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The effect of temperature on ammonia assimilation in a mycobacterium
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
71, 451-457
(1957)
The Effect of Temperature on Ammonia Assimilation in a Mycobacterium Frederick From
the Department School
Bemheim
and William
E. DeTurk
of Physiology and Pharmacology, Duke of Medicine, Durham, North Carolina Received
March
University
4, 1957
The assimilation of inorganic nitrogen by bacteria requires energy. This can be obtained by the oxidation of substrates in the cell, or of those supplied in the medium, and the question arises whether the mechanism of assimilation is the same for both cases. In the course of studying the effect of higher than normal temperatures on the metabolism of a mycobacterium, it was found that exposure to 51” for a few minutes inhibited assimilation, although, as previously shown (1)) endogenous respiration and oxidative phosphorylation were not affected. After a period of aerobic incubation the assimilation mechanism recovered and the recovery could be delayed by certain drugs. If, however, a substrate such as succinate was added at any time during the period when the endogenously controlled assimilation was inactive, immediate assimilation occurred. This assimilation was less sensitive to certain drugs, i.e., it was not inhibited by concentrations which delayed recovery of the endogenous mechanism. EXPERIMENTAL AND RESULTS Mycobacterium tuberculosis BCG A.T.C.C. No. 8420 was grown for 5-7 days on 20 ml. of Long’s synthetic medium at 35”. The cell masses were broken up and washed twice with water by centrifugation in Hopkins tubes and finally suspended in 0.05 Na-K phosphate buffer, 0.1 ml. of packed cells in 1.0 ml. of buffer. The suspension was placed in a water bath at 51” for 10 min. One-half milliliter was used in each Warburg vessel which had a final fluid volume of 2.0 ml. and contained 0.5 mg. (NHa)&SO, . At the end of the experiment 1.0 ml. of 2Oye trichloroacetic acid was added to each vessel, the cells were centrifuged down, and the ammonia in the supernatant was determined by direct nesslerization. Preliminary experiments showed that distillation of the ammonia prior to nesslerization was not necessary. 451
452
BERNHEIM
30
60
90
AND
DETURK
I20
150
160
210
240
2
MINUTES
FIG.
1. The effect of heating at 51” for 10 min. on the rate of ammonia assimilation. Both types of curves were obtained in about equal frequency.
Normal cells have a high endogenous respiration which is increased after heating. The increase is caused by the extracellular presence of oxidizable substrates produced, presumably, by the hydrolysis of high-molecular-weight storage compounds (1). After washing, the oxygen uptake of the heated cells is the same as that of the normal and is increased to the same extent by dinitrophenol. The heated cells were washed to remove these substrates.
After heating, the ammonia assimilation might be completely inhibited for a period of time before recovery began or there might be a small residual assimilation which stopped after about 60-90 min. No further assimilation occurred for 30-60 min., after which recovery began. These two effects are shown in Fig. 1. In neither case did the rate of assimilation through a period of 4 hr. after heating equal that of the control. There is apparently only partial recovery of the assimilation mechanism. Throughout this period the oxygen uptakes of control and heated cells were the same, but the increased uptake which accompanied assimilation in the control was absent in the heated cells until recovery occurred. The heat damage occurred rapidly; an effect could be seen after 1 min. at 51“ followed by immediate cooling. As shown in Fig. 2, the damage increased with further heating up to a period of 10 min., after which no further effects were seen. Raising the temperature to 53” for 10 min. did not significantly prolong the recovery period, but at 55” the damage was much more severe although recovery still occurred. The damage was the same in cells heated aerobically or anaerobically, but the recovery was dependent on oxygen. During the recovery period, addition of a substrate such as succinate, which was allowed to be completely oxidized
AMMONIA
ASSIMILATION
453
before the addition of ammonium sulfate, did not shorten the recovery period. Van Halteren (2) showed that this was also true for heated yeast cells, although in this case the endogenous respiration was depressed 40-60 %. On the other hand, addition of succinate with (NH&SOr at any time during the recovery period caused immediate assimilation of the ammonium ion, and this continued until the succinate was oxidized. This is shown in Fig. 3. Other substrates such as fumarate, pyruvate, or trehalose produced approximately the same effect, but acetate and certain other fatty acids, although oxidized, caused little assimilation. Assimilation could thus take place during the oxidation of an exogenous substrate, although the oxidation of endogenous substrates was ineffective. The following experiment shows that assimilation had occurred and that the ammonium ion disappearance was not the result of some transamination reaction, the product of which remained outside the cell. Heated cells were incubated for 120 mm. (a) alone, (b) with 117 pg. NH8 N as ammonium sulfate, and (c) with 117 pg. NH3 N and 2.0 mg. succinate. The trichloroacetic acid supernatants contained 0, 90, and 30 pg. of NH3 N, respectively, by direct nesslerization. By micro-Kjeldahl (b) 80
60
SO MINUTES
FIG.
2.
The effect of heating at 51” for different times on the rate of ammonia assimilation.
454
BERNHEIM
AND
DETURK
contained 112 pg. and (c) 36 pg. The cells were then stirred up in the remaining supernatant, heated for 10 min. in a boiling water bath, and centrifuged again. Micro-Kjeldahls on (b) and (c) supernatants gave 120 and 132 pg. NH, N, respectively. This showed that boiling in acid had extracted all the nitrogen which had been assimilated. The microKjeldahl values before and after boiling for (a) were 102 and 252 pg., respectively, and these were subtracted from the totals to obtain the above values for (b) and (c). The oxidation rate of succinate by heated cells was only slightly slower than that of normal cells, and the end points in both cases were the same, namely, 5 atoms of 02 were taken up and 3 molecules of CO2 were evolved. Complete oxidation did not occur in either case. If nitrogen assimilation was very active in normal cells, the addition of succinate had little effect. Apparently the oxidation of the endogenous substrates was sufficient to account for most of it. In heated cells the addition of succinate restored assimilation at a rate which approximated that of normal cells. Concentrations of drugs such as streptomycin, aureomycin, azide, and dinitrophenol, which did not affect normal assimilation, had also little effect on assimilation of heated cells in the presence of succinate. The same concentrations of these drugs did, however, inhibit assimilation in heated cells in the absence of succinate by prolonging the recovery of the endogenous mechanisms. This is shown for azide in Fig. 4. In order to determine in part the fate of the assimilated nitrogen, largescale experiments were done on the heated cells alone, in the presence of ammonia, and ammonia plus succinate. The experiments were carried
120 MINUTES
FIQ.
3. The
ammonia
effect
of two assimilation
concentrations of succinate on the rate in cells heated at 51” for 10 min.
of
AMMONIA
ASSIMILATION
455
MINUTES FIG. 4. The effect of 0.15 mg./ml. sodium azide on the ammonia assimilation by cells heated at 51” for 10 min. in the presence and absence of 1.0 mg./ml. succinate. The first two curves on the left represent assimilation by heated cells in the presence of succinate with and without azide. The next two curves represent assimilation by heated cells without succinate in the presence and absence of azide. The two curves on the right represent assimilation by unheated cells in the presence and absence of azide.
out in Erlenmeyer flasks with six times the usual amount of cells, (NHSzSO~ , and succinate in six times the usual volume. The incubation lasted 2 hr. so that some assimilation occurred in the absence of succinate. At the end of the incubation the cells were centrifuged from the suspending medium and washed twice with distilled water. They were then taken up in 1 N HCl and subjected to mild hydrolysis in a boiling water bath for 15 min. Ammonia nitrogen in the suspending medium was estimated by the method of Speck (3) to determine the amount of nitrogen assimilated, and labile nitrogen was estimated on the supernatants of the cells heated in acid. The precision of this method is =t3 %. From 1% to 2 % of glutamine is hydrolyzed during distillation and appears as free ammonia. Aliquots of the acid extracts were also chromatographed on paper using the following solvents: butanol-acetic acid-water (80: 20 : 20) ; water-saturated phenol; ethanol-ammonium hydroxide (95 : 5) ; and pyridine-water (65: 35). Such extracts contained glutamic acid, aspartic acid, lysine, alanine, and a spot not positively identified which may be -r-aminobutyric acid. The extracts were chromatographed on heavy paper in the butanol or phenol solvent, the paper was cut in strips and eluted with water, and the eluates were rechromatographed in the four solvents. Comparisons were made between the eluates, known compounds, and mixtures of the eluates and known amino acids.
456
BERNHEIM
AND
DETURK
Cells in the presence of (NHd)zSOd assimilated 64-141 pg. of nitrogen. In the presence of succinate the assimilation was increased to 201-336 pg. of nitrogen. The ammonia liberated by hot 1 N HCl was also increased. The increases in labile nitrogen were a function of the amount of ammonia assimilated whether succinate was present or not, and accounted for 10-23 % of the nitrogen taken up. This indicates an increase in glutamine in cells in both cases. For example, in a typical experiment, the labile nitrogen of control cells was 100 pg., cells incubated with ammonia assimilated 64 ,ug. of NH3 N and the labile N was 107 pg., and cells incubated with ammonium sulfate and succinate assimilated 336 pg. and the labile N was 155 pg. The labile ammonia might be present in the cell either as ammonia liberated by heating, or as a labile compound, hydrolyzable under these mild conditions. To determine this, cells were subjected to sonic vibration for 1 hr., acidified to pH 2.0, and centrifuged in the cold, and ammonia was estimated on the supernatant before and after heating in 1 N HCI. Free ammonia was present in small amounts, but accounted for only a fraction of the ammonia present after hydrolysis. Sonic extracts were subjected to chromatography and were found to contain glutamine, not present in the hot acid extracts. In addition, when the material was chromatographed on heavy paper which was cut in strips and eluted, labile ammonia was found only in the glutamine-containing eluate. Glutamine has not been previously found to be a constituent of acidfast organisms, though the other amino acids were found in hot water extracts of various mycobacteria by Pauletta and Defranceschi (4). DISCUSSION
Foulkes (5) has shown that there are two mechanisms in yeast for the assimilation of potassium ion: one, azide-sensitive and dependent on endogenous metabolism; the other, azide-insensitive which is activated by addition of glucose and phosphate. The ammonia assimilation in these mycobacteria is also effected by two independent systems, one of which is more temperature-sensitive than the other. Temperature affects assimilation by inactivating an unknown mechanism since the autorespiration of the heated cells and their oxidation of added substrates are normal, and there is no evidence of uncoupling of oxidation and phosphorylation. Since the addition of succinate to heated cells caused immediate assimilation, it is unlikely that its oxidation is providing more adenosine triphosphate (ATP) or glutamic acid since a lag period should
AMMONIA
ASSIMILATION
457
be evident before these compounds accumulated. The mechanism of assimilation in the presence of an exogenous substrate is therefore not clear. Once the ammonium ion enters the cell, glutamine is formed whether the energy source has been endogenous or exogenous. But addition of glutamic acid or glutamine has no direct effect because both are oxidized and deaminated, and ammonia assimilation occurs only as the result of the oxidation. SUMMARY
1. Mycobacterium A.T.C.C. No. 8420 heated to 51” for 10 mm. loses its ability to assimilate ammonium ion, although the autorespiration and oxidation of added substrates is normal and there is no evidence of uncoupling of oxidation and phosphorylation. Recovery occurs after 90-120 min., but the rate of assimilation is still slower than normal. Certain drugs prolong the recovery period. 2. Addition of succinate or certain other substrates when no assimilation is occuring causesimmediate assimilation which continues as long as there is substrate to be oxidized. This processis not sensitive to drugs in concentrations which prolong the recovery of the “endogenous” assimilation. 3. Apparently two separate mechanisms exist in these cells for ammonium ion assimilation: one activated by endogenous metabolism, the other by the metabolism of exogenous compounds. 4. Glutamine and a number of amino acids have been identified in the heated cells following assimilation both in the presence and absence of succinate. REFERENCES 1. BERNHEIM, F., Arch. B&hem. Biophys. 69, 252 (1955). 2. HALTEREN, P. VAN, Bull. sac. chim. biol. 32, 458 (1950). 3. SPECK, J. F., J. Biol. Chem. 149, 1387 (1949). 4. PAULETTA,G.,AND DEFRANCESCHI, A., Biochim.et Biophys.Acta9,271 5. FOULKES, E. C., J. Gen. Physiol. 39, 687 (1956).
(1952).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
71, 451-457
(1957)
The Effect of Temperature on Ammonia Assimilation in a Mycobacterium Frederick From
the Department School
Bemheim
and William
E. DeTurk
of Physiology and Pharmacology, Duke of Medicine, Durham, North Carolina Received
March
University
4, 1957
The assimilation of inorganic nitrogen by bacteria requires energy. This can be obtained by the oxidation of substrates in the cell, or of those supplied in the medium, and the question arises whether the mechanism of assimilation is the same for both cases. In the course of studying the effect of higher than normal temperatures on the metabolism of a mycobacterium, it was found that exposure to 51” for a few minutes inhibited assimilation, although, as previously shown (1)) endogenous respiration and oxidative phosphorylation were not affected. After a period of aerobic incubation the assimilation mechanism recovered and the recovery could be delayed by certain drugs. If, however, a substrate such as succinate was added at any time during the period when the endogenously controlled assimilation was inactive, immediate assimilation occurred. This assimilation was less sensitive to certain drugs, i.e., it was not inhibited by concentrations which delayed recovery of the endogenous mechanism. EXPERIMENTAL AND RESULTS Mycobacterium tuberculosis BCG A.T.C.C. No. 8420 was grown for 5-7 days on 20 ml. of Long’s synthetic medium at 35”. The cell masses were broken up and washed twice with water by centrifugation in Hopkins tubes and finally suspended in 0.05 Na-K phosphate buffer, 0.1 ml. of packed cells in 1.0 ml. of buffer. The suspension was placed in a water bath at 51” for 10 min. One-half milliliter was used in each Warburg vessel which had a final fluid volume of 2.0 ml. and contained 0.5 mg. (NHa)&SO, . At the end of the experiment 1.0 ml. of 2Oye trichloroacetic acid was added to each vessel, the cells were centrifuged down, and the ammonia in the supernatant was determined by direct nesslerization. Preliminary experiments showed that distillation of the ammonia prior to nesslerization was not necessary. 451
452
BERNHEIM
30
60
90
AND
DETURK
I20
150
160
210
240
2
MINUTES
FIG.
1. The effect of heating at 51” for 10 min. on the rate of ammonia assimilation. Both types of curves were obtained in about equal frequency.
Normal cells have a high endogenous respiration which is increased after heating. The increase is caused by the extracellular presence of oxidizable substrates produced, presumably, by the hydrolysis of high-molecular-weight storage compounds (1). After washing, the oxygen uptake of the heated cells is the same as that of the normal and is increased to the same extent by dinitrophenol. The heated cells were washed to remove these substrates.
After heating, the ammonia assimilation might be completely inhibited for a period of time before recovery began or there might be a small residual assimilation which stopped after about 60-90 min. No further assimilation occurred for 30-60 min., after which recovery began. These two effects are shown in Fig. 1. In neither case did the rate of assimilation through a period of 4 hr. after heating equal that of the control. There is apparently only partial recovery of the assimilation mechanism. Throughout this period the oxygen uptakes of control and heated cells were the same, but the increased uptake which accompanied assimilation in the control was absent in the heated cells until recovery occurred. The heat damage occurred rapidly; an effect could be seen after 1 min. at 51“ followed by immediate cooling. As shown in Fig. 2, the damage increased with further heating up to a period of 10 min., after which no further effects were seen. Raising the temperature to 53” for 10 min. did not significantly prolong the recovery period, but at 55” the damage was much more severe although recovery still occurred. The damage was the same in cells heated aerobically or anaerobically, but the recovery was dependent on oxygen. During the recovery period, addition of a substrate such as succinate, which was allowed to be completely oxidized
AMMONIA
ASSIMILATION
453
before the addition of ammonium sulfate, did not shorten the recovery period. Van Halteren (2) showed that this was also true for heated yeast cells, although in this case the endogenous respiration was depressed 40-60 %. On the other hand, addition of succinate with (NH&SOr at any time during the recovery period caused immediate assimilation of the ammonium ion, and this continued until the succinate was oxidized. This is shown in Fig. 3. Other substrates such as fumarate, pyruvate, or trehalose produced approximately the same effect, but acetate and certain other fatty acids, although oxidized, caused little assimilation. Assimilation could thus take place during the oxidation of an exogenous substrate, although the oxidation of endogenous substrates was ineffective. The following experiment shows that assimilation had occurred and that the ammonium ion disappearance was not the result of some transamination reaction, the product of which remained outside the cell. Heated cells were incubated for 120 mm. (a) alone, (b) with 117 pg. NH8 N as ammonium sulfate, and (c) with 117 pg. NH3 N and 2.0 mg. succinate. The trichloroacetic acid supernatants contained 0, 90, and 30 pg. of NH3 N, respectively, by direct nesslerization. By micro-Kjeldahl (b) 80
60
SO MINUTES
FIG.
2.
The effect of heating at 51” for different times on the rate of ammonia assimilation.
454
BERNHEIM
AND
DETURK
contained 112 pg. and (c) 36 pg. The cells were then stirred up in the remaining supernatant, heated for 10 min. in a boiling water bath, and centrifuged again. Micro-Kjeldahls on (b) and (c) supernatants gave 120 and 132 pg. NH, N, respectively. This showed that boiling in acid had extracted all the nitrogen which had been assimilated. The microKjeldahl values before and after boiling for (a) were 102 and 252 pg., respectively, and these were subtracted from the totals to obtain the above values for (b) and (c). The oxidation rate of succinate by heated cells was only slightly slower than that of normal cells, and the end points in both cases were the same, namely, 5 atoms of 02 were taken up and 3 molecules of CO2 were evolved. Complete oxidation did not occur in either case. If nitrogen assimilation was very active in normal cells, the addition of succinate had little effect. Apparently the oxidation of the endogenous substrates was sufficient to account for most of it. In heated cells the addition of succinate restored assimilation at a rate which approximated that of normal cells. Concentrations of drugs such as streptomycin, aureomycin, azide, and dinitrophenol, which did not affect normal assimilation, had also little effect on assimilation of heated cells in the presence of succinate. The same concentrations of these drugs did, however, inhibit assimilation in heated cells in the absence of succinate by prolonging the recovery of the endogenous mechanisms. This is shown for azide in Fig. 4. In order to determine in part the fate of the assimilated nitrogen, largescale experiments were done on the heated cells alone, in the presence of ammonia, and ammonia plus succinate. The experiments were carried
120 MINUTES
FIQ.
3. The
ammonia
effect
of two assimilation
concentrations of succinate on the rate in cells heated at 51” for 10 min.
of
AMMONIA
ASSIMILATION
455
MINUTES FIG. 4. The effect of 0.15 mg./ml. sodium azide on the ammonia assimilation by cells heated at 51” for 10 min. in the presence and absence of 1.0 mg./ml. succinate. The first two curves on the left represent assimilation by heated cells in the presence of succinate with and without azide. The next two curves represent assimilation by heated cells without succinate in the presence and absence of azide. The two curves on the right represent assimilation by unheated cells in the presence and absence of azide.
out in Erlenmeyer flasks with six times the usual amount of cells, (NHSzSO~ , and succinate in six times the usual volume. The incubation lasted 2 hr. so that some assimilation occurred in the absence of succinate. At the end of the incubation the cells were centrifuged from the suspending medium and washed twice with distilled water. They were then taken up in 1 N HCl and subjected to mild hydrolysis in a boiling water bath for 15 min. Ammonia nitrogen in the suspending medium was estimated by the method of Speck (3) to determine the amount of nitrogen assimilated, and labile nitrogen was estimated on the supernatants of the cells heated in acid. The precision of this method is =t3 %. From 1% to 2 % of glutamine is hydrolyzed during distillation and appears as free ammonia. Aliquots of the acid extracts were also chromatographed on paper using the following solvents: butanol-acetic acid-water (80: 20 : 20) ; water-saturated phenol; ethanol-ammonium hydroxide (95 : 5) ; and pyridine-water (65: 35). Such extracts contained glutamic acid, aspartic acid, lysine, alanine, and a spot not positively identified which may be -r-aminobutyric acid. The extracts were chromatographed on heavy paper in the butanol or phenol solvent, the paper was cut in strips and eluted with water, and the eluates were rechromatographed in the four solvents. Comparisons were made between the eluates, known compounds, and mixtures of the eluates and known amino acids.
456
BERNHEIM
AND
DETURK
Cells in the presence of (NHd)zSOd assimilated 64-141 pg. of nitrogen. In the presence of succinate the assimilation was increased to 201-336 pg. of nitrogen. The ammonia liberated by hot 1 N HCl was also increased. The increases in labile nitrogen were a function of the amount of ammonia assimilated whether succinate was present or not, and accounted for 10-23 % of the nitrogen taken up. This indicates an increase in glutamine in cells in both cases. For example, in a typical experiment, the labile nitrogen of control cells was 100 pg., cells incubated with ammonia assimilated 64 ,ug. of NH3 N and the labile N was 107 pg., and cells incubated with ammonium sulfate and succinate assimilated 336 pg. and the labile N was 155 pg. The labile ammonia might be present in the cell either as ammonia liberated by heating, or as a labile compound, hydrolyzable under these mild conditions. To determine this, cells were subjected to sonic vibration for 1 hr., acidified to pH 2.0, and centrifuged in the cold, and ammonia was estimated on the supernatant before and after heating in 1 N HCI. Free ammonia was present in small amounts, but accounted for only a fraction of the ammonia present after hydrolysis. Sonic extracts were subjected to chromatography and were found to contain glutamine, not present in the hot acid extracts. In addition, when the material was chromatographed on heavy paper which was cut in strips and eluted, labile ammonia was found only in the glutamine-containing eluate. Glutamine has not been previously found to be a constituent of acidfast organisms, though the other amino acids were found in hot water extracts of various mycobacteria by Pauletta and Defranceschi (4). DISCUSSION
Foulkes (5) has shown that there are two mechanisms in yeast for the assimilation of potassium ion: one, azide-sensitive and dependent on endogenous metabolism; the other, azide-insensitive which is activated by addition of glucose and phosphate. The ammonia assimilation in these mycobacteria is also effected by two independent systems, one of which is more temperature-sensitive than the other. Temperature affects assimilation by inactivating an unknown mechanism since the autorespiration of the heated cells and their oxidation of added substrates are normal, and there is no evidence of uncoupling of oxidation and phosphorylation. Since the addition of succinate to heated cells caused immediate assimilation, it is unlikely that its oxidation is providing more adenosine triphosphate (ATP) or glutamic acid since a lag period should
AMMONIA
ASSIMILATION
457
be evident before these compounds accumulated. The mechanism of assimilation in the presence of an exogenous substrate is therefore not clear. Once the ammonium ion enters the cell, glutamine is formed whether the energy source has been endogenous or exogenous. But addition of glutamic acid or glutamine has no direct effect because both are oxidized and deaminated, and ammonia assimilation occurs only as the result of the oxidation. SUMMARY
1. Mycobacterium A.T.C.C. No. 8420 heated to 51” for 10 mm. loses its ability to assimilate ammonium ion, although the autorespiration and oxidation of added substrates is normal and there is no evidence of uncoupling of oxidation and phosphorylation. Recovery occurs after 90-120 min., but the rate of assimilation is still slower than normal. Certain drugs prolong the recovery period. 2. Addition of succinate or certain other substrates when no assimilation is occuring causesimmediate assimilation which continues as long as there is substrate to be oxidized. This processis not sensitive to drugs in concentrations which prolong the recovery of the “endogenous” assimilation. 3. Apparently two separate mechanisms exist in these cells for ammonium ion assimilation: one activated by endogenous metabolism, the other by the metabolism of exogenous compounds. 4. Glutamine and a number of amino acids have been identified in the heated cells following assimilation both in the presence and absence of succinate. REFERENCES 1. BERNHEIM, F., Arch. B&hem. Biophys. 69, 252 (1955). 2. HALTEREN, P. VAN, Bull. sac. chim. biol. 32, 458 (1950). 3. SPECK, J. F., J. Biol. Chem. 149, 1387 (1949). 4. PAULETTA,G.,AND DEFRANCESCHI, A., Biochim.et Biophys.Acta9,271 5. FOULKES, E. C., J. Gen. Physiol. 39, 687 (1956).
(1952).