Vol. 127, No. 2 Printed in U.S.A.
JoURNAL oF BACTERIOLOGY, August 1976, p. 731-738 Copyright 0 1976 American Society for Microbiology
Hydrogenase and Ribulose Diphosphate Carboxylase During Autotrophic, Heterotrophic, and Mixotrophic Growth of Scotochromogenic Mycobacteria SANG S. PARK Arm B. T. DzCICCO* Department ofBiology, The Catholic University of America, Washington, D.C. 20064
Received for publication 27 February 1976
Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence ofcarbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.
Chemolithotrophy among the mycobacteria has been neglected compared with that in other hydrogen autotrophs (15, 21, 24). In 1954, Belajewa reported the autotrophic growth of Mycobacterium album (2) under an atmosphere of hydrogen, oxygen, and carbon dioxide. Four years later Dworkin and Foster (8) reported the isolation from soil of four mycobacterial strains that grew in a mineral salts medium under an atmosphere of 50% hydrogen, 10% carbon dioxide, and 40% air. In 1963 Lukins and Foster (14) reported that some known authentic species (M. fortuitum, M. marinum, and M. smegmatis) as well as a newly isolated Mycobacterium sp. grew autotrophically with hydrogen. They found the hydrogenase activity to reside in the clear supernatant fluid from a cell extract. One stock strain of M. phlei also has been reported to grow under autotrophic conditions (11). Most reports on hydrogen autotrophy in mycobacteria have involved rapidly growing strains. Upon the isolation of a slow-growing autotrophic Mycobacterium, we investigated the chemolithotrophic growth capacity of a number of mycobacteria, as previously reported (18). Among 34 mycobacterial strains tested, a distinctive autotrophic capacity was observed only with the scotochromogenic saprophytic species M. gordonae and two strains of an unclassified saprophytic scotochromogen. Eight 731
strains of the potentially pathogenic scotochromogen M. scrofulaceum were unable to grow autotrophically, suggesting that the capacity to grow autotrophically with hydrogen may be significant for differentiating the saprophytic and pathogenic scotochromogens. Although ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) is responsible for the assimilation of CO2 into RuDP in autotrophic organisms, studies of this enzyme in autotrophic mycobacteria have not been previously reported. The present report describes the activities of the RuDP carboxylase and hydrogenase systems during growth of several scotochromogenic mycobacteria under a number of different cultural conditions. MATERIALS AND METHODS Bacterial strains. The four mycobacterial strains employed in the present study were described and partially characterized in a previous report (18). M. gordonae E02 and Mycobacterium strains AU and 6Y grow autotrophically with hydrogen as the sole energy source; M. scrofulaceum E03 does not grow autotrophically. Chemolithotrophic cultivation. The medium employed for chemolithotrophic growth consisted of 0.1% (NH4)2SO4, 0.02% MgSO4-7H40, and 0.001% Fe(NH4)2(S04)2 * 6H20 in 0.04 M potassium phosphate buffer, pH 6.7. Erlenmeyer flasks (250-ml) containing 100 ml of mineral salts medium were
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inoculated and incubated under an atmosphere of either 50% H2, 10% CO2, 20% 02, and 20% N2 or 50% H2, 10% C02, and 40% air, as indicated. The gas mixture was continuously supplied to the culture flasks through a manifold from a gas reservoir and was maintained at slightly greater than atmospheric pressure using a water displacement system. Heterotrophic and mixotrophic cultivation. For heterotrophic growth in liquid media, either mineral salts medium containing a single organic substrate or Middlebrook 7H9 broth (Difco) enriched with oleic acid, bovine albumin fraction V, 1-glucose, beef catalase, sodium chloride (OADC, Difco), and 0.2% glycerol (Difco) was employed. Cells from Middlebrook 7H10 agar slants (enriched with glycerol and OADC as described by Difco) were inoculated into enriched 7H9 broth and grown for 6 to 10 days at the specified temperature with shaking. The cultures were harvested, washed, and resuspended in mineral salts medium and used as inocula. Cells were inoculated into 100 ml of either 7H9 broth or mineral salts solution containing 0.3% of the specified substrate in 250-ml Erlenmeyer flasks. The flasks were shaken at 300C under a gas mixture of 20% 02 and 80% N2 for heterotrophic growth and either 50% H2, 10% C02, and 40% air or 50% H2, 20% 02, 10% C02, and 20% N2 for mixotrophic growth. For determination of the specific effects of carbon dioxide and hydrogen, gas mixtures of 20% 02, 10% CO2, and 70% N2 or 50% H2, 20% O.,, and 30% N2, respectively, were used. The initial optical density at 540 nm (OD"O) was 0.01 to 0.05; however, for each set of experiments each flask had the same initial cell turbidity and incubation was at the same shaker speed and the same temperature (3000), unless otherwise indicated. Growth was monitored by following the increase in ODH,o using a Spectronic20 colorimeter (Bausch & Lomb). Hydrogenase assay. Hydrogenase levels were determined by the standard Warburg technique (28) at 2800, using an Aminco Warburg apparatus (American Instrument Co.). Cells grown under different cultural conditions were harvested by centrifugaton, washed, and resuspended in mineral salts medium. Each vessel contained 2.0 ml of the same mineral medium and 1.0 ml of cell suspension of OD 1.0. The Warburg vessels were flushed with a gas mixture containing 90% H2 and 10% 02 or 90% N2 and 10% 02 and allowed to equilibrate for 10 min. The amount of gas consumed by the cells under the H2 and 02 atmosphere was corrected for the endogenous level and was used as an indication of hydrogenase activity. Hydrogenase activity (specific activity) in expressed as microliters of hydrogen consumed per milligram (dry weight) of cells per hour. An ODs.0 of 0.3 corresponded to a cellular dry weight of 0.13 mg/ml for heterotrophically grown cells and 0.18 mg/ml for autotrophically grown cells of strains AU and 6Y, 0.17 mg/ml for heterotrophically grown cells and 0.27 mg/ml for autotrophically grown cells of M. gordonae E02, and 0.3 mg/ml for
heterotrophically grown cells of M. scrofulaceum.
The cellular dry weight determinations for heterotrophically grown cells were obtained with cells grown in 7H9 broth. Cellular dry weights were mea-
J. BACTERIOL.
sured in aluminum cups after the cells were washed with deionized water and dried at 80 to 8500 for 16 to 18 h. The disparity in the dry weight values of heterotrophic and autotrophic cultures was probably due to the granular nature of the autotrophic cultures, which produced a higher cell mass per OD unit than the smooth heterotrophic cells. RuDP carboxylase assay. The method employed for the RuDP carboxylase assay has been described previously (27). A unit of enzyme activity is defined as the amount catalyzing the formation of 1 nmol of 3-phosphoglyceric acid per min. Specific activity is defined as units per milligram of protein. Protein was determined by the biuret method (9).
RESULTS Cultures of M. gordonae E02 and strains AU and 6Y grown under chemolithotrophic conditions consumed large quantities of hydrogen when assayed manometrically. Rates of hydrogen consumption for the three strains varied from approximately 88 to 135 ,ul of H2/mg (dry weight) per h at 280C (Table 1). Cells grown heterotrophically in 7H9 broth (under 80%o N2 and 20% 02) showed low hydrogenase levels, varying from 2 to 5% of the autotrophic levels. Endogenous levels of oxygen consumption were low, ranging from 2 to 8 ,ul/mg (dry weight) per h. When cultures of M. gordonae E02 and strain AU were grown mixotrophically in 7H9 broth under a gas mixture of 50% H2, 40% air, and 10% C02, they exhibited hydrogenase levels approximately 83 and 66%, respectively, of the autotrophic cells. Strain 6Y showed relatively lower hydrogenase levels when grown mixotrophically, supporting earlier observations that this strain prefers heterotrophic growth conditions (18). RuDP carboxylase levels were influenced to a greater extent by the presence of organic substrates than were hydrogenase; mixotrophic levels of RuDP carboxylase were 10 to 26% of those present in autotrophic cultures. RuDP carboxylase levels were very low in heterotrophic cultures. Since M. scrofulaceum cannot grow autotrophically, hydrogenase and RuDP carboxylase assays were performed with cells incubated both heterotrophically and mixotrophically. No significant activity was detected for either enzyme with cells incubated under either set of conditions, indicating that the inability of this strain of M. scrofulaceum to grow autotrophically is due to a deficiency of both autotrophic enzyme systems. To study the inducibility of the hydrogenase and RuDP carboxylase systems, flasks containing mineral salts medium were inoculated with M. gordonae cells that had been grown heterotrophically in 7H9 broth for 6 days at 300C,
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TABLE 1. Hydrogenase and RuDP carboxylase levels of scotochromogenic mycobacteria grown heterotrophically and mixotrophically in 7H9 broth or chemolithotrophically with hydrogen Strain
Growth condition"
Incubation periodb
Hdoeae
ODsdo (sp act) (days)(sac) 6.1 0.50 6 0.69 89.3 6 135.0 28 0.82 6 1.53 4.5 6 1.80 18.7 97.9 50 1.10 1.4 7 3.00 73.2 2.80 7 88.5 0.31 21 0.1 6 0.47 0.51 0.0 6
abx acta ylase (sp yae(pct
RD
0.21 Heterotrophic 0.57 Mixotrophic 5.70 Chemolithotrophic 0.10 Heterotrophic 6Y 0.50 Mixotrophic 3.50 Chemolithotrophic 0.00 M. gordonae E02 Heterotrophic 0.76 Mixotrophic 2.89 Chemolithotrophic 0.00 Heterotrophic M. scrofulaceum 0.00 Mixotrophic E03 a For a description of heterotrophic, mixotrophic, and chemolithotrophic growth conditions, see the text. b Incubation was at room temperature (26 + 1°C). AU
harvested, washed, and suspended in mineral salts medium. The culture flasks were incubated at 300C with shaking under an H., O.,, CO2, and N2 atmosphere. As shown in Fig. 1, the very low levels of hydrogenase and RuDP carboxylase that were present in the heterotrophically grown inoculum increased rapidly during incubation under autotrophic conditions and remained high throughout the exponential growth phase. Specific activities of both enzymes declined rapidly during the stationary growth phase. Increased shaker speed lengthened the period of exponential growth and the corresponding period of high enzyme levels, as would be expected from the growth stimulation previously observed with increased oxygen levels (18), but the ratio of the specific activities of the two enzymes remained relatively constant throughout the different phases of autotrophic growth. Since the studied autotrophic mycobacteria exhibited regulation of the hydrogenase and RuDP carboxylase systems, it seemed desirable to observe the interaction of autotrophic and heterotrophic systems in one of these strains for comparison with other extensively studied hydrogen autotrophs (21, 24, 25). Thus, M. gordonae was inoculated into mineral salts medium containing selected individual organic substrates and incubated under various gas mixtures. When M. gordonae was grown mixotrophically with 0.3% glycerol under a gas mixture of H2, 02, C02, and N2, a dramatic increase in both growth rate and yield was observed as compared with autotrophic and heterotrophic incubation (Fig. 2). The increased growth rate and yield observed during mixotrophic growth were not due to C02 alone since the addition of C02 to the 02 and N2 gas mixture did not signif-
1.0-
6~ 75
m~~~~~~~2 2
E
0 ~~~~~~o
0 o
2
S
4
6
8 DAYS
10
12
14
FIG. 1. Hydrogenase and RuDP carboxylase specific activities during chemolithot rophic growth of M. gordonae E02.
icantly enhance growth. When hydrogen was added to the heterotrophic atmosphere, growth of M. gordonae was drastically inhibited. Since gas mixtures of 02-N., 09-N2-002, or H2 02-C02-N2 did not inhibit growth, the presence of hydrogen seemed to be essential for the inhibitory effect. When an autotrophically grown inoculum was used in similar experiments, the results were essentially the same (data not shown). To determine whether preadapted cells were inhibited by hydrogen, six flasks of mineral salts-glycerol medium were inoculated with M. gordonae (grown in 7H9 broth) and incubated under an atmosphere of 02 and N., After 7 days
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J. BACTERIOL.
E
0
0
2
4
6
10
8
12
14
16
DAYS
FIG. 2. Chemolithotrophic, heterotrophic, and mixotrophic growth of M. gordonae E02. Heterotrophic and mixotrophic incubations were in 0.3% glycerol-salts medium. Heterotrophic incubation was under gas atmospheres of either 20% 02 and 80% N2
(0) or 20% 02, 10% C02, and 70% N2 (A)- Chemolithotrophic (A) and mixotrophic (0) incubations were
under 50% H2, 20%
02,
10% CO., and 20% N2.
the atmosphere in two flasks was changed to H2, O,, and N2. Two other flasks were placed under H2, 02, C02, and N., whereas the last two flasks remained under the heterotrophic atmosphere of 02 and N.,. As shown in Fig. 3, growth in the presence of H2, O2, and N., continued at a rapid rate for 24 h, after which growth slowed to a very low rate similar to that observed previously. Growth with the other two gas mixtures was exponential and reached similar densities, but the mixotrophic culture grew much faster. Hydrogenase and RuDP carboxylase levels of M. gordonae cells grown in glycerol medium under the different gas mixtures are shown in Table 2. As previously observed, the levels of both enzymes were very low in cells grown under heterotrophic conditions (02-N2). It is evident that the addition of CO2 in the absence of H2 did not stimulate the production of high levels of either of the autotrophic enzyme systems, although moderate levels of carboxylase were consistently present for a brief time in glycerol cultures exposed to the 09-COo-N2 atmosphere (data not shown). Similarly, the presence of H2 without C02 did not stimulate high levels of both enzymes although, at 19 days, moderate levels of hydrogenase, compara-
ble to mixotrophic cultures, were present under an atmosphere of H2, 02, and N2. Hydrogenase and RuDP carboxylase levels increased during exponential mixotrophic growth and then declined with decreasing growth rates. The relationship between enzyme levels and growth closely paralleled that shown with autotrophic cultures in Fig. 1, except that the RuDP carboxylase levels in mixotrophic cultures were consistently much lower. When mineral salts media containing sodium acetate, sodium pyruvate, glucose, or fructose were incubated under different gas mixtures, increased growth rates and yields were noted with all substrates under mixotrophic conditions. The growth curves observed with acetate and pyruvate media closely resembled those observed with glycerol (Fig. 2). With glucose and fructose, the stimulatory effect of the autotrophic atmosphere was even more apparent because no growth occurred under heterotrophic conditions (with or without CO2) during the times indicated (Fig. 4). Heterotrophic growth did commence with glucose and fructose after several weeks of incubation. RuDP carboxylase levels of cells grown under mixotrophic conditions with glycerol, glucose, sodium acetate, sodium pyruvate, or fructose were relatively low compared with those of autotrophically grown cells (Table 2) and were similar to those observed after mixotrophic growth in 7H9 broth (Table 1). RuDP carboxylase levels were insignificant after heterotrophic growth with all substrates (Table 2). During mixotrophic incubation, hydrogenase levels remained comparatively high; in fact, M. gordonae cells grown mixotrophically with acetate, pyruvate, or fructose contained levels of hydrogenase that significantly exceeded the levels present in autotrophically grown cells (Table 2), which were never found to exceed a specific activity of 107. In contrast with the studies with single organic substrates, the growth curves obtained when M. gordonae was grown heterotrophically and mixotrophically in 7H9 broth were almost identical; however, the levels of hydrogenase and RuDP carboxylase were much higher in cells incubated mixotrophically (Tables 1 and 2). Since hydrogenase and RuDP carboxylase levels were lower in mixotrophically and heterotrophically grown cells as compared with autotrophically grown cells, it was possible that a form of catabolic repression, reversible with cyclic adenosine monophosphate (cAMP), might be operative (19). To determine this, M. gordonae was grown mixotrophically with glyc-
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HYDROGENASE AND CARBOXYLASE IN MYCOBACTERIA
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10.0
1.0 E 0
0. 1 _
t
I
0
2
4
FIG. 3. Inhibitory effect of hydrogen
6 on
8
I
I
10 12 14 16 18 20 22 DAYS
the heterotrophic growth of M. gordonae E02 with glycerol. Three
sets of cultures were grown in 0.3% glycerol-salts medium under an atmosphere of20% O., and 80% N2. At the indicated time the atmosphere in one set was changed to 50% H., 20% O2, 10% CO. and 20% N., (A) and in the second set it was changed to 50% H2, 20% 02, and 30% N2 (0). The third set remained under 02 and N2 (@) .
TABLE 2. Hydrogenase and RuDP carboxylase levels in M. gordonae E02 grown with various substrates and gas atmospheres IncubaRuDP carOrganic subHydrogen- boxlas Incubation condition Atmosphere strate ase (sp act) bosyase) riod (pat (days) None Chemolithotrophic 12 H,-0{,-C0,-N.,N 0.23 75.3 2.49 02 N.b Glycerol Heterotrophic 14 0.21 3.7 0 16 0.84 1.7 0.01 14 0.25 10.6 0 O.,-CO2-N2,' 19 0.37 46.7 0.10 30 0.31 5.3 0 0 Mixotrophic H2-02--C02-N2 0.03 4.0 0.1 8 0.67 62.3 0.65 14 1.68 37.4 0.53 Glucose Heterotrophic 02-N2 32 0.04 Nae NA 32 NA 0.03 NA 02-C02-N2 12 0.42 54.7 H2-,0---C02- N2 0.25 Mixotrophic Acetate Heterotrophic 0.,-N., 12 0.29 8.1 0 Mixotrophic 12 H2-02--C02,-N., 0.40 175.5 0.55 02- N2 Pyruvate Heterotrophic 24 0.44 3.3 0 Mixotrophic H2-02--C02-N2 12 0.54 122.4 0.76 Fructose Heterotrophic 02-N2 12 0.02 NA NA Mixotrophic H2,-0--C02,-N2 12 0.20 145.7 0.54 7H9 broth Heterotrophic 0.,-CO2-N2 7 3.07 4.4 0 a 50% H.,-20% O.,-10% CO.,-20% N.,. b 20% 02-80% N2. 20% 0°2-10% CO,-70% N2. (I 50%o H2-20% 02-30% N2. "NA, Not assayed due to lack of growth under these conditions.
tionpe
erol in the presence of cAMP at concentrations of 5 x 10-4 M, 5 x 10-5M, and 5 x 10-6 M. At a concentration of 5 x 10-4 M cAMP, a transient inhibition of growth occurred, followed by a
OD54,
growth rate paralleling that of cultures without cAMP. Concentrations of 5 x 10-; M and 5 x 10-'" M cAMP produced no significant effect. When cAMP at 5 x 10-, M was added to expo-
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E
6
8 DAYS
FIG. 4. Chemolithotrophic, heterotrophic, and mixotrophic growth of M. gordonae E02. Heterotrophic and mixotrophic incubations were in 0.3% glucose-salts medium. Heterotrophic incubation was under gas atmospheres (A) or 20% 02, 10%
of either 20% 02 and 80% N2
CO2, and 70% N2 (A). Chemo-
lithotrophic (0) and mixotrophic (C) incubations were under 50% H2, 20% 02, 10% CO2, and 20% N2.
nential heterotrophic glycerol cultures (02N2) and to repressed glycerol cultures under a H2-02-N2 atmosphere, the heterotrophic growth rates increased slightly, whereas the hydrogen-repressed cultures showed no effect. Hydrogenase and RuDP carboxylase levels of heterotrophic and mixotrophic cultures exposed to 5 x 10-5 M cAMP were compared with cells grown in the absence of cAMP. No significant hydrogenase or RuDP carboxylase activity was observed with cells grown heterotrophically with glycerol, either in the absence of cAMP or after exposure of growing cells to 5 x 10-5 M cAMP. Cells exposed to 5 x 10-5 M cAMP either for 20 h or 4 days during mixotrophic cultivation had 20 to 25% higher hydrogenase and 30 to 45% higher RuDP carboxylase levels than did cells not exposed to cAMP. However, since the cells exposed to cAMP were harvested at turbidities somewhat lower than the corresponding unexposed cells and an inverse relation between cell density and enzyme levels was consistently observed with mixotrophic glycerol cultures, the increases are probably not significant since the levels of both enzymes in cAMPexposed cultures were within the normal range for unexposed mixotrophic cells.
J. BACTERIOL.
DISCUSSION Both of the chemolithotrophic enzyme systems studied in the three autotrophic mycobacteria were strongly influenced by the growth conditions employed. When cultures were grown in 7H9 broth, the levels of both enzymes were negligible, but after transfer into chemolithotrophic conditions the enzyme levels increased throughout the exponential growth phase and then declined during stationary phase. During mixotrophic growth with glycerol, a similar pattern was observed, except that hydrogenase levels were somewhat lower and RuDP carboxylase levels were much lower than in autotrophic cells. In general, a consistent pattern was observed with the autotrophic mycobacteria studied; cells grown heterotrophically with either single substrates or 7H9 broth possessed negligible levels of hydrogenase and carboxylase, whereas mixotrophically grown cells contained carboxylase levels that were internediate between heterotrophic and autotrophic cells. Hydrogenase levels of mixotrophically grown M. gordonae were much higher relative to autotrophic cells than were carboxylase levels and, in cells grown with acetate, pyruvate, or fructose, were actually considerably higher than in cells grown autotrophically. Mixotrophic incubation with acetate and pyruvate elicited very different responses in M. gordonae as compared with Alcaligenes eutrophus (Hydrogenomonas eutropha). With A. eutrophus the presence of acetate or pyruvate completely repressed RuDP carboxylase synthesis (27). In M. gordonae cells grown mixotrophically, these substrates yielded carboxylase levels comparable to those in cells grown in the presence of other organic substrates, and both autotrophic and heterotrophic systems functioned simultaneously, producing greater growth rates and yields than with either heterotrophic or autotrophic cultures. Fructose and glucose were poor substrates for heterotrophic growth of M. gordonae; however, under mixotrophic conditions growth was rapid. It appears likely that not only do autotrophic and heterotrophic systems operate simultaneously in M. gordonae but also the autotrophic systems or substrates have the capacity to facilitate the utilization of normally unfavorable organic substrates. The mechanism for this facilitation is not known, but the rapid uptake of autotrophic gases could provide the energy for increased permeation of organic substrates. Although we know of no other reports describing RuDP carboxylase in mycobacteria, several other mycobacterial strains have been
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HYDROGENASE AND CARBOXYLASE IN MYCOBACTERIA
analyzed for hydrogenase activity. One, designated Mycobacterium "tap water," was reported to oxidize both hydrocarbons and hydrogen adaptively and independent of each other (14). Dworkin and Foster (8) described two mycobacterial isolates, one of which contained a constitutive hydrogenase system, whereas the second contained an inducible system. Among the hydrogen autotrophs other than mycobacteria, some possess inducible (1, 4, 12, 17, 22) and some constitutive hydrogen-oxidizing systems (7, 20). The RuDP carboxylase systems in the facultative autotrophs all seem to be inducible or repressible and appear to be a major mechanism regulating autotrophic metabolism. Physical factors such as oxygen tension (13, 23, 29) as well as the type of organic substrate available (13, 16, 21, 27) can affect the levels of hydrogenase and RuDP carboxylase in these organisms. The repressive effect of acetate and pyruvate for A. eutrophus was shown to be at the level of enzyme synthesis and not a direct inhibition of enzyme activity (27). DeCicco and Umbreit (6) suggested that those organic substrates that are utilized relatively slowly allow simultaneous autotrophic and heterotrophic metabolism, whereas more readily utilizable organic substrates repress autotrophic metabolism. With the autotrophic mycobacteria no increase in growth rate or yield was observed in 7H9 broth under mixotrophic conditions. Since 7H9 broth allows much faster heterotrophic growth than any single organic substrate tested, this supports those observations (6) as well as growth results obtained with A. eutrophus (5). However, cultures of all three autotrophic mycobacteria grown mixotrophically in 7H9 broth contained moderate levels of both autotrophic enzymes, in contrast to the severely repressed levels of RuDP carboxylase in A. eutrophus during rapid heterotrophic growth (27). Reducing the oxygen tension did not stimulate either enzyme system in mycobacteria, as it does in some other hydrogen autotrophs. Our studies with autotrophic mycobacteria indicate that each autotrophic system is specifically induced by the substrate of the enzyme; hydrogenase is only produced in the presence of H2 and RuDP carboxylase is produced in the presence of C02. The complete autotrophic gas mixture (H2-2O---02) was necessary for the maintenance of high levels of carboxylase, supporting an obligatory coupling of H2 oxidation and CO2 assimilation, as was suggested by Schlegel (24) while studying other hydrogen autotrophs. The presence of H2 in the absence of CO., produced a severe inhibition of growth of M.
737
gordonae in glycerol medium. The inhibitory effect of H2 was observed regardless of whether the inoculum was grown heterotrophically with glycerol or autotrophically; thus, glycerol utilization was specifically being inhibited, even after being fully functional (Fig. 3). Assuming the obligatory coupling of H2 oxidation and CO2 assimilation occurs through the mediation of adenosine 5'-triphosphate (ATP), in the absence of C02 the ratios of ATP/adenosine 5'diphosphate and reduced nicotinamide adenine dinucleotide (NADH2)/NAD would be expected to increase. The latter ratio could directly affect the activity of glycerol-phosphate-NAD-oxidoreductase, the enzyme catalyzing the conversion of glycerol phosphate to dihydroxyacetone phosphate. A situation that may be analogous has been reported in other hydrogen autotrophs (10, 26), in which the presence of H2 in the absence of C02 inhibited the utilization of fructose. Schlegel and Eberhardt (25) proposed that ATP, NADH2, or other metabolites in equilibrium with these may act as corepressors of catabolic enzymes, and glucose-6-phosphate dehydrogenase was shown to be sensitive to ATP and NADH2 (3), which could explain the inhibition of fructose utilization by H2. ATP and/or NADH2 may affect the catabolism of glycerol via a similar mechanism. M. scrofulaceum apparently cannot grow autotrophically because it lacks both hydrogenase and RuDP carboxylase. This organism may also lack the other major autotrophic enzyme, phosphoribulokinase, but this enzyme was not included in these studies. Many of the facultative hydrogen-oxidizing autotrophs either lose their ability to grow autotrophically or must be readapted to autotrophic growth after prolonged heterotrophic cultivation. However, even though the scotochromogenic mycobacteria do not possess significant levels of hydrogenase and RuDP carboxylase when grown heterotrophically, the strains used in this study have been maintained on heterotrophic agar for a number of years without any diminution in their autotrophic capacity. ACKNOWLEDGMENT Some materials used in this study were provided by the U.S.-Japan Cooperative Medical Science Program, National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Aggag, M., and H. G. Schlegel. 1973. Studies on a gram positive hydrogen bacterium. Nocardia opaca strain lb. I. Description and characterization. Arch. Microbiol. 88:299-318.
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2. Beijajewa, M. I. 1954. Chemismus der C02-Assimilation und die Rolle der Phosphate bei der autotrophen Assimilation von Kohlenwasserstoffen durch wasserstoffoxydierende Bakterien. Mitt. Univ. Kasan 114: 3-12. 3. Blackkolb, F., and H. G. Schlegel. 1968. Regulation der Glucose-6-Phosphate-Dehydrogenase aus Hydrogenomonas H16 durch ATP and NADH2. Arch. Mikrobiol. 63:177-196. 4. Canevascini, G., and U. Eberhardt. 1975. Chemolithotrophic growth and regulation of hydrogenase formation in the coryneform hydrogen bacterium strain 11/ x. Arch. Microbiol. 103:283-291. 5. DeCicco, B. T., and P. E. Stukus. 1968. Autotrophic and heterotrophic metabolism of Hydrogenomonas. I. Growth yields and patterns under dual substrate conditions. J. Bacteriol. 95:1469-1475. 6. DeCicco, B. T., and W. W. Umbreit. 1964. Utilization of aromatic amino acids by Hydrogenomonas facilis. J. Bacteriol. 88:1590-1594. 7. Doudoroff, M. 1940. The oxidative assimilation of glucose and related substances by Pseudomonas saccharophilia. Enzymologia 9:59-72. 8. Dworkin, M., and J. W. Foster. 1958. Experiments with some microorganisms which utilize ethane and hydrogen. J. Bacteriol. 75:592-603. 9. Gornall, A. G., C. J. Bardawill, and M. M. Davis. 1949. Determination of serum proteins by means of the Biuret reaction. J. Biol. Chem. 177:751-.766. 10. Gottschalk, G., U. Eberhardt, and H. G. Schlegel. 1964. Verwertung von Fructose durch Hydrogenomonas H16. Arch. Mikrobiol. 48:95-108. 11. Hirsch, P. 1961. Wasserstoffactivierung und Chemoautotrophie bei Actinomyceten. Arch. Mikrobiol. 39:360-373. 12. Kluyver, H. J., and A. Manten. 1942. Some observations on the metabolism of bacteria oxidizing molecular hydrogen. Antonie van Leeuwenhoek J. Microbiol. Serol. 8:71-85. 13. Kuehn, G. D., and B. A. McFadden. 1968. Factors affecting the synthesis and degradation of ribulose-1,5diphosphate carboxylase in Hydrogenomonas facilis and Hydrogenomonas eutropha. J. Bacteriol. 95:937946. 14. Lukins, H. B., and J. W. Foster. 1963. Utilization of hydrocarbons and hydrogen by mycobacteria. Z. Allg. Mikrobiol. 3:251-264.
J. BACTERIOL. 15. McFadden, B. 1973. Autotrophic CO2 assimilation and the evolution of ribulose diphosphate carboxylase. Bacteriol. Rev. 37:289-319. 16. McFadden, B. A., and C. L. Tu. 1967. Regulation of autotrophic and heterotrophic carbon dioxide fixation in Hydrogenomonas facilis. J. Bacteriol. 93:886-893. 17. Packer, L., and W. Vishniac. 1955. Chemosynthetic fixation of carbon dioxide and characteristics of hydrogenase in resting cell suspensions of Hydrogenomonas ruhlandii nov. spec. J. Bacteriol. 70:216-223. 18. Park, S. S., and B. T. DeCicco. 1974. Autotrophic growth with hydrogen of Mycobacterium gordonae and another scotochromogenic Mycobacterium. Int. J. Syst. Bacteriol. 24:338-345. 19. Pastan, I., and R. Perlman. 1970. Cyclic adenosine monophosphate in bacteria. Science 169.339-344. 20. Repaske, R. 1962. Nutritional requirements for Hydrogenomonas eutropha. J. Bacteriol. 83:418-422. 21. Rittenberg, S. C. 1969. The roles of exogenous organic matter in the physiology of chemolithotrophic bacteria. Adv. Microbiol. Physiol. 3:159-196. 22. Ruhland, W. 1924. Beitrage zur Physiologie der Knallgasbakterien. Jahrb. Wiss. Bot. 63:321-389. 23. Schatz, A., and C. R. Bovell. 1952. Growth and hydrogenase activity of a new bacterium. Hydrogenomonas facillis. J. Bacteriol. 63:87-98. 24. Schlegel, H. G. 1966. Physiology and biochemistry of knallgasbacteria. Adv. Comp. Physiol. Biochem. 2:185-236. 25. Schlegel, H. G., and U. Eberhardt. 1972. Regulatory phenomena in the metabolism of knallgasbacteria. Adv. Microb. Physiol. 7:205-242. 26. Schlegel, H. G., and H. G. Truper. 1966. Repression of enzyme formation in Hydrogenomonas strain H16+ by molecular hydrogen and by fructose. Antonie van Leeuwenhoek J. Microbiol. Serol. 32:277-292. 27. Stukus, P. E., and B. T. DeCicco. 1970. Autotrophic and heterotrophic metabolism of Hydrogenomonas: regulation of autotrophic growth by organic substrates. J. Bacteriol. 101:339-345. 28. Umbreit, W. W., R. H. Bums, and J. P. Stauffer. 1957. Manometric techniques, 3rd ed. Burgess Publishing Co., Minneapolis. 29. Wilson, E., H. A. Stout, D. Powelson, and H. Koffler. 1953. Comparative biochemistry of the hydrogen bacteria. I. The simultaneous oxidation of hydrogen and lactate. J. Bacteriol. 65:283-287.
JoURNAL oF BACTERIOLOGY, August 1976, p. 731-738 Copyright 0 1976 American Society for Microbiology
Hydrogenase and Ribulose Diphosphate Carboxylase During Autotrophic, Heterotrophic, and Mixotrophic Growth of Scotochromogenic Mycobacteria SANG S. PARK Arm B. T. DzCICCO* Department ofBiology, The Catholic University of America, Washington, D.C. 20064
Received for publication 27 February 1976
Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence ofcarbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.
Chemolithotrophy among the mycobacteria has been neglected compared with that in other hydrogen autotrophs (15, 21, 24). In 1954, Belajewa reported the autotrophic growth of Mycobacterium album (2) under an atmosphere of hydrogen, oxygen, and carbon dioxide. Four years later Dworkin and Foster (8) reported the isolation from soil of four mycobacterial strains that grew in a mineral salts medium under an atmosphere of 50% hydrogen, 10% carbon dioxide, and 40% air. In 1963 Lukins and Foster (14) reported that some known authentic species (M. fortuitum, M. marinum, and M. smegmatis) as well as a newly isolated Mycobacterium sp. grew autotrophically with hydrogen. They found the hydrogenase activity to reside in the clear supernatant fluid from a cell extract. One stock strain of M. phlei also has been reported to grow under autotrophic conditions (11). Most reports on hydrogen autotrophy in mycobacteria have involved rapidly growing strains. Upon the isolation of a slow-growing autotrophic Mycobacterium, we investigated the chemolithotrophic growth capacity of a number of mycobacteria, as previously reported (18). Among 34 mycobacterial strains tested, a distinctive autotrophic capacity was observed only with the scotochromogenic saprophytic species M. gordonae and two strains of an unclassified saprophytic scotochromogen. Eight 731
strains of the potentially pathogenic scotochromogen M. scrofulaceum were unable to grow autotrophically, suggesting that the capacity to grow autotrophically with hydrogen may be significant for differentiating the saprophytic and pathogenic scotochromogens. Although ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) is responsible for the assimilation of CO2 into RuDP in autotrophic organisms, studies of this enzyme in autotrophic mycobacteria have not been previously reported. The present report describes the activities of the RuDP carboxylase and hydrogenase systems during growth of several scotochromogenic mycobacteria under a number of different cultural conditions. MATERIALS AND METHODS Bacterial strains. The four mycobacterial strains employed in the present study were described and partially characterized in a previous report (18). M. gordonae E02 and Mycobacterium strains AU and 6Y grow autotrophically with hydrogen as the sole energy source; M. scrofulaceum E03 does not grow autotrophically. Chemolithotrophic cultivation. The medium employed for chemolithotrophic growth consisted of 0.1% (NH4)2SO4, 0.02% MgSO4-7H40, and 0.001% Fe(NH4)2(S04)2 * 6H20 in 0.04 M potassium phosphate buffer, pH 6.7. Erlenmeyer flasks (250-ml) containing 100 ml of mineral salts medium were
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inoculated and incubated under an atmosphere of either 50% H2, 10% CO2, 20% 02, and 20% N2 or 50% H2, 10% C02, and 40% air, as indicated. The gas mixture was continuously supplied to the culture flasks through a manifold from a gas reservoir and was maintained at slightly greater than atmospheric pressure using a water displacement system. Heterotrophic and mixotrophic cultivation. For heterotrophic growth in liquid media, either mineral salts medium containing a single organic substrate or Middlebrook 7H9 broth (Difco) enriched with oleic acid, bovine albumin fraction V, 1-glucose, beef catalase, sodium chloride (OADC, Difco), and 0.2% glycerol (Difco) was employed. Cells from Middlebrook 7H10 agar slants (enriched with glycerol and OADC as described by Difco) were inoculated into enriched 7H9 broth and grown for 6 to 10 days at the specified temperature with shaking. The cultures were harvested, washed, and resuspended in mineral salts medium and used as inocula. Cells were inoculated into 100 ml of either 7H9 broth or mineral salts solution containing 0.3% of the specified substrate in 250-ml Erlenmeyer flasks. The flasks were shaken at 300C under a gas mixture of 20% 02 and 80% N2 for heterotrophic growth and either 50% H2, 10% C02, and 40% air or 50% H2, 20% 02, 10% C02, and 20% N2 for mixotrophic growth. For determination of the specific effects of carbon dioxide and hydrogen, gas mixtures of 20% 02, 10% CO2, and 70% N2 or 50% H2, 20% O.,, and 30% N2, respectively, were used. The initial optical density at 540 nm (OD"O) was 0.01 to 0.05; however, for each set of experiments each flask had the same initial cell turbidity and incubation was at the same shaker speed and the same temperature (3000), unless otherwise indicated. Growth was monitored by following the increase in ODH,o using a Spectronic20 colorimeter (Bausch & Lomb). Hydrogenase assay. Hydrogenase levels were determined by the standard Warburg technique (28) at 2800, using an Aminco Warburg apparatus (American Instrument Co.). Cells grown under different cultural conditions were harvested by centrifugaton, washed, and resuspended in mineral salts medium. Each vessel contained 2.0 ml of the same mineral medium and 1.0 ml of cell suspension of OD 1.0. The Warburg vessels were flushed with a gas mixture containing 90% H2 and 10% 02 or 90% N2 and 10% 02 and allowed to equilibrate for 10 min. The amount of gas consumed by the cells under the H2 and 02 atmosphere was corrected for the endogenous level and was used as an indication of hydrogenase activity. Hydrogenase activity (specific activity) in expressed as microliters of hydrogen consumed per milligram (dry weight) of cells per hour. An ODs.0 of 0.3 corresponded to a cellular dry weight of 0.13 mg/ml for heterotrophically grown cells and 0.18 mg/ml for autotrophically grown cells of strains AU and 6Y, 0.17 mg/ml for heterotrophically grown cells and 0.27 mg/ml for autotrophically grown cells of M. gordonae E02, and 0.3 mg/ml for
heterotrophically grown cells of M. scrofulaceum.
The cellular dry weight determinations for heterotrophically grown cells were obtained with cells grown in 7H9 broth. Cellular dry weights were mea-
J. BACTERIOL.
sured in aluminum cups after the cells were washed with deionized water and dried at 80 to 8500 for 16 to 18 h. The disparity in the dry weight values of heterotrophic and autotrophic cultures was probably due to the granular nature of the autotrophic cultures, which produced a higher cell mass per OD unit than the smooth heterotrophic cells. RuDP carboxylase assay. The method employed for the RuDP carboxylase assay has been described previously (27). A unit of enzyme activity is defined as the amount catalyzing the formation of 1 nmol of 3-phosphoglyceric acid per min. Specific activity is defined as units per milligram of protein. Protein was determined by the biuret method (9).
RESULTS Cultures of M. gordonae E02 and strains AU and 6Y grown under chemolithotrophic conditions consumed large quantities of hydrogen when assayed manometrically. Rates of hydrogen consumption for the three strains varied from approximately 88 to 135 ,ul of H2/mg (dry weight) per h at 280C (Table 1). Cells grown heterotrophically in 7H9 broth (under 80%o N2 and 20% 02) showed low hydrogenase levels, varying from 2 to 5% of the autotrophic levels. Endogenous levels of oxygen consumption were low, ranging from 2 to 8 ,ul/mg (dry weight) per h. When cultures of M. gordonae E02 and strain AU were grown mixotrophically in 7H9 broth under a gas mixture of 50% H2, 40% air, and 10% C02, they exhibited hydrogenase levels approximately 83 and 66%, respectively, of the autotrophic cells. Strain 6Y showed relatively lower hydrogenase levels when grown mixotrophically, supporting earlier observations that this strain prefers heterotrophic growth conditions (18). RuDP carboxylase levels were influenced to a greater extent by the presence of organic substrates than were hydrogenase; mixotrophic levels of RuDP carboxylase were 10 to 26% of those present in autotrophic cultures. RuDP carboxylase levels were very low in heterotrophic cultures. Since M. scrofulaceum cannot grow autotrophically, hydrogenase and RuDP carboxylase assays were performed with cells incubated both heterotrophically and mixotrophically. No significant activity was detected for either enzyme with cells incubated under either set of conditions, indicating that the inability of this strain of M. scrofulaceum to grow autotrophically is due to a deficiency of both autotrophic enzyme systems. To study the inducibility of the hydrogenase and RuDP carboxylase systems, flasks containing mineral salts medium were inoculated with M. gordonae cells that had been grown heterotrophically in 7H9 broth for 6 days at 300C,
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TABLE 1. Hydrogenase and RuDP carboxylase levels of scotochromogenic mycobacteria grown heterotrophically and mixotrophically in 7H9 broth or chemolithotrophically with hydrogen Strain
Growth condition"
Incubation periodb
Hdoeae
ODsdo (sp act) (days)(sac) 6.1 0.50 6 0.69 89.3 6 135.0 28 0.82 6 1.53 4.5 6 1.80 18.7 97.9 50 1.10 1.4 7 3.00 73.2 2.80 7 88.5 0.31 21 0.1 6 0.47 0.51 0.0 6
abx acta ylase (sp yae(pct
RD
0.21 Heterotrophic 0.57 Mixotrophic 5.70 Chemolithotrophic 0.10 Heterotrophic 6Y 0.50 Mixotrophic 3.50 Chemolithotrophic 0.00 M. gordonae E02 Heterotrophic 0.76 Mixotrophic 2.89 Chemolithotrophic 0.00 Heterotrophic M. scrofulaceum 0.00 Mixotrophic E03 a For a description of heterotrophic, mixotrophic, and chemolithotrophic growth conditions, see the text. b Incubation was at room temperature (26 + 1°C). AU
harvested, washed, and suspended in mineral salts medium. The culture flasks were incubated at 300C with shaking under an H., O.,, CO2, and N2 atmosphere. As shown in Fig. 1, the very low levels of hydrogenase and RuDP carboxylase that were present in the heterotrophically grown inoculum increased rapidly during incubation under autotrophic conditions and remained high throughout the exponential growth phase. Specific activities of both enzymes declined rapidly during the stationary growth phase. Increased shaker speed lengthened the period of exponential growth and the corresponding period of high enzyme levels, as would be expected from the growth stimulation previously observed with increased oxygen levels (18), but the ratio of the specific activities of the two enzymes remained relatively constant throughout the different phases of autotrophic growth. Since the studied autotrophic mycobacteria exhibited regulation of the hydrogenase and RuDP carboxylase systems, it seemed desirable to observe the interaction of autotrophic and heterotrophic systems in one of these strains for comparison with other extensively studied hydrogen autotrophs (21, 24, 25). Thus, M. gordonae was inoculated into mineral salts medium containing selected individual organic substrates and incubated under various gas mixtures. When M. gordonae was grown mixotrophically with 0.3% glycerol under a gas mixture of H2, 02, C02, and N2, a dramatic increase in both growth rate and yield was observed as compared with autotrophic and heterotrophic incubation (Fig. 2). The increased growth rate and yield observed during mixotrophic growth were not due to C02 alone since the addition of C02 to the 02 and N2 gas mixture did not signif-
1.0-
6~ 75
m~~~~~~~2 2
E
0 ~~~~~~o
0 o
2
S
4
6
8 DAYS
10
12
14
FIG. 1. Hydrogenase and RuDP carboxylase specific activities during chemolithot rophic growth of M. gordonae E02.
icantly enhance growth. When hydrogen was added to the heterotrophic atmosphere, growth of M. gordonae was drastically inhibited. Since gas mixtures of 02-N., 09-N2-002, or H2 02-C02-N2 did not inhibit growth, the presence of hydrogen seemed to be essential for the inhibitory effect. When an autotrophically grown inoculum was used in similar experiments, the results were essentially the same (data not shown). To determine whether preadapted cells were inhibited by hydrogen, six flasks of mineral salts-glycerol medium were inoculated with M. gordonae (grown in 7H9 broth) and incubated under an atmosphere of 02 and N., After 7 days
734
PARK AND DECICCO
J. BACTERIOL.
E
0
0
2
4
6
10
8
12
14
16
DAYS
FIG. 2. Chemolithotrophic, heterotrophic, and mixotrophic growth of M. gordonae E02. Heterotrophic and mixotrophic incubations were in 0.3% glycerol-salts medium. Heterotrophic incubation was under gas atmospheres of either 20% 02 and 80% N2
(0) or 20% 02, 10% C02, and 70% N2 (A)- Chemolithotrophic (A) and mixotrophic (0) incubations were
under 50% H2, 20%
02,
10% CO., and 20% N2.
the atmosphere in two flasks was changed to H2, O,, and N2. Two other flasks were placed under H2, 02, C02, and N., whereas the last two flasks remained under the heterotrophic atmosphere of 02 and N.,. As shown in Fig. 3, growth in the presence of H2, O2, and N., continued at a rapid rate for 24 h, after which growth slowed to a very low rate similar to that observed previously. Growth with the other two gas mixtures was exponential and reached similar densities, but the mixotrophic culture grew much faster. Hydrogenase and RuDP carboxylase levels of M. gordonae cells grown in glycerol medium under the different gas mixtures are shown in Table 2. As previously observed, the levels of both enzymes were very low in cells grown under heterotrophic conditions (02-N2). It is evident that the addition of CO2 in the absence of H2 did not stimulate the production of high levels of either of the autotrophic enzyme systems, although moderate levels of carboxylase were consistently present for a brief time in glycerol cultures exposed to the 09-COo-N2 atmosphere (data not shown). Similarly, the presence of H2 without C02 did not stimulate high levels of both enzymes although, at 19 days, moderate levels of hydrogenase, compara-
ble to mixotrophic cultures, were present under an atmosphere of H2, 02, and N2. Hydrogenase and RuDP carboxylase levels increased during exponential mixotrophic growth and then declined with decreasing growth rates. The relationship between enzyme levels and growth closely paralleled that shown with autotrophic cultures in Fig. 1, except that the RuDP carboxylase levels in mixotrophic cultures were consistently much lower. When mineral salts media containing sodium acetate, sodium pyruvate, glucose, or fructose were incubated under different gas mixtures, increased growth rates and yields were noted with all substrates under mixotrophic conditions. The growth curves observed with acetate and pyruvate media closely resembled those observed with glycerol (Fig. 2). With glucose and fructose, the stimulatory effect of the autotrophic atmosphere was even more apparent because no growth occurred under heterotrophic conditions (with or without CO2) during the times indicated (Fig. 4). Heterotrophic growth did commence with glucose and fructose after several weeks of incubation. RuDP carboxylase levels of cells grown under mixotrophic conditions with glycerol, glucose, sodium acetate, sodium pyruvate, or fructose were relatively low compared with those of autotrophically grown cells (Table 2) and were similar to those observed after mixotrophic growth in 7H9 broth (Table 1). RuDP carboxylase levels were insignificant after heterotrophic growth with all substrates (Table 2). During mixotrophic incubation, hydrogenase levels remained comparatively high; in fact, M. gordonae cells grown mixotrophically with acetate, pyruvate, or fructose contained levels of hydrogenase that significantly exceeded the levels present in autotrophically grown cells (Table 2), which were never found to exceed a specific activity of 107. In contrast with the studies with single organic substrates, the growth curves obtained when M. gordonae was grown heterotrophically and mixotrophically in 7H9 broth were almost identical; however, the levels of hydrogenase and RuDP carboxylase were much higher in cells incubated mixotrophically (Tables 1 and 2). Since hydrogenase and RuDP carboxylase levels were lower in mixotrophically and heterotrophically grown cells as compared with autotrophically grown cells, it was possible that a form of catabolic repression, reversible with cyclic adenosine monophosphate (cAMP), might be operative (19). To determine this, M. gordonae was grown mixotrophically with glyc-
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HYDROGENASE AND CARBOXYLASE IN MYCOBACTERIA
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10.0
1.0 E 0
0. 1 _
t
I
0
2
4
FIG. 3. Inhibitory effect of hydrogen
6 on
8
I
I
10 12 14 16 18 20 22 DAYS
the heterotrophic growth of M. gordonae E02 with glycerol. Three
sets of cultures were grown in 0.3% glycerol-salts medium under an atmosphere of20% O., and 80% N2. At the indicated time the atmosphere in one set was changed to 50% H., 20% O2, 10% CO. and 20% N., (A) and in the second set it was changed to 50% H2, 20% 02, and 30% N2 (0). The third set remained under 02 and N2 (@) .
TABLE 2. Hydrogenase and RuDP carboxylase levels in M. gordonae E02 grown with various substrates and gas atmospheres IncubaRuDP carOrganic subHydrogen- boxlas Incubation condition Atmosphere strate ase (sp act) bosyase) riod (pat (days) None Chemolithotrophic 12 H,-0{,-C0,-N.,N 0.23 75.3 2.49 02 N.b Glycerol Heterotrophic 14 0.21 3.7 0 16 0.84 1.7 0.01 14 0.25 10.6 0 O.,-CO2-N2,' 19 0.37 46.7 0.10 30 0.31 5.3 0 0 Mixotrophic H2-02--C02-N2 0.03 4.0 0.1 8 0.67 62.3 0.65 14 1.68 37.4 0.53 Glucose Heterotrophic 02-N2 32 0.04 Nae NA 32 NA 0.03 NA 02-C02-N2 12 0.42 54.7 H2-,0---C02- N2 0.25 Mixotrophic Acetate Heterotrophic 0.,-N., 12 0.29 8.1 0 Mixotrophic 12 H2-02--C02,-N., 0.40 175.5 0.55 02- N2 Pyruvate Heterotrophic 24 0.44 3.3 0 Mixotrophic H2-02--C02-N2 12 0.54 122.4 0.76 Fructose Heterotrophic 02-N2 12 0.02 NA NA Mixotrophic H2,-0--C02,-N2 12 0.20 145.7 0.54 7H9 broth Heterotrophic 0.,-CO2-N2 7 3.07 4.4 0 a 50% H.,-20% O.,-10% CO.,-20% N.,. b 20% 02-80% N2. 20% 0°2-10% CO,-70% N2. (I 50%o H2-20% 02-30% N2. "NA, Not assayed due to lack of growth under these conditions.
tionpe
erol in the presence of cAMP at concentrations of 5 x 10-4 M, 5 x 10-5M, and 5 x 10-6 M. At a concentration of 5 x 10-4 M cAMP, a transient inhibition of growth occurred, followed by a
OD54,
growth rate paralleling that of cultures without cAMP. Concentrations of 5 x 10-; M and 5 x 10-'" M cAMP produced no significant effect. When cAMP at 5 x 10-, M was added to expo-
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PARK AND DECICCO
E
6
8 DAYS
FIG. 4. Chemolithotrophic, heterotrophic, and mixotrophic growth of M. gordonae E02. Heterotrophic and mixotrophic incubations were in 0.3% glucose-salts medium. Heterotrophic incubation was under gas atmospheres (A) or 20% 02, 10%
of either 20% 02 and 80% N2
CO2, and 70% N2 (A). Chemo-
lithotrophic (0) and mixotrophic (C) incubations were under 50% H2, 20% 02, 10% CO2, and 20% N2.
nential heterotrophic glycerol cultures (02N2) and to repressed glycerol cultures under a H2-02-N2 atmosphere, the heterotrophic growth rates increased slightly, whereas the hydrogen-repressed cultures showed no effect. Hydrogenase and RuDP carboxylase levels of heterotrophic and mixotrophic cultures exposed to 5 x 10-5 M cAMP were compared with cells grown in the absence of cAMP. No significant hydrogenase or RuDP carboxylase activity was observed with cells grown heterotrophically with glycerol, either in the absence of cAMP or after exposure of growing cells to 5 x 10-5 M cAMP. Cells exposed to 5 x 10-5 M cAMP either for 20 h or 4 days during mixotrophic cultivation had 20 to 25% higher hydrogenase and 30 to 45% higher RuDP carboxylase levels than did cells not exposed to cAMP. However, since the cells exposed to cAMP were harvested at turbidities somewhat lower than the corresponding unexposed cells and an inverse relation between cell density and enzyme levels was consistently observed with mixotrophic glycerol cultures, the increases are probably not significant since the levels of both enzymes in cAMPexposed cultures were within the normal range for unexposed mixotrophic cells.
J. BACTERIOL.
DISCUSSION Both of the chemolithotrophic enzyme systems studied in the three autotrophic mycobacteria were strongly influenced by the growth conditions employed. When cultures were grown in 7H9 broth, the levels of both enzymes were negligible, but after transfer into chemolithotrophic conditions the enzyme levels increased throughout the exponential growth phase and then declined during stationary phase. During mixotrophic growth with glycerol, a similar pattern was observed, except that hydrogenase levels were somewhat lower and RuDP carboxylase levels were much lower than in autotrophic cells. In general, a consistent pattern was observed with the autotrophic mycobacteria studied; cells grown heterotrophically with either single substrates or 7H9 broth possessed negligible levels of hydrogenase and carboxylase, whereas mixotrophically grown cells contained carboxylase levels that were internediate between heterotrophic and autotrophic cells. Hydrogenase levels of mixotrophically grown M. gordonae were much higher relative to autotrophic cells than were carboxylase levels and, in cells grown with acetate, pyruvate, or fructose, were actually considerably higher than in cells grown autotrophically. Mixotrophic incubation with acetate and pyruvate elicited very different responses in M. gordonae as compared with Alcaligenes eutrophus (Hydrogenomonas eutropha). With A. eutrophus the presence of acetate or pyruvate completely repressed RuDP carboxylase synthesis (27). In M. gordonae cells grown mixotrophically, these substrates yielded carboxylase levels comparable to those in cells grown in the presence of other organic substrates, and both autotrophic and heterotrophic systems functioned simultaneously, producing greater growth rates and yields than with either heterotrophic or autotrophic cultures. Fructose and glucose were poor substrates for heterotrophic growth of M. gordonae; however, under mixotrophic conditions growth was rapid. It appears likely that not only do autotrophic and heterotrophic systems operate simultaneously in M. gordonae but also the autotrophic systems or substrates have the capacity to facilitate the utilization of normally unfavorable organic substrates. The mechanism for this facilitation is not known, but the rapid uptake of autotrophic gases could provide the energy for increased permeation of organic substrates. Although we know of no other reports describing RuDP carboxylase in mycobacteria, several other mycobacterial strains have been
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HYDROGENASE AND CARBOXYLASE IN MYCOBACTERIA
analyzed for hydrogenase activity. One, designated Mycobacterium "tap water," was reported to oxidize both hydrocarbons and hydrogen adaptively and independent of each other (14). Dworkin and Foster (8) described two mycobacterial isolates, one of which contained a constitutive hydrogenase system, whereas the second contained an inducible system. Among the hydrogen autotrophs other than mycobacteria, some possess inducible (1, 4, 12, 17, 22) and some constitutive hydrogen-oxidizing systems (7, 20). The RuDP carboxylase systems in the facultative autotrophs all seem to be inducible or repressible and appear to be a major mechanism regulating autotrophic metabolism. Physical factors such as oxygen tension (13, 23, 29) as well as the type of organic substrate available (13, 16, 21, 27) can affect the levels of hydrogenase and RuDP carboxylase in these organisms. The repressive effect of acetate and pyruvate for A. eutrophus was shown to be at the level of enzyme synthesis and not a direct inhibition of enzyme activity (27). DeCicco and Umbreit (6) suggested that those organic substrates that are utilized relatively slowly allow simultaneous autotrophic and heterotrophic metabolism, whereas more readily utilizable organic substrates repress autotrophic metabolism. With the autotrophic mycobacteria no increase in growth rate or yield was observed in 7H9 broth under mixotrophic conditions. Since 7H9 broth allows much faster heterotrophic growth than any single organic substrate tested, this supports those observations (6) as well as growth results obtained with A. eutrophus (5). However, cultures of all three autotrophic mycobacteria grown mixotrophically in 7H9 broth contained moderate levels of both autotrophic enzymes, in contrast to the severely repressed levels of RuDP carboxylase in A. eutrophus during rapid heterotrophic growth (27). Reducing the oxygen tension did not stimulate either enzyme system in mycobacteria, as it does in some other hydrogen autotrophs. Our studies with autotrophic mycobacteria indicate that each autotrophic system is specifically induced by the substrate of the enzyme; hydrogenase is only produced in the presence of H2 and RuDP carboxylase is produced in the presence of C02. The complete autotrophic gas mixture (H2-2O---02) was necessary for the maintenance of high levels of carboxylase, supporting an obligatory coupling of H2 oxidation and CO2 assimilation, as was suggested by Schlegel (24) while studying other hydrogen autotrophs. The presence of H2 in the absence of CO., produced a severe inhibition of growth of M.
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gordonae in glycerol medium. The inhibitory effect of H2 was observed regardless of whether the inoculum was grown heterotrophically with glycerol or autotrophically; thus, glycerol utilization was specifically being inhibited, even after being fully functional (Fig. 3). Assuming the obligatory coupling of H2 oxidation and CO2 assimilation occurs through the mediation of adenosine 5'-triphosphate (ATP), in the absence of C02 the ratios of ATP/adenosine 5'diphosphate and reduced nicotinamide adenine dinucleotide (NADH2)/NAD would be expected to increase. The latter ratio could directly affect the activity of glycerol-phosphate-NAD-oxidoreductase, the enzyme catalyzing the conversion of glycerol phosphate to dihydroxyacetone phosphate. A situation that may be analogous has been reported in other hydrogen autotrophs (10, 26), in which the presence of H2 in the absence of C02 inhibited the utilization of fructose. Schlegel and Eberhardt (25) proposed that ATP, NADH2, or other metabolites in equilibrium with these may act as corepressors of catabolic enzymes, and glucose-6-phosphate dehydrogenase was shown to be sensitive to ATP and NADH2 (3), which could explain the inhibition of fructose utilization by H2. ATP and/or NADH2 may affect the catabolism of glycerol via a similar mechanism. M. scrofulaceum apparently cannot grow autotrophically because it lacks both hydrogenase and RuDP carboxylase. This organism may also lack the other major autotrophic enzyme, phosphoribulokinase, but this enzyme was not included in these studies. Many of the facultative hydrogen-oxidizing autotrophs either lose their ability to grow autotrophically or must be readapted to autotrophic growth after prolonged heterotrophic cultivation. However, even though the scotochromogenic mycobacteria do not possess significant levels of hydrogenase and RuDP carboxylase when grown heterotrophically, the strains used in this study have been maintained on heterotrophic agar for a number of years without any diminution in their autotrophic capacity. ACKNOWLEDGMENT Some materials used in this study were provided by the U.S.-Japan Cooperative Medical Science Program, National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Aggag, M., and H. G. Schlegel. 1973. Studies on a gram positive hydrogen bacterium. Nocardia opaca strain lb. I. Description and characterization. Arch. Microbiol. 88:299-318.
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