Antonie van Leeuwenhoek 45 (1979) 499 511
499
[1-14C] A c e t a t e a s s i m i l a t i o n b y o b l i g a t e m e t h y l o t r o p h s ,
Pseudomonas methanica a n d Methylosinus trichosporium R . N . PATEL 1, S. LOUISE HOARE z, AND D . S. HOARE 3
Department of Microbiology, University of Texas Austin, Texas 78712, USA
PATEL, R. N., HOAR~, S. L. and HOARE, D. S. 1979.1-14C Acetate assimilation by methylotrophs, Pseudomonas methanica and Methylosinus trichosporium. Antonie van Leeuwenhoek 45:499-511. The oxidation of one carbon compounds (methane, methanol, formaldehyde, formate) and primary alcohols (ethanol, propanol, butanol) supported the assimilation of [1-14C]acetate by cell suspensions of type I obligate methylotroph, Pseudomonas methanica,Texas strain,and type II obligate methylotroph, Methylosinus trichosporium, strain PG. The amount of oxygen consumed and substrate oxidized correlated with the amount of [1-14C]acetate assimilated during oxidation of C-1 compounds and primary alcohols. Oxidation of methanol, formaldehyde, and primary alcohols in extracts of Pseudomonas methanica, Texas strain, and Methylosinus trichosporium, strain PG, was catalyzed by a phenazine methosulfate linked, ammonium ion dependent methanol dehydrogenase. The oxidation of aldehydes was catalyzed by a phenazine methosulfate linked, ammonium ion independent aldehyde dehydrogenase. Formate was oxidized by a N A D + linked formate dehydrogenase.
INTRODUCTION Methylotrophs are microorganisms that grow non-autotrophically on compounds containing one or more carbon atoms but no carbon-carbon bonds (Colby and Zatman, 1975), There are both obligate and facultative methylotrophs. Many methane-utilizing bacteria are obligately dependent on methane, methanol (obligate methylotrophs) as sole sources of carbon and energy for growth 1Present address: Corporate Research Laboratories, Exxon Research and EngineeringCo., P.O. Box 45, Linden,N.J. 07036. 2Present Address: Department of Bacteriology,Universityof California, Los Angeles,CA 90024. 3Deceased.
500
R.N. PATEL, S. L. HOARE AND D. S. HOARE
(Brown, Strawinski and McCleskey, 1964; Dworkin and Foster, 1956; Foster and Davis, 1966; Leadbetter and Foster, 1958 ; Whittenbury, Phillip and Wilkinson, 1970). Recently, Patt et al. (1974) isolated facultative methane-utilizing bacteria that can also utilize more complex organic molecules for carbon and energy sources. All methane-utilizing bacteria so far described possess extensive intracytoplasmic membranes (Davis andWhittenbury, 1970 ; Ribbons, Harrison and Wadzinski, 1970). On the basis of the structural organization of their intracytoplasmic membranes and the pathway of carbon assimilation, the obligate methylotrophs are divided into two distinct groups. The methane-utilizing bacteria with a type I membrane structure utilize a hexulose phosphate pathway of formaldehyde fixation for carbon assimilation while type II membrane bacteria utilize a serine pathway for carbon assimilation (Davis and Whittenbury, 1970; Lawrence and Quayle, 1970). However, some organisms with the type I membrane structure do possess key enzymes of both the hexulose phosphate pathway and the serine pathway (Whittenbury et al., 1974). Furthermore, Cox and Quayle (1975) have reported that Micrococcus denitrificans can also grow autotrophically on methanol and ribulose diphosphate carboxylase is present in a high level in extracts of the organism. The oblig~,te methylotrophs are in many respects similar to other obligate autotrophs such as some strains of nitrifying bacteria, thiobacilli, photosynthetic bacteria of the genus Chlorobium, and blue-green algae in that they are unable to utilize organic compounds as sources of carbon and energy for growth and possess extensive intracytoplasmic membrane structure (Clark and Schmidt, 1967; Delwiche and Finstein, 1965; Hoare and Gibson, 1964; Kelly, 1971; Smith, London and Stanier, 1967; Taylor and Hoare, 1972). One of the major biochemical defects of some obligate autotrophs is a metabolic block at the ~-ketoglutarate dehydrogenase. They also possess low levels of some of the enzymes of the tricarboxylic acid cycle (Hoare and Gibson, 1964; Kelly, 1971 ; Smith and Hoare, 1.968 ; Smith et al., 1967 ; Taylor and Hoare, 1972; Taylor and Anthony, 1976). We first reported absence of a ~-ketoglutarate dehydrogenase in a type I obligate methylotroph, Methylococcus capsulatus, based on enzymatic studies and the fate of assimilated [1-1~C]acetate during the growth of organisms on methane (Patel, Hoare and Taylor, 1969; Patel et al., 1975). Subsequently, Davey, Whittenbury and Wilkinson (1972) reported the absence of a complete tricarboxylic acid cycle in all type I obligate metylotrophs. However, all type lI obligate methylotrophs and facultative methane-utilizing organisms do possess a complete tricarboxylic acid cycle including a low activity of ~-ketoglutarate dehydrogenase (Davey et al., 1972; Colby and Zatman, 1975; Patt et al., 1974). In this report, ~ve describe the effect of various C-1 compounds and primary alcohols on assimilation of [1-14C]acetate by cell suspensions of type I obligate methylotroph, Pseudomonas rnethanica and type II obligate methylotroph,
Methylosinus trichosporium.
ACETATE ASSIMILATIONBY OBLIGATE METHYLOTROPHS
501
MATERIALS AND METHODS
Bacterial strains Pseudomonas methanica, Texas strain (Leadbetter and Foster, 1958), was kindly provided by Dr J. J. Perry. Microbiology Department, North Carolina State University, Raleigh, N. C. Methylosinus trichosporium, strain PG (Whittenbury et al., 1970), was kindly supplied by Professor R. Whittenbury, Department of Biological Sciences, University of Warwick, Coventry, Great Britain. The salt medium of Foster and Davis (1966) plus methane as a sole carbon source was used for growing the organisms. Cultures were maintained on agar plates in a desiccator under an atmosphere of methane and air (I: 1, v/v) and were transferred every two weeks. For routine use, the cultures were maintained in liquid medium under methane and air (1: 1, v/v).
Growth of organisms Cultures were grown in 2-liter Erlenmeyer flasks containing 300 ml salt medium with methane as sole carbon and energy sources as described previously (Patel and Hoare, 1971).
Experiments with cell suspensions Cells were harvested during the logarithmic growth phase by centrifuging for 15 rain at 15000 x g and washed twice in phosphate buffer, pH 7.0. Cells were then suspended in the same buffer to a turbidity of 400 500 in the Klett colorimeter. Standard manometric techniques were used to follow.the respiration in cell suspension at 30 °C. Warburg flasks contained in a total volume of 3.0 ml, 100 ~mol of potassium phosphate buffer (pH 7.0), 0.5 ml of cell suspensions (3 mg of protein/ml), and substrates in side bulbs. The center well contained 0.2 ml of 20 ~ potassium hydroxide. [14C]acetat e assimilation experiments were carried out with cell suspensions of Ps. methanica and M. trichosporium in a Warburg constant volume respirometer at 30°C. The reaction mixture contained 100 ~tmoles of potassium phosphate buffer (pH 7.0), cells suspensions (1.6 mg of protein), non-radioactive substrate in one side bulb, and sodium [14C]acetate in the other side bulb in a total volume of 3.0 ml. Reactions were terminated by immersing the Warburg flasks in crushed ice. Samples of cell suspensions were filtered through a membrane filter (pore size 0.45 tam, Millipore Corp.) and washed three times with a 15 ml portion of cold 50 mM sodium acetate and three times with cold distilled water. The filters were then glued on aluminium planchets, dried, and assayed in a model D47 gas flow planchet counter (Nuclear Chicago Corp., Des Plaines, Ill.).
Preparation of cell-extracts Cell-extracts were prepared by sonic disintegration as described previously (Patel and Hoare, 1971). Cell-debris and large particles were removed by cen-
502
R . N . PATEL, S. L. HOARE AND D. S. HOARE
trifuging for 10 min at 12000 x g. The supernatant obtained after centrifugation was used for measuring methanol dehydrogenase, aldehyde dehydrogenase, and formate dehydrogenase activities.
Enzyme assays Methanol dehydrogenase activity was assayed spectrophotometrically as described previously (Patel et al., 1972; Patel and Felix, 1976). Aldehyde dehydrogenase was assayed spectrophotometrically by measuring the oxidation of various aldehydes in the presence of an auxiliary electron acceptor, phenazine methosulfate (PMS). The reaction mixture contained 50 mN Tris-HC1 buffer, pH 7.0; 0.5 gmole 2,6-dichlorophenolindophenol (DCPIP), 2 gmoles PMS, and the soluble fraction in a total volume of 3.0 ml. Initially, the reaction was started by the addition of 50 gmoles ofsubstrate followed by measuring the reduction of DCPIP at 600 nm. Specific activity was expressed as nmoles of DCPIP reduced per min and per mg of protein. Formate dehydrogenase was assayed spectrophotometrically using N A D ÷ as an electron acceptor as described previously (Patel and Hoare, 1971). Protein was estimated by Folin phenol reagent method (Lowry et al., 1951).
RESULTS
Experiments with cell suspensions Cell suspensions of methane- or methanol-grown Pseudomonas methanica and Methylosinus trichosporium oxidized C-1 compounds, methanol, formaldehyde, and formate. The amount of oxygen consumed was 65 70 ~ of the value expected if the substrate supplied were completely oxidized to carbon dioxide and water. Primary alcohols (ethanol, propan-l-ol, butan-l-ol tested) were incompletely oxidized. The ~mole of oxygen consumed per ~tmole of various alcohols oxidized varied from 0.63-0.67 for both organisms.
Oxidation ofjormaldehyde, methanol, and primary alcohols The oxidation of formaldehyde, methanol, and primary alcohols (ethanol, propan-l-ol, butan-l-ol tested) in extracts of Ps. methanica and M. trichosporium was catalyzed by an ammonium ion dependent soluble methanol dehydrogenase. There was no catalytic activity detected in the absence of ammonium chloride. The activity could be enhanced by increasing the concentration of ammonium chloride up to 30 ~tmoles per 3.0 ml assay system. Among various artificial electron acceptors tested, phenazine methosulfate and phenazine ethosulfate could act as electron acceptors. Many natural biological electron acceptots, such as N A D +, N A D phosphate, flavin adenine dinucleotide and cytochrome c, and artificial acceptors such as 2,6-dichlorophenolindophenol, benzyl viologen and potassium ferricyanide could not act as electron carriers. The op-
A C E T A T E A S S I M I L A T I O N BY O B L I G A T E M E T H Y L O T R O P H S
503
timum pH for oxidation of various alcohols by methanol dehydrogenase was found to be 9.0. No catalytic activity was detected at pH 6.5 in the presence or absence of ammonium chloride. A specific activity (nmoles of DCPIP reduced per min and per mg of protein) of 271 and 343 was obtained with extracts of Ps. methanica and M. trichosporium, respectively, with methanol as a substrate. A specific activity of 216 and 274 was obtained with extracts of Ps. methanica and M. trichosporium, respectively, with formaldehyde or other alcohols as substrate. Secondary alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized by methanol dehydrogenase at pH 9.0. In aqueous solution formaldehyde is predominantly hydrated (99 %) and presumably appears as an analogue of methanol for the dehydrogenase (Sperl, Forrest and Gibson, 1974) and this may account for oxidation of formaldehyde bymethanol dehydrogenase.
Oxidation of aldehydes Oxidation of aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde tested) in extracts of Ps. methanica and M. trichosporium was catalized by an ammonium ion independent, soluble aldehyde dehydrogenase. The optimum pH for the oxidation of various aldehydes by aldehyde dehydrogenase was found to be 6.5 for catalysis. Phenazine methosulfate and 2,6dichlorophenolindophenol (DCPIP) could act as electron acceptors. Activity with DCPIP was only 15 ~ to that of 100 ~ activity with PMS as an electron carrier. NAD +, NADP, FAD and F M N could not act as an electron acceptor. A specific activity (nmoles of DCPIP reduced per rain and per mg of protein) of 110 and 89 was obtained with extracts of Ps. methanica and M. trichosporium, respectively, with propionaldehyde as a substrate. The activity with formaldehyde, acetaldehyde and butyraldehyde was 80 90 ~o to that of 100 ~ activity with propionaldehyde as a substrate. Oxidation ojformate Extracts of Ps. methanica and M. trichosporium contained a soluble formate dehydrogenase which catalyzed the oxidation of formate. Enzyme activity was demonstrated spectrophotometrically with NAD + as an electron acceptor. A specific activity (nmoles o f N A D + reduced per rain and per mg of protein) of 130 and 890 was obtained with extracts of Ps. methanica and M. trichosporium, respectively. [1-14C]acetate assimilation experiments The data presented in the following section reflect minimal assimilation values in acetate [1-14C] assimilation coupled with oxidation of various unlabeled substrates. This may be because the labeled acetate (or 14C-labeled intermediate derived from acetate) may be diluted by nonlabeled metabolite derived from the substrate undergoing oxidation and also by unlabeled acetate provided during
504
R. N. PATEL, S. L. HOARE AND D. S. HOARE
14 Cn
80
12
60 8
g
40
6
x o
20 __
__ 15
30
I 45
I 60
I 75
I 90
I 105
TIME (MINUTES]
Fig. la. Time course of [1- t4C]acetate assimilation and oxygen consumption by cell suspensions of M. trichosporium oxidizing methanol Warburg flasks contained in 3.0 mh potassium phosphate buffer (pH 7.0), 100 gmoles; sodium [14C]acetate 2 gCi and 10 gmoles; methanol, 20 gmoles; cell suspensions, 1.5 mg of protein. Flasks were incubated aerobically at 30 °C. Symbols: (El), [1-14C]acetat e assimilated; (o), oxygen uptake. oxidation studies. T h e t i m e c o u r s e o f a c e t a t e a s s i m i l a t i o n s u p p o r t e d b y m e t h a n o l o x i d a t i o n in cell s u s p e n s i o n s o f M. trichospoHum a n d Ps. methanica is s h o w n i n F i g s l a a n d I
I
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-410
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o 10 w
? x o
8~0
100
TIME [MINUTES)
Fig. lb. Time course of [1-14C]acetate assimilation and oxygen consumption by cell suspensions of Ps. methanica oxidizing methanol Warburg flasks contained in 3.0 ml: potassium phosphate buffer (pH 7.0), 100 Ilmoles ; methanol, 20 lamoles; sodium [1-~4C]acetate 2 p.Ci and l 0 gmoles; cell suspensions, 1.0 mg of protein. Flasks were incubated aerobically at 30 °C. Symbols: ([::]), [1-t ¢C]acetate assimilated; (o), oxygen uptake.
ACETATE ASSIMILATION BY OBLIGATE METHYLOTROPHS
505
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499
[1-14C] A c e t a t e a s s i m i l a t i o n b y o b l i g a t e m e t h y l o t r o p h s ,
Pseudomonas methanica a n d Methylosinus trichosporium R . N . PATEL 1, S. LOUISE HOARE z, AND D . S. HOARE 3
Department of Microbiology, University of Texas Austin, Texas 78712, USA
PATEL, R. N., HOAR~, S. L. and HOARE, D. S. 1979.1-14C Acetate assimilation by methylotrophs, Pseudomonas methanica and Methylosinus trichosporium. Antonie van Leeuwenhoek 45:499-511. The oxidation of one carbon compounds (methane, methanol, formaldehyde, formate) and primary alcohols (ethanol, propanol, butanol) supported the assimilation of [1-14C]acetate by cell suspensions of type I obligate methylotroph, Pseudomonas methanica,Texas strain,and type II obligate methylotroph, Methylosinus trichosporium, strain PG. The amount of oxygen consumed and substrate oxidized correlated with the amount of [1-14C]acetate assimilated during oxidation of C-1 compounds and primary alcohols. Oxidation of methanol, formaldehyde, and primary alcohols in extracts of Pseudomonas methanica, Texas strain, and Methylosinus trichosporium, strain PG, was catalyzed by a phenazine methosulfate linked, ammonium ion dependent methanol dehydrogenase. The oxidation of aldehydes was catalyzed by a phenazine methosulfate linked, ammonium ion independent aldehyde dehydrogenase. Formate was oxidized by a N A D + linked formate dehydrogenase.
INTRODUCTION Methylotrophs are microorganisms that grow non-autotrophically on compounds containing one or more carbon atoms but no carbon-carbon bonds (Colby and Zatman, 1975), There are both obligate and facultative methylotrophs. Many methane-utilizing bacteria are obligately dependent on methane, methanol (obligate methylotrophs) as sole sources of carbon and energy for growth 1Present address: Corporate Research Laboratories, Exxon Research and EngineeringCo., P.O. Box 45, Linden,N.J. 07036. 2Present Address: Department of Bacteriology,Universityof California, Los Angeles,CA 90024. 3Deceased.
500
R.N. PATEL, S. L. HOARE AND D. S. HOARE
(Brown, Strawinski and McCleskey, 1964; Dworkin and Foster, 1956; Foster and Davis, 1966; Leadbetter and Foster, 1958 ; Whittenbury, Phillip and Wilkinson, 1970). Recently, Patt et al. (1974) isolated facultative methane-utilizing bacteria that can also utilize more complex organic molecules for carbon and energy sources. All methane-utilizing bacteria so far described possess extensive intracytoplasmic membranes (Davis andWhittenbury, 1970 ; Ribbons, Harrison and Wadzinski, 1970). On the basis of the structural organization of their intracytoplasmic membranes and the pathway of carbon assimilation, the obligate methylotrophs are divided into two distinct groups. The methane-utilizing bacteria with a type I membrane structure utilize a hexulose phosphate pathway of formaldehyde fixation for carbon assimilation while type II membrane bacteria utilize a serine pathway for carbon assimilation (Davis and Whittenbury, 1970; Lawrence and Quayle, 1970). However, some organisms with the type I membrane structure do possess key enzymes of both the hexulose phosphate pathway and the serine pathway (Whittenbury et al., 1974). Furthermore, Cox and Quayle (1975) have reported that Micrococcus denitrificans can also grow autotrophically on methanol and ribulose diphosphate carboxylase is present in a high level in extracts of the organism. The oblig~,te methylotrophs are in many respects similar to other obligate autotrophs such as some strains of nitrifying bacteria, thiobacilli, photosynthetic bacteria of the genus Chlorobium, and blue-green algae in that they are unable to utilize organic compounds as sources of carbon and energy for growth and possess extensive intracytoplasmic membrane structure (Clark and Schmidt, 1967; Delwiche and Finstein, 1965; Hoare and Gibson, 1964; Kelly, 1971; Smith, London and Stanier, 1967; Taylor and Hoare, 1972). One of the major biochemical defects of some obligate autotrophs is a metabolic block at the ~-ketoglutarate dehydrogenase. They also possess low levels of some of the enzymes of the tricarboxylic acid cycle (Hoare and Gibson, 1964; Kelly, 1971 ; Smith and Hoare, 1.968 ; Smith et al., 1967 ; Taylor and Hoare, 1972; Taylor and Anthony, 1976). We first reported absence of a ~-ketoglutarate dehydrogenase in a type I obligate methylotroph, Methylococcus capsulatus, based on enzymatic studies and the fate of assimilated [1-1~C]acetate during the growth of organisms on methane (Patel, Hoare and Taylor, 1969; Patel et al., 1975). Subsequently, Davey, Whittenbury and Wilkinson (1972) reported the absence of a complete tricarboxylic acid cycle in all type I obligate metylotrophs. However, all type lI obligate methylotrophs and facultative methane-utilizing organisms do possess a complete tricarboxylic acid cycle including a low activity of ~-ketoglutarate dehydrogenase (Davey et al., 1972; Colby and Zatman, 1975; Patt et al., 1974). In this report, ~ve describe the effect of various C-1 compounds and primary alcohols on assimilation of [1-14C]acetate by cell suspensions of type I obligate methylotroph, Pseudomonas rnethanica and type II obligate methylotroph,
Methylosinus trichosporium.
ACETATE ASSIMILATIONBY OBLIGATE METHYLOTROPHS
501
MATERIALS AND METHODS
Bacterial strains Pseudomonas methanica, Texas strain (Leadbetter and Foster, 1958), was kindly provided by Dr J. J. Perry. Microbiology Department, North Carolina State University, Raleigh, N. C. Methylosinus trichosporium, strain PG (Whittenbury et al., 1970), was kindly supplied by Professor R. Whittenbury, Department of Biological Sciences, University of Warwick, Coventry, Great Britain. The salt medium of Foster and Davis (1966) plus methane as a sole carbon source was used for growing the organisms. Cultures were maintained on agar plates in a desiccator under an atmosphere of methane and air (I: 1, v/v) and were transferred every two weeks. For routine use, the cultures were maintained in liquid medium under methane and air (1: 1, v/v).
Growth of organisms Cultures were grown in 2-liter Erlenmeyer flasks containing 300 ml salt medium with methane as sole carbon and energy sources as described previously (Patel and Hoare, 1971).
Experiments with cell suspensions Cells were harvested during the logarithmic growth phase by centrifuging for 15 rain at 15000 x g and washed twice in phosphate buffer, pH 7.0. Cells were then suspended in the same buffer to a turbidity of 400 500 in the Klett colorimeter. Standard manometric techniques were used to follow.the respiration in cell suspension at 30 °C. Warburg flasks contained in a total volume of 3.0 ml, 100 ~mol of potassium phosphate buffer (pH 7.0), 0.5 ml of cell suspensions (3 mg of protein/ml), and substrates in side bulbs. The center well contained 0.2 ml of 20 ~ potassium hydroxide. [14C]acetat e assimilation experiments were carried out with cell suspensions of Ps. methanica and M. trichosporium in a Warburg constant volume respirometer at 30°C. The reaction mixture contained 100 ~tmoles of potassium phosphate buffer (pH 7.0), cells suspensions (1.6 mg of protein), non-radioactive substrate in one side bulb, and sodium [14C]acetate in the other side bulb in a total volume of 3.0 ml. Reactions were terminated by immersing the Warburg flasks in crushed ice. Samples of cell suspensions were filtered through a membrane filter (pore size 0.45 tam, Millipore Corp.) and washed three times with a 15 ml portion of cold 50 mM sodium acetate and three times with cold distilled water. The filters were then glued on aluminium planchets, dried, and assayed in a model D47 gas flow planchet counter (Nuclear Chicago Corp., Des Plaines, Ill.).
Preparation of cell-extracts Cell-extracts were prepared by sonic disintegration as described previously (Patel and Hoare, 1971). Cell-debris and large particles were removed by cen-
502
R . N . PATEL, S. L. HOARE AND D. S. HOARE
trifuging for 10 min at 12000 x g. The supernatant obtained after centrifugation was used for measuring methanol dehydrogenase, aldehyde dehydrogenase, and formate dehydrogenase activities.
Enzyme assays Methanol dehydrogenase activity was assayed spectrophotometrically as described previously (Patel et al., 1972; Patel and Felix, 1976). Aldehyde dehydrogenase was assayed spectrophotometrically by measuring the oxidation of various aldehydes in the presence of an auxiliary electron acceptor, phenazine methosulfate (PMS). The reaction mixture contained 50 mN Tris-HC1 buffer, pH 7.0; 0.5 gmole 2,6-dichlorophenolindophenol (DCPIP), 2 gmoles PMS, and the soluble fraction in a total volume of 3.0 ml. Initially, the reaction was started by the addition of 50 gmoles ofsubstrate followed by measuring the reduction of DCPIP at 600 nm. Specific activity was expressed as nmoles of DCPIP reduced per min and per mg of protein. Formate dehydrogenase was assayed spectrophotometrically using N A D ÷ as an electron acceptor as described previously (Patel and Hoare, 1971). Protein was estimated by Folin phenol reagent method (Lowry et al., 1951).
RESULTS
Experiments with cell suspensions Cell suspensions of methane- or methanol-grown Pseudomonas methanica and Methylosinus trichosporium oxidized C-1 compounds, methanol, formaldehyde, and formate. The amount of oxygen consumed was 65 70 ~ of the value expected if the substrate supplied were completely oxidized to carbon dioxide and water. Primary alcohols (ethanol, propan-l-ol, butan-l-ol tested) were incompletely oxidized. The ~mole of oxygen consumed per ~tmole of various alcohols oxidized varied from 0.63-0.67 for both organisms.
Oxidation ofjormaldehyde, methanol, and primary alcohols The oxidation of formaldehyde, methanol, and primary alcohols (ethanol, propan-l-ol, butan-l-ol tested) in extracts of Ps. methanica and M. trichosporium was catalyzed by an ammonium ion dependent soluble methanol dehydrogenase. There was no catalytic activity detected in the absence of ammonium chloride. The activity could be enhanced by increasing the concentration of ammonium chloride up to 30 ~tmoles per 3.0 ml assay system. Among various artificial electron acceptors tested, phenazine methosulfate and phenazine ethosulfate could act as electron acceptors. Many natural biological electron acceptots, such as N A D +, N A D phosphate, flavin adenine dinucleotide and cytochrome c, and artificial acceptors such as 2,6-dichlorophenolindophenol, benzyl viologen and potassium ferricyanide could not act as electron carriers. The op-
A C E T A T E A S S I M I L A T I O N BY O B L I G A T E M E T H Y L O T R O P H S
503
timum pH for oxidation of various alcohols by methanol dehydrogenase was found to be 9.0. No catalytic activity was detected at pH 6.5 in the presence or absence of ammonium chloride. A specific activity (nmoles of DCPIP reduced per min and per mg of protein) of 271 and 343 was obtained with extracts of Ps. methanica and M. trichosporium, respectively, with methanol as a substrate. A specific activity of 216 and 274 was obtained with extracts of Ps. methanica and M. trichosporium, respectively, with formaldehyde or other alcohols as substrate. Secondary alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized by methanol dehydrogenase at pH 9.0. In aqueous solution formaldehyde is predominantly hydrated (99 %) and presumably appears as an analogue of methanol for the dehydrogenase (Sperl, Forrest and Gibson, 1974) and this may account for oxidation of formaldehyde bymethanol dehydrogenase.
Oxidation of aldehydes Oxidation of aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde tested) in extracts of Ps. methanica and M. trichosporium was catalized by an ammonium ion independent, soluble aldehyde dehydrogenase. The optimum pH for the oxidation of various aldehydes by aldehyde dehydrogenase was found to be 6.5 for catalysis. Phenazine methosulfate and 2,6dichlorophenolindophenol (DCPIP) could act as electron acceptors. Activity with DCPIP was only 15 ~ to that of 100 ~ activity with PMS as an electron carrier. NAD +, NADP, FAD and F M N could not act as an electron acceptor. A specific activity (nmoles of DCPIP reduced per rain and per mg of protein) of 110 and 89 was obtained with extracts of Ps. methanica and M. trichosporium, respectively, with propionaldehyde as a substrate. The activity with formaldehyde, acetaldehyde and butyraldehyde was 80 90 ~o to that of 100 ~ activity with propionaldehyde as a substrate. Oxidation ojformate Extracts of Ps. methanica and M. trichosporium contained a soluble formate dehydrogenase which catalyzed the oxidation of formate. Enzyme activity was demonstrated spectrophotometrically with NAD + as an electron acceptor. A specific activity (nmoles o f N A D + reduced per rain and per mg of protein) of 130 and 890 was obtained with extracts of Ps. methanica and M. trichosporium, respectively. [1-14C]acetate assimilation experiments The data presented in the following section reflect minimal assimilation values in acetate [1-14C] assimilation coupled with oxidation of various unlabeled substrates. This may be because the labeled acetate (or 14C-labeled intermediate derived from acetate) may be diluted by nonlabeled metabolite derived from the substrate undergoing oxidation and also by unlabeled acetate provided during
504
R. N. PATEL, S. L. HOARE AND D. S. HOARE
14 Cn
80
12
60 8
g
40
6
x o
20 __
__ 15
30
I 45
I 60
I 75
I 90
I 105
TIME (MINUTES]
Fig. la. Time course of [1- t4C]acetate assimilation and oxygen consumption by cell suspensions of M. trichosporium oxidizing methanol Warburg flasks contained in 3.0 mh potassium phosphate buffer (pH 7.0), 100 gmoles; sodium [14C]acetate 2 gCi and 10 gmoles; methanol, 20 gmoles; cell suspensions, 1.5 mg of protein. Flasks were incubated aerobically at 30 °C. Symbols: (El), [1-14C]acetat e assimilated; (o), oxygen uptake. oxidation studies. T h e t i m e c o u r s e o f a c e t a t e a s s i m i l a t i o n s u p p o r t e d b y m e t h a n o l o x i d a t i o n in cell s u s p e n s i o n s o f M. trichospoHum a n d Ps. methanica is s h o w n i n F i g s l a a n d I
I
I
I
I
16 150
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'~
90
i-
-:° ° J
// 210
-410
60
14
12
G,
o 10 w
? x o
8~0
100
TIME [MINUTES)
Fig. lb. Time course of [1-14C]acetate assimilation and oxygen consumption by cell suspensions of Ps. methanica oxidizing methanol Warburg flasks contained in 3.0 ml: potassium phosphate buffer (pH 7.0), 100 Ilmoles ; methanol, 20 lamoles; sodium [1-~4C]acetate 2 p.Ci and l 0 gmoles; cell suspensions, 1.0 mg of protein. Flasks were incubated aerobically at 30 °C. Symbols: ([::]), [1-t ¢C]acetate assimilated; (o), oxygen uptake.
ACETATE ASSIMILATION BY OBLIGATE METHYLOTROPHS
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