APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1977, p. 607-610
Copyright X) 1977 American Society for Microbiology
Vol. 34, No.5 Printed in U.S.A.
['4C]Acetate Assimilation by a Type I Obligate Methylotroph, Methylococcus capsulatus RAMESH N. PATEL,t* S. LOUISE HOARE,ff D. S. HOARE,t#t AND B. F. TAYLORtttt Department of Microbiology, University of Texas, Austin, Texas 78712 Received for publication 20 June 1977
Methanol and formate oxidation supported the assimilation of ['4C]acetate by cell suspensions of Methylococcus capsulatus; oxidation of other primary alcohols, except ethanol, did not. The extent of [1-14C]acetate assimilation supported by methanol oxidation was decreased in the presence of primary alcohols, except ethanol. Potassium cyanide (0.33 mM) completely inhibited the oxidation of formate and its stimulation of [1-'4C]acetate assimilation. The amount of [1-l4C] acetate amiltion supported by methanol oxidation was significantly inhibited by cyanide.
The methane-utilizing bacteria are obligately dependent upon methane, methanol, or dimethyl ether as sole sources of carbon and energy (8, 17, 18, 25). Recently, Patt et al. (16) isolated facultative methane-utilizing bacteria that utilize methane as well as the more complex organic carbon and energy sources. All methaneutilizing bacteria so far described possess extensive intracytoplasmic membranes (6, 16-18). On the basis of structural organization of their intracytoplasmic membranes and pathway of carbon assimilation, the methane-utilizing bacteria are divisible into two distinct groups (6, 17). The obligate methylotrophs are in many respects similar to other "obligate autotrophs" such as some of the blue-green algae, thiobacilli, photosynthetic bacteria of the genus Chlorobium, and nitrifying bacteria in that they are unable to use organic compounds as sources of carbon and energy for growth (2, 7, 9, 11, 20, 22). Recent work in a number of laboratories (9-11, 19, 20) has led to the proposal of a biochemical basis for obligate autotrophy in various bacteria. One of the major biochemical defects of some obligate autotrophs is a metabolic block at the a-ketoglutarate dehydrogenase step of the tricarboxylic acid cycle. One immediate consequence of this enzymatic deficiency is an inability to oxidize acetate, a key internediate in the breakdown of a wide range of organic compounds. We first reported the absence of a a-ketoglutarate dehydrogenase in a type I methylotroph, Methylococcus capsulatus, based on enzymatic t Present address: Corporate Research Laboratories, Exxon Research and Engineering Co., Linden, NJ 07036. tt Present address: Department of Bacteriology, University of California, Los Angeles, CA 90024. m Deceased. Itlt Present address: Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149.
assays and the fate of assimnilated [1-'4C]acetate during growth of organisms on methane (R. Patel, D. S. Hoare, and B. F. Taylor, Bacterial Proc., p. 128, 1969) (15). Subsequently, Davey et al. (5) reported the absence of a complete tricarboxylic acid cycle in all type I obligate methane utilizers. However, all type II obligate and facultative methane utilizers possess a complete tricarboxylic acid cycle, including a-keto glutarate dehydrogenase (5, 16, 23, 24). In this report, we describe the effect of various C-1 compounds and primary alcohols on assimilation of ['4C]acetate by cell suspensions of M. capsulatus. M. capsulatus (Texas strain) (18) was reisolated from an old contaminated culture. Organisms were maintained on salt agar plates in a desiccator under an atmosphere of methane and air (1:1, vol/vol) and were transferred every 15 days. Cultures were grown in Erlenmeyer flasks (2 liters) containing 300 ml of salt medium (8) with methane as sole carbon and energy sources as described previously (14). Cells were harvested by centrifuging for 15 min at 12,000 x g and were washed twice in 50 mM potassium phosphate buffer, pH 7.0. Cells were suspended in the same buffer to a turbidity of 500 in the Klett colorimeter. Standard manometric techniques were used to follow respiration in cell suspensions at 37°C. Warburg flasks contained, in a total volume of 3.0 ml, 50 ,umol of potassium phosphate buffer, pH 7.0, and 0.5 ml of cell suspension (5.0 mg of protein/ml). Substrates were put in a side bulb; center wells contained 0.2 ml of 20% potassium hydroxide. [1-'4C]acetate and [2-'4C]acetate assimilation experiments were carried out with cell suspensions of M. capsulatus in a Warburg constantvolume respirometer at 37°C. Reaction mixtures contained, in 3.0 ml: 100 ,umol of potassium 607
608
APPL. ENVIRON. MICROBIOL.
NOTES
phosphate buffer (pH 7.0); cell suspensions (2 to 5 mg of protein); nonradioactive substrate in a side bulb; sodium [14C]acetate in a second side bulb; and 20% (wt/vol) KOH, 0.1 ml in center well. Reactions were terminated by immersing the Warburg flasks in crushed ice. Samples of cell suspensions were filtered on to membrane fiters (pore size, 0.45 ,um; Millipore Corp.) and washed three times with 15-ml portions of cold 50 mM sodium acetate and three times with cold distilled water. The filters were then glued on aluminum planchets, dried, and assayed for radioactivity in a model D47 gas-flow planchet counter (Nuclear Chicago Corp., Des Plaines, Ill.). The effect of acetate concentration on [1-'4C] acetate assimilation by a cell suspension of M. capsulatus is shown in Table 1. The extent of [1-'4C]acetate assimilation supported by methanol oxidation was independent of acetate concentration in the range of 0.83 to 3.3 mM, but was depressed at higher levels of acetate. The rate of methanol oxidation was decreased about 50% by 10 mM acetate. Subsequent experiments were usually performed with an acetate concentration of 3.3 mM or lower. The time course of [1-'4C]acetate assimilation supported by methanol oxidation is shown in Fig. 1. The amount of [1-'4C]acetate assimilated correlated with the amount of oxygen consumed during methanol oxidation. Similar results were obtained in an experiment with [2-'4C]acetate assimilation. The effect of varying the concentration of methanol on assimilation of [1-'4C] acetate was examined. There was a direct relationship between the amount of methanol oxidized and the assimilation of [1-'4C]acetate by cell suspensions of M. capsulatus (Fig. 2). The amount of [1-'4C]acetate assimilated varied from 4.5 to 6.5 ,umol per ,imol of methanol oxidized or oxygen consumed. TABLE 1. Effect of acetate concentration on the assimilation of [1-14C]acetate by M. capsulatus
oxidizing methanola
Acetate concn (MM)
0.83 1.67 2.50 3.33 5.00 10.00
Oxygen up-
~~take"
(,omol) 5.7 5.6 5.5 5.7 5.4 5.3
assimiAcetate latedb
(nmol)
39.1 33.6 43.2 42.7 22.6 20.6
aWarburg flasks contained, in 3.0 ml: potassium phosphate (pH 7.0), 100 ,tmol; sodium [1_-4C]acetate, 5 ,tCi; methanol, 5 ,umol; cell suspensions, 1.33 mg of protein; 20% (wt/vol) NaOH, 0.1 ml (center well). Flasks were incubated for 90 min at 37°C. b Corrected for endogenous activity.
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Copyright X) 1977 American Society for Microbiology
Vol. 34, No.5 Printed in U.S.A.
['4C]Acetate Assimilation by a Type I Obligate Methylotroph, Methylococcus capsulatus RAMESH N. PATEL,t* S. LOUISE HOARE,ff D. S. HOARE,t#t AND B. F. TAYLORtttt Department of Microbiology, University of Texas, Austin, Texas 78712 Received for publication 20 June 1977
Methanol and formate oxidation supported the assimilation of ['4C]acetate by cell suspensions of Methylococcus capsulatus; oxidation of other primary alcohols, except ethanol, did not. The extent of [1-14C]acetate assimilation supported by methanol oxidation was decreased in the presence of primary alcohols, except ethanol. Potassium cyanide (0.33 mM) completely inhibited the oxidation of formate and its stimulation of [1-'4C]acetate assimilation. The amount of [1-l4C] acetate amiltion supported by methanol oxidation was significantly inhibited by cyanide.
The methane-utilizing bacteria are obligately dependent upon methane, methanol, or dimethyl ether as sole sources of carbon and energy (8, 17, 18, 25). Recently, Patt et al. (16) isolated facultative methane-utilizing bacteria that utilize methane as well as the more complex organic carbon and energy sources. All methaneutilizing bacteria so far described possess extensive intracytoplasmic membranes (6, 16-18). On the basis of structural organization of their intracytoplasmic membranes and pathway of carbon assimilation, the methane-utilizing bacteria are divisible into two distinct groups (6, 17). The obligate methylotrophs are in many respects similar to other "obligate autotrophs" such as some of the blue-green algae, thiobacilli, photosynthetic bacteria of the genus Chlorobium, and nitrifying bacteria in that they are unable to use organic compounds as sources of carbon and energy for growth (2, 7, 9, 11, 20, 22). Recent work in a number of laboratories (9-11, 19, 20) has led to the proposal of a biochemical basis for obligate autotrophy in various bacteria. One of the major biochemical defects of some obligate autotrophs is a metabolic block at the a-ketoglutarate dehydrogenase step of the tricarboxylic acid cycle. One immediate consequence of this enzymatic deficiency is an inability to oxidize acetate, a key internediate in the breakdown of a wide range of organic compounds. We first reported the absence of a a-ketoglutarate dehydrogenase in a type I methylotroph, Methylococcus capsulatus, based on enzymatic t Present address: Corporate Research Laboratories, Exxon Research and Engineering Co., Linden, NJ 07036. tt Present address: Department of Bacteriology, University of California, Los Angeles, CA 90024. m Deceased. Itlt Present address: Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149.
assays and the fate of assimnilated [1-'4C]acetate during growth of organisms on methane (R. Patel, D. S. Hoare, and B. F. Taylor, Bacterial Proc., p. 128, 1969) (15). Subsequently, Davey et al. (5) reported the absence of a complete tricarboxylic acid cycle in all type I obligate methane utilizers. However, all type II obligate and facultative methane utilizers possess a complete tricarboxylic acid cycle, including a-keto glutarate dehydrogenase (5, 16, 23, 24). In this report, we describe the effect of various C-1 compounds and primary alcohols on assimilation of ['4C]acetate by cell suspensions of M. capsulatus. M. capsulatus (Texas strain) (18) was reisolated from an old contaminated culture. Organisms were maintained on salt agar plates in a desiccator under an atmosphere of methane and air (1:1, vol/vol) and were transferred every 15 days. Cultures were grown in Erlenmeyer flasks (2 liters) containing 300 ml of salt medium (8) with methane as sole carbon and energy sources as described previously (14). Cells were harvested by centrifuging for 15 min at 12,000 x g and were washed twice in 50 mM potassium phosphate buffer, pH 7.0. Cells were suspended in the same buffer to a turbidity of 500 in the Klett colorimeter. Standard manometric techniques were used to follow respiration in cell suspensions at 37°C. Warburg flasks contained, in a total volume of 3.0 ml, 50 ,umol of potassium phosphate buffer, pH 7.0, and 0.5 ml of cell suspension (5.0 mg of protein/ml). Substrates were put in a side bulb; center wells contained 0.2 ml of 20% potassium hydroxide. [1-'4C]acetate and [2-'4C]acetate assimilation experiments were carried out with cell suspensions of M. capsulatus in a Warburg constantvolume respirometer at 37°C. Reaction mixtures contained, in 3.0 ml: 100 ,umol of potassium 607
608
APPL. ENVIRON. MICROBIOL.
NOTES
phosphate buffer (pH 7.0); cell suspensions (2 to 5 mg of protein); nonradioactive substrate in a side bulb; sodium [14C]acetate in a second side bulb; and 20% (wt/vol) KOH, 0.1 ml in center well. Reactions were terminated by immersing the Warburg flasks in crushed ice. Samples of cell suspensions were filtered on to membrane fiters (pore size, 0.45 ,um; Millipore Corp.) and washed three times with 15-ml portions of cold 50 mM sodium acetate and three times with cold distilled water. The filters were then glued on aluminum planchets, dried, and assayed for radioactivity in a model D47 gas-flow planchet counter (Nuclear Chicago Corp., Des Plaines, Ill.). The effect of acetate concentration on [1-'4C] acetate assimilation by a cell suspension of M. capsulatus is shown in Table 1. The extent of [1-'4C]acetate assimilation supported by methanol oxidation was independent of acetate concentration in the range of 0.83 to 3.3 mM, but was depressed at higher levels of acetate. The rate of methanol oxidation was decreased about 50% by 10 mM acetate. Subsequent experiments were usually performed with an acetate concentration of 3.3 mM or lower. The time course of [1-'4C]acetate assimilation supported by methanol oxidation is shown in Fig. 1. The amount of [1-'4C]acetate assimilated correlated with the amount of oxygen consumed during methanol oxidation. Similar results were obtained in an experiment with [2-'4C]acetate assimilation. The effect of varying the concentration of methanol on assimilation of [1-'4C] acetate was examined. There was a direct relationship between the amount of methanol oxidized and the assimilation of [1-'4C]acetate by cell suspensions of M. capsulatus (Fig. 2). The amount of [1-'4C]acetate assimilated varied from 4.5 to 6.5 ,umol per ,imol of methanol oxidized or oxygen consumed. TABLE 1. Effect of acetate concentration on the assimilation of [1-14C]acetate by M. capsulatus
oxidizing methanola
Acetate concn (MM)
0.83 1.67 2.50 3.33 5.00 10.00
Oxygen up-
~~take"
(,omol) 5.7 5.6 5.5 5.7 5.4 5.3
assimiAcetate latedb
(nmol)
39.1 33.6 43.2 42.7 22.6 20.6
aWarburg flasks contained, in 3.0 ml: potassium phosphate (pH 7.0), 100 ,tmol; sodium [1_-4C]acetate, 5 ,tCi; methanol, 5 ,umol; cell suspensions, 1.33 mg of protein; 20% (wt/vol) NaOH, 0.1 ml (center well). Flasks were incubated for 90 min at 37°C. b Corrected for endogenous activity.
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