Vol. 122, No. 3 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, June 1975, p. 905-910 Copyright 0 1975 American Society for Microbiology
Enzymes Involved in the Assimilation of One-Carbon Units by Pseudomonas MS CONRAD WAGNER* AND MARK E. LEVITCH Research Laboratory, Veterans Administration Hospital,* and the Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37203, and the Department of Microbiology, Meharry Medical College, Nashville, Tennessee 37208 Received for publication 26 February 1975
Pseudomonas MS can grow on methylamine and a number of other compounds containing Cl units as a sole source of carbon and energy. Assimilation of carbon into cell material occurs via the "serine pathway" since enzymes of this pathway are induced after growth on methylamine, but not malate or acetate. A mutant has been isolated which is unable to grow on methylamine or any other related substrate providing Cl units. This mutant is also unable to grow on acetate. Measurment of enzyme activities in cell-free extracts of wild-type cells showed that growth on methylamine caused induction of isocitrate lyase, a key enzyme in the glyoxylate cycle. The mutant organism lacks malate lyase, a key enzyme of the serine pathway, and isocitrate lyase as well. These results suggest that utilization of Cl units by Pseudomonas MS results in the net accumulation of acetate which is then assimilated into cell material via the glyoxylate cycle. Pseudomonas MS was isolated from soil by enrichment culture on trimethylsulfonium chloride (C. Wagner, Bacteriol. Proc., p. 91, 1964). The organism will also grow on methylamine, dimethylamine, trimethylamine, a number of sugars, and various C2, C,, and C, compounds (8). With respect to the types of reduced C, growth substrates, it is similar to a number of other facultative methylotrophs described in the literature (13). There is increasing evidence to indicate that many of these organisms assimilate C1 compounds into cell material by a pathway elucidated primarily by Quayle and his group (13). This is shown in Fig. 1 and is referred to as the serine pathway because incubation of ["4C]methanol or ["4C]methylamine with intact cells of Pseudomonas AM1 results in the appearance of the label in serine as the earliest identified product (9). This pathway has been vigorously proven in Pseudomonas AM1 by a variety of enzymatic and genetic techniques. The first reaction involves the hydroxymethylation of glycine by a C, folate derivative originating from methylamine or methanol to form serine. The C, intermediates of the serine pathway must be converted to C4 compounds for the synthesis of a number of cell constituents. In Pseudomonas AM1 a single enzyme, phosphoenolpyruvate carboxylase (10), is responsible for that conversion. Glycine is regenerated from glyoxylate, which is formed by two reactions comprising the malate lyase system, as follows:
Malate + ATP + CoA = malyl CoA + ADP + P1 (malyl CoA synthase) (1) Malyl CoA = acetyl CoA + glyoxylate (malyl CoA lyase) (2) Net: Malate + ATP + CoA = acetyl CoA + glyoxylate + ADP + P1 (malate lyase) (3)
The malyl CoA lyase has been found in Pseudomonas AM1 (14) and the complete reaction has been demonstrated in Pseudomonas MA (1) and in bacterium 5H2 (2). In contrast to the results obtained with Pseudomonas AM1, when Pseudomonas MS is incubated with [4C ]methylamine the initial products of methylamine assimilation are two unusual amino acids, N-methylglutamate and y-glutamylmethylamide, whereas serine is formed much later (H. F. Kung, Ph.D. thesis, Vanderbilt Univ., Nashville, Tenn., 1969). These observations suggested that the serine pathway might not be operative for the assimilation of methylamine by Pseudomonas MS or that N-methylglutamate and 'y-glutamylmethylamide might be early products of methylamine metabolism unrelated to the main pathway of carbon assimilation. In a previous study, Wagner and Quayle (17) measured the specific activities of two key enzymes of the serine
905
906
WAGNER AND LEVITCH CH30H -
@GLYCINE ACETYL-COA
GLYOXYLATE
_
N2C0CH3-NH2
OH-PYRUVATE
MALATE --0OAA
CO2
A
I GLYCERATE
PEPR,'
defects in one of these mutants tend to support the operation of the serine pathway during growth of Pseudomonas MS on C1 substrates.
{C
SERINE
A
J. BACTROL.
V
® P-G>CERATE
C, + CO2 -C2
FiG. 1. Serine pathway. The enzymatic reactions indicated by the numbers are: 1, serine hydroxymethyltransferase; 2, serine-glyoxy late aminotransferase; 3, hydroxypyruvate reductase; 4, glycerate kinase; 5, several enzymatic steps; 6, phosphoenolpyruvate carboxylase; 7, malate dehydrogenase; 8, malate Iyase.
pathway in extracts of Pseudomonas MS grown on methylamine. One of these enzymes, hydroxypyruvate reductase, was found to be absent from Pseudomonas MS and it was concluded at that time that the serine pathway might not operate in Pseudomonas MS under these conditions. In this paper, we present the results of a more extensive study of a number of enzymes of the+ serine pathway in Pseudomonas MS. These data indicate that key enzymes of the serine pathway, including hydroxypyruvate reductase, are indeed present in this organism and are induced during growth on methylamine. During the course of this study, it was found that the pH optimum of hydroxypyruvate reductase measured in Pseudomonas MS is much higher than that for the same enzyme obtained from Pseudomonas AMi. This explains the failure of the earlier study to detect the presence of this enzyme in Pseudomonas MS. A corollary to the operation of the serine pathway is the net synthesis of C. units in the form of acetyl CoA. Therefore, one would expect the growth of Pseudomonas MS on methylamine to necessarily involve growth on acetate. Evidence is presented to show that growth of Pseudomonas MS on acetate involves the induction of isocitrate lyase, a key enzyme of the glyoxylate cycle, suggesting that this cycle is operative during growth of Pseudomonas MS on acetate. Furthermore, growth of Pseudomonas MS on methylamine results in the induction of isocitrate lyase as well as enzymes of the serine pathway. Mutants have also been isolated which have simultaneously lost the ability to grow on any C2 substrate or acetate. The specific enzymatic
MATERIALS AND METHODS Bacterial strains. Pseudomonas MS was originally isolated from soil and is maintained as described previously (8). All mutant strains were derived from Pseudomonas MS. Mutagenesis and isolation of mutant strains. The procedure was a modification of the method of Harder and Quayle (4). Pseudomonas MS was grown in 50 ml of methylamine medium to a final optical density (540 nm) of 0.5. Cells were harvested by centrifugation and washed twice with sterile saline. Cells were suspended in maleate buffer (pH 6.1) to an optical density of 1.5. An equal volume of N-methylN'-nitro-N-nitrosoguanidine (4 mg/ml) was added to the cell suspension, and the mixture was incubated at 30 C with shaking for 2 h. Cells were then centrifuged and washed twice with sterile saline. The washed cells were suspended in 20 ml of pyruvate medium, and incubated with shaking until growth occurred, as evidenced by an increase in optical density (about 50 h). The suspension was then centrifuged, and the cells were washed twice with saline and suspended in methylamine medium to an optical density of 0.2 to 0.4. Cells were incubated for 4 h, and a solution of penicillin G (10,000 U/ml) was added to a final concentration of 250 U/ml. The mixture was incubated for approximately 20 h. The mixture was centrifuged, and the cells were washed twice with saline and suspended in pyruvate medium. The penicillin selection procedure was repeated twice, with penicillin concentrations of 500 and 750 U/ml, respectively. The cells were centrifuged and washed twice with sterile saline, and serial 10-fold dilutions were made in sterile saline. Aliquots of 0.1 ml of the appropriate dilutions were plated on pyruvate agar medium and incubated until colonies appeared. The colonies were then replica plated on methylamine agar and pyruvate agar plates and incubated. Presumptive mutant colonies were picked, suspended in a drop of saline, and streaked onto fresh plates for single-colony isolation. Growth media. Liquid media consisted of an inorganic salts medium (6) to which had been added the designated growth substrate (pyruvate, methylamine, etc.) at a concentration of 0.3% (wt/vol). Solid media were as described for liquid media with the addition of 1.5% agar. Culture conditions. Stock cultures were carried on agar slants of the appropriate medium incubated at 30 C. Liquid cultures were incubated at 30 C on a rotary shaker. Growth was measured turbidimetrically in a Bausch & Lomb Spectronic 20 spectrophotometer at 540 nm. Growth measurements were made in test tubes of 13 mm diameter, or in flasks with side arms of the same diameter. Cells to be used for enzymatic assays were harvested in the logarithmic growth phase. Preparation of cell extracts. Cells were harvested by centrifugation and suspended in 2 volumes of cold 50 mM potassium phosphate buffer, pH 7.0 The cell
suspension was
907
ASSIMILATION OF C, UNITS
VOL. 122, 1975
sonically treated for five 30-s intervals
at 4 C. The unbroken cells and debris were removed by centrifugation at 32,000 g for 30 min at 4 C. The clear supernatant was passed through a Sephadex G-25 column to remove low-molecular-weight material. This extract was used for enzymatic assays.
TABLE 1. Mutant selection: growth response of isolates to various substrates
x
Induction of enzyme synthesis. Cells which had been grown in malate were centrifuged, washed twice with saline, and suspended in the growth medium containing the particular substrate to be used as an inducer. Cells were incubated overnight at 30 C with shaking. Mutant organisms were always tested after induction or growth to make certain they had not reverted. Enzyme assays. Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was assayed by the radioactive method of Salem et al. (15), except the concentration of magnesium chloride in the reaction mixture was increased from 0.5 to 3.0 mM. Isocitrate lyase (EC 4.1.3.1) was determined by the method of Dixon and Komberg (G. H. Dixon and H. L. Kornberg, Biochem. J. 72:30, 1959) with the following changes. The concentration of potassium phosphate buffer (pH 6.85) was increased to 100 mM, 2-mercaptoethanol was substituted for cysteine, and the concentration of isocitrate was increased to 4 mM. The volume of the reaction mixture was changed to 1.0 ml. Malate lyase was assayed by a modification of the method of Bellion and Hersh (1), with the following concentrations of reactants: malate, 40 mM; adenosine 5'-triphosphate, 20 mM; coenzyme A (CoA), 0.15 mM; phenylhydrazine, 2.5 mM; 2-mercaptoethanol, 6 mM. Hydroxypyruvate reductase (EC 1.1.1.29) was assayed by the method of Large and Quayle (11), adapted to a volume of 1 ml with pH 7.5 potassium phosphate buffer. Serine hydroxymethyltransferase (EC 2.1.2.1) was assayed by the method of Scrimegour and Huennekens (16). Malate synthase (EC 4.1.3.2) was assayed by the method of Dixon and Kornberg. Protein was determined by the method of Lowry et al. (12).
RESULTS Mutant production. Pseudomonas MS is a facultative methylotroph which grows on a number of C, compounds, as well as more complex organic compounds, such as pyruvate, malate, and glucose (8). In an attempt to define those reactions that are essential to Cl metabolism, mutants were selected which are unable to grow on methylamine. Mutants were produced by chemical mutagenesis, and a penicillin selection procedure was used, as described above. A total of 30 mutants were isolated. The growth response of isolates to various C, compounds is shown in Table 1. The mutants were classified as follows: type I lack the ability to grow on methylamine only; type II lack the ability to grow on any C1 compound tested; type III will not grow on N-methyl compounds, but will grow on S-methyl compounds. Because type II mutants lack a general ability to grow on C,
Substrate
Type I Type II Type III
Pyruvate ................. Methylamine ............. Dimethylamine ........... Trimethylamine .......... Trimethylamine oxide .... Trimethylsulfonium ......
+
Numbers ...............
5
+ + + +
+
+
-
-
-
+
6
20
compounds, a mutant from this group was investigated in more detail. Figure 2 shows the growth curves of both Pseudomonas MS wild type and mutant on pyruvate, methylamine, and acetate. The wild-type organism grows well on acetate and methylamine but the mutant exhibits no growth on either acetate or methylamine. Both the wild type and the mutant grow well on pyruvate. Mutants grown on pyruvate were tested for growth on methylamine, to insure that reversion was not occurring. Mutant organisms did exhibit some growth on methylamine and acetate after 96 h, and tests indicated that this growth was due to reversion. Enzyme studies. Four key enzymes of the serine pathway were assayed in both the wildtype and mutant organisms, grown on malate. Assays were performed also on malate-grown cells which were then incubated overnight with either methylamine or acetate. The results of such an experiment are shown in Table 2. In wild-type cells, the specific acitivity of each of the four enzymes of the serine pathway increases after incubation with methylamine, whereas there is no change in specific activity of any of the four enzymes after incubation with acetate. The mutant, however, exhibits striking changes in enzymatic activities. Serine hydroxymethyltransferase is not induced by methylamine, although uninduced (not exposed to methylamine) mutant and wild-type cells exhibit similar specific activities of this enzyme. Hydroxypyruvate reductase and phosphoenolpyruvate carboxylase, on the other hand, are both induced by methylamine, as in wild type; malate lyase (reaction 3) activity is essentially absent in the mutant under all conditions. The net result of the operation of one turn of the cyclic serine pathway is the condensation of a Cl unit (in the form of 5,10-methylene tetrahydrofolate) with carbon dioxide to form acetyl CoA. A consequence of the operation of the serine pathway in an organism growing on Cl compounds is that the organism must also contain the enzymatic apparatus for growth on
908
J. BACTERIOL.
WAGNER AND LEVITCH
organism and is essentially absent in the
acetate. Growth on acetate normally requires the operation of the glyoxylate cycle (7). Therefore, the two enzymes which are re-
The presence of hydroxypyruvate reductase in extracts of methylamine-grown Pseudomornas MS is in conflict with earlier studies which failed to detect any activity of this enzyme (17). The earlier studies were carried out at pH 4.5 by using the method of Large and Quayle (11) which was shown to be the pH optimum for this enzyme in Pseudomonas AML. Initial studies carried out at pH 7.5 during the present study indicated that substantial enzymatic activity was present. This reaction was then measured at various pH values (Fig. 3). This indicates that considerable activity for this enzyme from Pseudomonas MS is found at pH 6.3 and that no detectable activity was present at 4.7, thus resolving the apparent discrepancy with earlier work.
quired for operation of the glyoxylate cycle, malate synthase and isocitrate lyase, were assayed in wild-type and mutant cells. Growth and induction conditions were the same as for the cells used in the assays of the serine pathway enzymes. The results are shown in Table 3. Malate synthase appears to be a constitutive enzyme in both wild-type and mutant organisms. Isocitrate lyase is induced by either methylamine or acetate in the wild-type l.0
PYRUVATE
mu-
tant.
ACETATE
rMEHYLAMINE 0.5-
0
z
~I0.1 0
TIME (Hr)
FIG. 2. Growth of wild-type (0) and mutalit (A) Pseudomonas MS on various substrates.
DISCUSSION In those organisms growing on C1 compounds which utilize the serine pathway for assimilation, the first reaction is a transfer of a Cl unit from 5,10-methylenetetrahydrofolate to glycine. In order that Cl metabolism continue, the glycine must be regenerated. In Pseudomonas AM1, glyoxylate was found to be the precursor of glycine (13). Bellion and Hersh (1), working with Pseudomonas MA, demonstrated that glyoxylate was derived from malate and isocitrate, catalyzed by the respective lyases. Cox and Zatman (2) obtained similar results with another facultative methylotroph, bacterium 5H2. Oddly enough, Salem and co-workers (14) were unable to demonstrate the presence of malate lyase in Pseudomonas AML. Malyl CoA lyase is present in this organism, but malyl CoA synthase is apparently absent. In this study, we have demonstrated the presence of key enzymes of the serine pathway in wild-type Pseudomonas MS and that each of
TABLE 2. Activities of enzymes of the serine pathway' Activity'
Wild type
Enzyme
Serine-hydroxymethyltransferase Hydroxypyruvate reductase Phosphoenolpyruvate carboxylase Malate lyase
Mutant
Malate
Methylamine
Acetate
Malate
Methylamine
Acetate
21.7 48.3 1.17 1.00
93.3 800 167 5.17
16.0
28.3 21.7 0.33 0'
18.3 160 80 0'
16.0 16.7 0.50 0'
0.33 0.50
a Cells were grown in malate medium until log phase was reached, transferred to the indicated medium, and incubated an additional 24 h to permit induction to take place. 'All activities are expressed as nanomoles of products formed per minute per milligram of protein. 'Less than 0.14 nmol/min per milligram.
VOL. 122, 1975
909
ASSIMILATION OF C1 UNITS TABLE 3. Activities of enzymes of the glyoxylate cyclea Activityb Wild type
Enzyme Malate
Methylamine
Acetate
Malate
Methylamine
Acetate
31.7 75
48.3 81.7
23.3
20.0
46.7
0^
0'
55.0 0.065
Malate synthase ........... Isocitrate lyase .............
Mutant
a Cells were grown in malate medium until log phase was reached, transferred to the indicated medium, and incubated an additional 18 h to permit induction to take place. I All activities are expressed as nanomoles of product formed per minute per milligram of protein. ('Less than 0.03 nmol/min per milligram.
> 600-
'II 2
400-
N
200 -
4
5
6
8
7
9
10
pH
FIG. 3. Effect of pH on the activity of hydroxpyruuate reductase. Acetate buffer was used for the measurement at pH 4.7; phosphate buffers were used for measurement at pH 6.3 and 7.6; Tris buffers were used for measurements at pH 8.1 and 9.2. Control
experiments indicated that the absence of activity at pH 4.7 was not due to any inhibitory action of acetate. Enzyme activity is expressed as nanomoles per
minute per milligram of protein.
these activities is inducible by methylamine. We have also demonstrated that one of the enzymes involved in the production of glyoxylate, isocitrate lyase, is induced by acetate. A mutant of Pseudomonas MS which is unable to grow on C1 compounds or acetate has been studied which contains neither the isocitrate lyase nor the malate lyase. These results are consistent with a scheme for utilization of C1 units in which acetyl CoA derived from malate lyase activity is utilized in the glyoxylate cycle to produce metabolic intermediates (Fig. 4). This scheme employs the glyoxylate cycle for the assimilation of the acetate units which are derived in net fashion from the serine pathway. Bellion and Hersh (1) pr^-posed a modified scheme for the incorporation of the acetate units into cell material which they suggested would be appropriate for Pseudomonas MA. This latter scheme did not utilize malate synthesis for the regeneration of
Co units. When grown on C1 units, those organisms utilizing the serine pathway can regenerate C4 units for the synthesis of citrate by the carboxylation of phosphoenolpyruvate. When Pseudomonas MA was grown on methylamine, the activity of malate synthase was repressed to a level much lower than was present when grown on acetate or succinate (1), indicating a lack of participation of this enzyme in growth of Pseudomonas MA on C1 compounds. Malate synthase activity is not repressed, however, when Pseudomonas MS is grown on methylamine (Table 3) and its activity is at least equal to that of malate lyase. Whether or not malate synthase actually functions in the way it is written in Fig. 4 cannot be determined at this time. All that can be said is that the enzyme activity is present. One precaution to be aware of concerning the activity of malate synthase is that reversal of the malyl CoA lyase (reaction 2) followed by hydrolysis of malyl CoA could result in the formation of malate from glyoxylate and acetyl CoA, apparently as a result of malate synthase activity (14). That this is probably not the case in these studies is shown by the fact that malate synthase activity is roughly the same whether cells are grown on malate, methylamine, or acetate (Table 3), but malate lyase SUCCINATE
ISOCITRATE
(ACETATE CITRATE
GLYCINE
GLYOXALATE
MALATE OXALOACETATE CO PEP
C1
JOURNAL OF BACTERIOLOGY, June 1975, p. 905-910 Copyright 0 1975 American Society for Microbiology
Enzymes Involved in the Assimilation of One-Carbon Units by Pseudomonas MS CONRAD WAGNER* AND MARK E. LEVITCH Research Laboratory, Veterans Administration Hospital,* and the Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37203, and the Department of Microbiology, Meharry Medical College, Nashville, Tennessee 37208 Received for publication 26 February 1975
Pseudomonas MS can grow on methylamine and a number of other compounds containing Cl units as a sole source of carbon and energy. Assimilation of carbon into cell material occurs via the "serine pathway" since enzymes of this pathway are induced after growth on methylamine, but not malate or acetate. A mutant has been isolated which is unable to grow on methylamine or any other related substrate providing Cl units. This mutant is also unable to grow on acetate. Measurment of enzyme activities in cell-free extracts of wild-type cells showed that growth on methylamine caused induction of isocitrate lyase, a key enzyme in the glyoxylate cycle. The mutant organism lacks malate lyase, a key enzyme of the serine pathway, and isocitrate lyase as well. These results suggest that utilization of Cl units by Pseudomonas MS results in the net accumulation of acetate which is then assimilated into cell material via the glyoxylate cycle. Pseudomonas MS was isolated from soil by enrichment culture on trimethylsulfonium chloride (C. Wagner, Bacteriol. Proc., p. 91, 1964). The organism will also grow on methylamine, dimethylamine, trimethylamine, a number of sugars, and various C2, C,, and C, compounds (8). With respect to the types of reduced C, growth substrates, it is similar to a number of other facultative methylotrophs described in the literature (13). There is increasing evidence to indicate that many of these organisms assimilate C1 compounds into cell material by a pathway elucidated primarily by Quayle and his group (13). This is shown in Fig. 1 and is referred to as the serine pathway because incubation of ["4C]methanol or ["4C]methylamine with intact cells of Pseudomonas AM1 results in the appearance of the label in serine as the earliest identified product (9). This pathway has been vigorously proven in Pseudomonas AM1 by a variety of enzymatic and genetic techniques. The first reaction involves the hydroxymethylation of glycine by a C, folate derivative originating from methylamine or methanol to form serine. The C, intermediates of the serine pathway must be converted to C4 compounds for the synthesis of a number of cell constituents. In Pseudomonas AM1 a single enzyme, phosphoenolpyruvate carboxylase (10), is responsible for that conversion. Glycine is regenerated from glyoxylate, which is formed by two reactions comprising the malate lyase system, as follows:
Malate + ATP + CoA = malyl CoA + ADP + P1 (malyl CoA synthase) (1) Malyl CoA = acetyl CoA + glyoxylate (malyl CoA lyase) (2) Net: Malate + ATP + CoA = acetyl CoA + glyoxylate + ADP + P1 (malate lyase) (3)
The malyl CoA lyase has been found in Pseudomonas AM1 (14) and the complete reaction has been demonstrated in Pseudomonas MA (1) and in bacterium 5H2 (2). In contrast to the results obtained with Pseudomonas AM1, when Pseudomonas MS is incubated with [4C ]methylamine the initial products of methylamine assimilation are two unusual amino acids, N-methylglutamate and y-glutamylmethylamide, whereas serine is formed much later (H. F. Kung, Ph.D. thesis, Vanderbilt Univ., Nashville, Tenn., 1969). These observations suggested that the serine pathway might not be operative for the assimilation of methylamine by Pseudomonas MS or that N-methylglutamate and 'y-glutamylmethylamide might be early products of methylamine metabolism unrelated to the main pathway of carbon assimilation. In a previous study, Wagner and Quayle (17) measured the specific activities of two key enzymes of the serine
905
906
WAGNER AND LEVITCH CH30H -
@GLYCINE ACETYL-COA
GLYOXYLATE
_
N2C0CH3-NH2
OH-PYRUVATE
MALATE --0OAA
CO2
A
I GLYCERATE
PEPR,'
defects in one of these mutants tend to support the operation of the serine pathway during growth of Pseudomonas MS on C1 substrates.
{C
SERINE
A
J. BACTROL.
V
® P-G>CERATE
C, + CO2 -C2
FiG. 1. Serine pathway. The enzymatic reactions indicated by the numbers are: 1, serine hydroxymethyltransferase; 2, serine-glyoxy late aminotransferase; 3, hydroxypyruvate reductase; 4, glycerate kinase; 5, several enzymatic steps; 6, phosphoenolpyruvate carboxylase; 7, malate dehydrogenase; 8, malate Iyase.
pathway in extracts of Pseudomonas MS grown on methylamine. One of these enzymes, hydroxypyruvate reductase, was found to be absent from Pseudomonas MS and it was concluded at that time that the serine pathway might not operate in Pseudomonas MS under these conditions. In this paper, we present the results of a more extensive study of a number of enzymes of the+ serine pathway in Pseudomonas MS. These data indicate that key enzymes of the serine pathway, including hydroxypyruvate reductase, are indeed present in this organism and are induced during growth on methylamine. During the course of this study, it was found that the pH optimum of hydroxypyruvate reductase measured in Pseudomonas MS is much higher than that for the same enzyme obtained from Pseudomonas AMi. This explains the failure of the earlier study to detect the presence of this enzyme in Pseudomonas MS. A corollary to the operation of the serine pathway is the net synthesis of C. units in the form of acetyl CoA. Therefore, one would expect the growth of Pseudomonas MS on methylamine to necessarily involve growth on acetate. Evidence is presented to show that growth of Pseudomonas MS on acetate involves the induction of isocitrate lyase, a key enzyme of the glyoxylate cycle, suggesting that this cycle is operative during growth of Pseudomonas MS on acetate. Furthermore, growth of Pseudomonas MS on methylamine results in the induction of isocitrate lyase as well as enzymes of the serine pathway. Mutants have also been isolated which have simultaneously lost the ability to grow on any C2 substrate or acetate. The specific enzymatic
MATERIALS AND METHODS Bacterial strains. Pseudomonas MS was originally isolated from soil and is maintained as described previously (8). All mutant strains were derived from Pseudomonas MS. Mutagenesis and isolation of mutant strains. The procedure was a modification of the method of Harder and Quayle (4). Pseudomonas MS was grown in 50 ml of methylamine medium to a final optical density (540 nm) of 0.5. Cells were harvested by centrifugation and washed twice with sterile saline. Cells were suspended in maleate buffer (pH 6.1) to an optical density of 1.5. An equal volume of N-methylN'-nitro-N-nitrosoguanidine (4 mg/ml) was added to the cell suspension, and the mixture was incubated at 30 C with shaking for 2 h. Cells were then centrifuged and washed twice with sterile saline. The washed cells were suspended in 20 ml of pyruvate medium, and incubated with shaking until growth occurred, as evidenced by an increase in optical density (about 50 h). The suspension was then centrifuged, and the cells were washed twice with saline and suspended in methylamine medium to an optical density of 0.2 to 0.4. Cells were incubated for 4 h, and a solution of penicillin G (10,000 U/ml) was added to a final concentration of 250 U/ml. The mixture was incubated for approximately 20 h. The mixture was centrifuged, and the cells were washed twice with saline and suspended in pyruvate medium. The penicillin selection procedure was repeated twice, with penicillin concentrations of 500 and 750 U/ml, respectively. The cells were centrifuged and washed twice with sterile saline, and serial 10-fold dilutions were made in sterile saline. Aliquots of 0.1 ml of the appropriate dilutions were plated on pyruvate agar medium and incubated until colonies appeared. The colonies were then replica plated on methylamine agar and pyruvate agar plates and incubated. Presumptive mutant colonies were picked, suspended in a drop of saline, and streaked onto fresh plates for single-colony isolation. Growth media. Liquid media consisted of an inorganic salts medium (6) to which had been added the designated growth substrate (pyruvate, methylamine, etc.) at a concentration of 0.3% (wt/vol). Solid media were as described for liquid media with the addition of 1.5% agar. Culture conditions. Stock cultures were carried on agar slants of the appropriate medium incubated at 30 C. Liquid cultures were incubated at 30 C on a rotary shaker. Growth was measured turbidimetrically in a Bausch & Lomb Spectronic 20 spectrophotometer at 540 nm. Growth measurements were made in test tubes of 13 mm diameter, or in flasks with side arms of the same diameter. Cells to be used for enzymatic assays were harvested in the logarithmic growth phase. Preparation of cell extracts. Cells were harvested by centrifugation and suspended in 2 volumes of cold 50 mM potassium phosphate buffer, pH 7.0 The cell
suspension was
907
ASSIMILATION OF C, UNITS
VOL. 122, 1975
sonically treated for five 30-s intervals
at 4 C. The unbroken cells and debris were removed by centrifugation at 32,000 g for 30 min at 4 C. The clear supernatant was passed through a Sephadex G-25 column to remove low-molecular-weight material. This extract was used for enzymatic assays.
TABLE 1. Mutant selection: growth response of isolates to various substrates
x
Induction of enzyme synthesis. Cells which had been grown in malate were centrifuged, washed twice with saline, and suspended in the growth medium containing the particular substrate to be used as an inducer. Cells were incubated overnight at 30 C with shaking. Mutant organisms were always tested after induction or growth to make certain they had not reverted. Enzyme assays. Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was assayed by the radioactive method of Salem et al. (15), except the concentration of magnesium chloride in the reaction mixture was increased from 0.5 to 3.0 mM. Isocitrate lyase (EC 4.1.3.1) was determined by the method of Dixon and Komberg (G. H. Dixon and H. L. Kornberg, Biochem. J. 72:30, 1959) with the following changes. The concentration of potassium phosphate buffer (pH 6.85) was increased to 100 mM, 2-mercaptoethanol was substituted for cysteine, and the concentration of isocitrate was increased to 4 mM. The volume of the reaction mixture was changed to 1.0 ml. Malate lyase was assayed by a modification of the method of Bellion and Hersh (1), with the following concentrations of reactants: malate, 40 mM; adenosine 5'-triphosphate, 20 mM; coenzyme A (CoA), 0.15 mM; phenylhydrazine, 2.5 mM; 2-mercaptoethanol, 6 mM. Hydroxypyruvate reductase (EC 1.1.1.29) was assayed by the method of Large and Quayle (11), adapted to a volume of 1 ml with pH 7.5 potassium phosphate buffer. Serine hydroxymethyltransferase (EC 2.1.2.1) was assayed by the method of Scrimegour and Huennekens (16). Malate synthase (EC 4.1.3.2) was assayed by the method of Dixon and Kornberg. Protein was determined by the method of Lowry et al. (12).
RESULTS Mutant production. Pseudomonas MS is a facultative methylotroph which grows on a number of C, compounds, as well as more complex organic compounds, such as pyruvate, malate, and glucose (8). In an attempt to define those reactions that are essential to Cl metabolism, mutants were selected which are unable to grow on methylamine. Mutants were produced by chemical mutagenesis, and a penicillin selection procedure was used, as described above. A total of 30 mutants were isolated. The growth response of isolates to various C, compounds is shown in Table 1. The mutants were classified as follows: type I lack the ability to grow on methylamine only; type II lack the ability to grow on any C1 compound tested; type III will not grow on N-methyl compounds, but will grow on S-methyl compounds. Because type II mutants lack a general ability to grow on C,
Substrate
Type I Type II Type III
Pyruvate ................. Methylamine ............. Dimethylamine ........... Trimethylamine .......... Trimethylamine oxide .... Trimethylsulfonium ......
+
Numbers ...............
5
+ + + +
+
+
-
-
-
+
6
20
compounds, a mutant from this group was investigated in more detail. Figure 2 shows the growth curves of both Pseudomonas MS wild type and mutant on pyruvate, methylamine, and acetate. The wild-type organism grows well on acetate and methylamine but the mutant exhibits no growth on either acetate or methylamine. Both the wild type and the mutant grow well on pyruvate. Mutants grown on pyruvate were tested for growth on methylamine, to insure that reversion was not occurring. Mutant organisms did exhibit some growth on methylamine and acetate after 96 h, and tests indicated that this growth was due to reversion. Enzyme studies. Four key enzymes of the serine pathway were assayed in both the wildtype and mutant organisms, grown on malate. Assays were performed also on malate-grown cells which were then incubated overnight with either methylamine or acetate. The results of such an experiment are shown in Table 2. In wild-type cells, the specific acitivity of each of the four enzymes of the serine pathway increases after incubation with methylamine, whereas there is no change in specific activity of any of the four enzymes after incubation with acetate. The mutant, however, exhibits striking changes in enzymatic activities. Serine hydroxymethyltransferase is not induced by methylamine, although uninduced (not exposed to methylamine) mutant and wild-type cells exhibit similar specific activities of this enzyme. Hydroxypyruvate reductase and phosphoenolpyruvate carboxylase, on the other hand, are both induced by methylamine, as in wild type; malate lyase (reaction 3) activity is essentially absent in the mutant under all conditions. The net result of the operation of one turn of the cyclic serine pathway is the condensation of a Cl unit (in the form of 5,10-methylene tetrahydrofolate) with carbon dioxide to form acetyl CoA. A consequence of the operation of the serine pathway in an organism growing on Cl compounds is that the organism must also contain the enzymatic apparatus for growth on
908
J. BACTERIOL.
WAGNER AND LEVITCH
organism and is essentially absent in the
acetate. Growth on acetate normally requires the operation of the glyoxylate cycle (7). Therefore, the two enzymes which are re-
The presence of hydroxypyruvate reductase in extracts of methylamine-grown Pseudomornas MS is in conflict with earlier studies which failed to detect any activity of this enzyme (17). The earlier studies were carried out at pH 4.5 by using the method of Large and Quayle (11) which was shown to be the pH optimum for this enzyme in Pseudomonas AML. Initial studies carried out at pH 7.5 during the present study indicated that substantial enzymatic activity was present. This reaction was then measured at various pH values (Fig. 3). This indicates that considerable activity for this enzyme from Pseudomonas MS is found at pH 6.3 and that no detectable activity was present at 4.7, thus resolving the apparent discrepancy with earlier work.
quired for operation of the glyoxylate cycle, malate synthase and isocitrate lyase, were assayed in wild-type and mutant cells. Growth and induction conditions were the same as for the cells used in the assays of the serine pathway enzymes. The results are shown in Table 3. Malate synthase appears to be a constitutive enzyme in both wild-type and mutant organisms. Isocitrate lyase is induced by either methylamine or acetate in the wild-type l.0
PYRUVATE
mu-
tant.
ACETATE
rMEHYLAMINE 0.5-
0
z
~I0.1 0
TIME (Hr)
FIG. 2. Growth of wild-type (0) and mutalit (A) Pseudomonas MS on various substrates.
DISCUSSION In those organisms growing on C1 compounds which utilize the serine pathway for assimilation, the first reaction is a transfer of a Cl unit from 5,10-methylenetetrahydrofolate to glycine. In order that Cl metabolism continue, the glycine must be regenerated. In Pseudomonas AM1, glyoxylate was found to be the precursor of glycine (13). Bellion and Hersh (1), working with Pseudomonas MA, demonstrated that glyoxylate was derived from malate and isocitrate, catalyzed by the respective lyases. Cox and Zatman (2) obtained similar results with another facultative methylotroph, bacterium 5H2. Oddly enough, Salem and co-workers (14) were unable to demonstrate the presence of malate lyase in Pseudomonas AML. Malyl CoA lyase is present in this organism, but malyl CoA synthase is apparently absent. In this study, we have demonstrated the presence of key enzymes of the serine pathway in wild-type Pseudomonas MS and that each of
TABLE 2. Activities of enzymes of the serine pathway' Activity'
Wild type
Enzyme
Serine-hydroxymethyltransferase Hydroxypyruvate reductase Phosphoenolpyruvate carboxylase Malate lyase
Mutant
Malate
Methylamine
Acetate
Malate
Methylamine
Acetate
21.7 48.3 1.17 1.00
93.3 800 167 5.17
16.0
28.3 21.7 0.33 0'
18.3 160 80 0'
16.0 16.7 0.50 0'
0.33 0.50
a Cells were grown in malate medium until log phase was reached, transferred to the indicated medium, and incubated an additional 24 h to permit induction to take place. 'All activities are expressed as nanomoles of products formed per minute per milligram of protein. 'Less than 0.14 nmol/min per milligram.
VOL. 122, 1975
909
ASSIMILATION OF C1 UNITS TABLE 3. Activities of enzymes of the glyoxylate cyclea Activityb Wild type
Enzyme Malate
Methylamine
Acetate
Malate
Methylamine
Acetate
31.7 75
48.3 81.7
23.3
20.0
46.7
0^
0'
55.0 0.065
Malate synthase ........... Isocitrate lyase .............
Mutant
a Cells were grown in malate medium until log phase was reached, transferred to the indicated medium, and incubated an additional 18 h to permit induction to take place. I All activities are expressed as nanomoles of product formed per minute per milligram of protein. ('Less than 0.03 nmol/min per milligram.
> 600-
'II 2
400-
N
200 -
4
5
6
8
7
9
10
pH
FIG. 3. Effect of pH on the activity of hydroxpyruuate reductase. Acetate buffer was used for the measurement at pH 4.7; phosphate buffers were used for measurement at pH 6.3 and 7.6; Tris buffers were used for measurements at pH 8.1 and 9.2. Control
experiments indicated that the absence of activity at pH 4.7 was not due to any inhibitory action of acetate. Enzyme activity is expressed as nanomoles per
minute per milligram of protein.
these activities is inducible by methylamine. We have also demonstrated that one of the enzymes involved in the production of glyoxylate, isocitrate lyase, is induced by acetate. A mutant of Pseudomonas MS which is unable to grow on C1 compounds or acetate has been studied which contains neither the isocitrate lyase nor the malate lyase. These results are consistent with a scheme for utilization of C1 units in which acetyl CoA derived from malate lyase activity is utilized in the glyoxylate cycle to produce metabolic intermediates (Fig. 4). This scheme employs the glyoxylate cycle for the assimilation of the acetate units which are derived in net fashion from the serine pathway. Bellion and Hersh (1) pr^-posed a modified scheme for the incorporation of the acetate units into cell material which they suggested would be appropriate for Pseudomonas MA. This latter scheme did not utilize malate synthesis for the regeneration of
Co units. When grown on C1 units, those organisms utilizing the serine pathway can regenerate C4 units for the synthesis of citrate by the carboxylation of phosphoenolpyruvate. When Pseudomonas MA was grown on methylamine, the activity of malate synthase was repressed to a level much lower than was present when grown on acetate or succinate (1), indicating a lack of participation of this enzyme in growth of Pseudomonas MA on C1 compounds. Malate synthase activity is not repressed, however, when Pseudomonas MS is grown on methylamine (Table 3) and its activity is at least equal to that of malate lyase. Whether or not malate synthase actually functions in the way it is written in Fig. 4 cannot be determined at this time. All that can be said is that the enzyme activity is present. One precaution to be aware of concerning the activity of malate synthase is that reversal of the malyl CoA lyase (reaction 2) followed by hydrolysis of malyl CoA could result in the formation of malate from glyoxylate and acetyl CoA, apparently as a result of malate synthase activity (14). That this is probably not the case in these studies is shown by the fact that malate synthase activity is roughly the same whether cells are grown on malate, methylamine, or acetate (Table 3), but malate lyase SUCCINATE
ISOCITRATE
(ACETATE CITRATE
GLYCINE
GLYOXALATE
MALATE OXALOACETATE CO PEP
C1
