs based on the size of citrate synthase is evident. Furthermore, the relative size of the citrate synthases of facultative methylotrophs was independent of the nature of the growth substrate (Table 1). It is known that this difference in M r coincides with a difference in sensitivity to NADH. Contrary to the enzymes from Gram-positive bacteria, the citrate synthases from Gram-negative bacteria are sensitive to feedback inhibition by N A D H [1]. This difference was also observed in Bacillus sp. S2A1 and Arthrobacter sp. 2B2; the citrate synthases of these 2 Gram-positive methylotrophs were unaffected by 5 mM N A D H , but of all the Gram-negatives tested, only the citrate synthase of the facultative methylotroph Hyphomicrobium sp. strain X showed appreciable inhibition by N A D H . The citrate synthases from M. methylotrophus and Hyphomicrobium sp. strain Hanham were insensitive, and are therefore clear exceptions, this probably reflects the highly specialised life style of these organisms. Despite this, the results together with literature data [3-6] indicate that the regulatory and molecular properties of the citrate synthases of methylotrophs follow the same pattern and division, as in so many other non-methylotrophs [1]. However, the different responses of the citrate synthases from the 2 Hyphomicrobium strains to N A D H presents an interesting example of enzyme diversity within a single genus and the
193 Table 1 Molecular sizes of citrate synthases from methylotrophic organisms Organism
C.S. (C1)
C.S. (H)
Gram
Ass.
Type
M. methylotrophus Hyphomicrobium sp. strain Hanham Hyphomicrobium sp. strain X Arthrobacter sp. strain 2B2 Bacillus sp. strain S2A1
L
N.A. N.A. L S S
+ +
RuMP serine serine RuMP RuMP
obl obl fac fac fac
L L S S
Abbreviations: First column: C.S. (C1), citrate synthase expressed during methylotrophic growth (Methylophilus methylotrophus was grown on methanol, all other strains were grown on methylamine as carbon and energy source); L, "large" citrate synthase; S, "small" citrate synthase. Second column; C.S. (H), citrate synthase expressed during growth on multicarbon substrates (Bacillus S2A1 and Arthrobacter 2B2 were grown on glucose, Hyphomicrobium X was grown on ethanol); N.A., not applicable. Third column; Gram character. Fourth column; Pathway of formaldehyde fixation; ribulose monophosphate cycle (RUMP), serine pathway (serine). Fifth column; Nutritional type; obligate (obl), facultative methyiotroph (fac).
kinetic properties of these enzymes were studied in more detail. The citrate synthase of Hyphomicrobium sp. strain X was almost completely inhibited even by low NADH concentrations, whilst the enzyme of the obligate methylotroph Hyphomicrobiurn sp. strain Hanham was unaffected (Fig. 1). The inhibition of the citrate synthase of Hyphomicrobium sp. strain X by NADH was non-competitive with respect to acetyl-CoA (Table 2). The inhibitor constant (Ki) of the citrate
loc z 0 t.-
z
o
o
o
o
Do mM NADH Fig. 1. Relation between degree of inhibition of citrate synthase and NADH concentration. O, Hyphomicrobium sp..strain X; O, Hyphomicrobium sp. strain Hanham. The solid line representing the theoretical relationship was calculated by assuming that K i (NADH) is 0.4 mM.
synthase of Hyphomicrobiumsp. strain X (0.4 mM) was considerably lower than that of Hyphornicrobium sp. strain Hanham (12 mM) (Table 2). This type of inhibition suggests the presence of a binding site for NADH separate from the active site; presumably this site has been modified in the obligate methylotroph Hyphomicrobium sp. strain Hanham. The inhibition of the citrate synthase by 0.75 mM NADH could be overcome by the addition of 1 mM AMP (Table 2). In this respect the citrate synthase of Hyphomicrobiumsp. strain X is very similar to the citrate synthases of other aerobic Gram-negative bacteria [1]. The insensitivity of the citrate synthase of Hyphomicrobium sp. strain Hanham to NADH is also interesting for another reason. This organism contains low levels of oxoglutarate dehydrogenase activit2( (0.03 nkat. mg-1), and hence, TCA-cycle activity. Zatman's hypothesis [2] concerning the regulation of citrate synthase in methylotrophs stipulates that under these circumstances an organism should have a NADH-sensitive citrate synthase. This is apparently not always the case, and in this respect Hyphomicrobium sp. strain Hanham is similar to another serine-pathway obligate methylotroph Methylococcus trichosporum OB3b, which was also shown to possess a NADHinsensitive citrate synthase [3]. This makes it unlikely that the reason for the presence of a NADH-sensitive citrate synthase in other serinepathway Gram-negative facultative methylotrophs is to prevent unwanted dissimilation of metabo-
194 Table 2 Kinetic parameters of the citrate synthases of Hyphomicrobium sp. strain X and Hyphomicrobium sp. strain Hanham. Kinetic parameters were determined using a single batch of enzyme; standard errors were calculated according to Cornish-Bowden and Eisenthal [15] Strain
X
Hanham
Km ( # M acetyl-CoA) Vmax (nkat- mg protein- l) in the presence of 0.75 mM N A D H K m (/~M acetyl-CoA) Vma~ (nkat- mg protein- 1) in the presence of 5 mM N A D H K m ( ~ M acetyl-CoA) Vm~X (nkat. mg protein- 1 ) in the presence of 0.75 mM N A D H and 1 mM AMP K m (/~M acetyl-CoA) Vm~x (nkat. mg protein- 1 ) K i (mM NADH)
161 _+22 0.68-+ 0.04
143 +11 0.3 + 0.01
159 -+ 34 0.23 -+ 0.03
N.T. N.T.
N.T. N.T.
156 _+41 0.23 _+ 0.05
214 _+40 0.73 -4- 0.10 0.4
N.T. N.T. 12
N.T. = not tested.
lites derived from the serine pathway. It is more likely that the presence of a NADH-sensitive citrate synthase is inherent to the more nutritionally versatile life style of these organisms. In their natural habitat, which is usually very poor in nutrients, facultative methylotrophs probably grow on a mixture of different substrates rather than a single substrate, and this involves both one-carbon as well as multicarbon substrates. Under these circumstances, regulation of the citrate synthase and hence the TCA-cycle is essential. Moreover, multicarbon and one-carbon substrates do not occur sequentially in natural milieux but are released simultaneously as a result of the slow decomposition of organic matter. Therefore, a facultative methylotroph has little to gain by expressing 2 different citrate synthases, one during methylotrophic growth and one (NADH-sensitive) during growth on multicarbon substrates [5].
ACKNOWLEDGEMENTS I gratefully acknowledge the help of Dr. M.M. Attwood, Prof. J.R. Quayle, Prof. P.D.J. Weitzman and Dr. L.J. Zatman in providing both materials and experimental facilities, and the Science and Engineering Research Council for financial support under grant number GR/C/71644.
REFERENCES [1] Weitzman, P.D.J. (1981) Adv. Microb. Physiol. 22, 185-244. [2] Zatman, L.J. (1981) in Microbial growth on C-1 Compounds, (Dalton, H., Ed.) pp. 42-54. Heyden, London. [3] Colby, J. and Zatman, L.J. (1975) Biochem. J. 150, 141-144. [4] Cox, R.B. and Quayle, J.R. (1976) J. Gen. Microbiol. 95, 121-133. [5] Babel, W. and Muller-Kraft, G. (1979) Z. Allg. Mikrobiol. 19, 687-693. [6] Anthony, C. and Taylor, I.J. (1975) Proc. Soc. Gen. Microbiol. 4, 67. [7] Colby, J. and Zatman, L.J. (1973) Biochem. J. 132, 101-112. [8] Attwood, M.M. and Harder, W. (1972) Antonie van Leeuwenhoek 39, 369-378. [9] Large, P.J. and Haywood, G.W. (1981) FEMS Microbiol. Lett. 11,207-209. [10] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [11] Srere, P. (1969) in Methods in Enzymology, Vol. 13 (Lowenstein, J.M., Ed.), pp. 3-11. Academic Press, New York and London. [12] Bergmeyer, H.U. (1974) Methods of Enzymatic Analysis, Vol. 1, pp. 481-482. Academic Press, New York and London. [13] Reed, L.J. and Mukherjee, B.B. (1969) Methods in Enzymology, Vol. 13 (Lowenstein, J.M., Ed.), pp. 55-61. Academic Press, New York and London. [14] Eisenthal, R. and Cornish-Bowden, A. (1974) Biochem. J. 139, 715-720. [15] Cornish-Bowden, A. and Eisenthal, R. (1974) Biochem. J. 139, 721-730.