Influence of Glutamic Acid on the Endogenous

Transcription

1 JOURNAL OF BACTERIOLOGY, Feb., American Society for Microbiology Influence of Glutamic Acid on the Endogenous Respiration of Bacillus subtilis C. E. CLIFTON AND JOHN CHERRY Department of Medical Microbiology, Stanford University School ofmedicine, Stanford, California Received for publication 23 August 1965 ABSTRACT CLIFTON, C. E. (Stanford University, Stanford, Calif.), AND JOHN CHERRY. Influence of glutamic acid on the endogenous respiration of Bacillus subtilis. J. Bacteriol. 91: Amino acids serve as the major initial endogenous substrate for Bacillus subtilis. The endogenous activity of freshly harvested washed cells is high and falls off rapidly with time of shaking at 30 C to lower but still significant levels. The rate of 02 consumption after the addition of glutamic acid also decreases as the cells age, but more slowly than noted for endogenous respiration. When cells were fed glutamate as soon as possible after harvesting, an apparent stimulation of endogenous respiration was noted. However, endogenous activity was inhibited if the cell suspensions were shaken for at least 1 hr before addition of the glutamate. Similar results were obtained with glycerol or glucose as exogenous substrates. Variation in rates of respiration with age of the cells, inherent instability of B. subtilis, and possible utilization of substances initially excreted by the cells appear to account for the variations noted regarding the influence of an exogenous substrate on endogenous respiration. Studies of endogenous metabolism, reviewed by Dawes and Ribbons (5, 6) lead to the conclusions that different cell constituents serve as the substrates of endogenous respiration in different organisms, and that the extent of degradation of individual substrates appears to depend on their initial concentrations in the cells. The concentration of each substrate is determined by the nutritional status of the organism at the time of harvest and by subsequent changes which occur on washing and aging the cells. One problem in studies on endogenous respiration is the extent to which endogenous respiration continues in the presence of an exogenous substrate. Various procedures have been employed in attempts to solve this problem. The combined manometric-isotopic technique described by Blumenthal (1) has generally proved to be the most satisfactory. Variable results, however, were obtained by using this method in the present study with Bacillus subtilis as the test organism and glutamate as the exogenous substrate. Cell suspensions frequently are aerated for various periods of time to reduce endogenous respiration before the addition of an exogenous substrate. Therefore, the influence of aeration, and hence short periods of aging (starvation) of the cells, on endogenous respiration in the 546 Vol. 91, No. 2 Printed In US.A. absence or presence of glutamate was studied in more detail. MATERIALS AND METHODS The methods employed in this study were for the most part the same as previously described (3, 4). B. subtilis D76 was grown for 16 hr at 30 C on nutrient agar (Difco). Cells were washed twice at room temperature in a buffered salts solution of the following composition: NaCl, 1.0 g; K2HP04, 8.4 g; KH2PO4, 1.7 g; MgSO4.7H20, 0.2 g; ethylenediaminetetraacetic acid, 0.2 g; Fe2(SO4)3nH20, 10 mg; CaCI2, 10 mg; trace salts (10), 5 ml; and water to 1,000 ml (ph adjusted to 7.2). The final suspension in buffered salts solution was adjusted to a Klett- Summerson colorimeter (54 filter) reading of 125 for a 1:10 dilution, which corresponds to 2.6 mg (dry weight) of cells per ml of the freshly prepared suspension. All tests were conducted at 30C with air as the gas phase in either Warburg or Gilson respirometers. A 2-ml amount of the cell suspension, 0.2 ml of 0.04 M uniformly labeled glutamic acid (8 jimoles) or of water (unless otherwise noted), 0.2 ml of 10% sulfuric acid (if the suspensions were to be acidified), and 0.2 ml of 20% KOH were added to two-arm Warburg vessels of around 18-ml volume. Alkali was omitted from the center well in the vessels in which total CO2 production was to be determined. Acid was tipped into the main compartment to halt respiration and to release bound C02 from the liquid and metabolic pool compounds from the cells. If

2 VOL. 91, 1966 ENDOGENOUS RESPIRATION OF B. SUBTILIS v, 140 b. p X,, _+ 14 TABLE 1. Oxygen consumption, ammonzia production, decrease in cellular nitrogen, and increase in pentose nucleic acid (PNA) content of supernatant fluids per hour per 2 ml of a suspension of Bacillus subtilis Z 120 ef o120- I00 1 (ioo- 0tota NH30nC 50~~~~~~~~~~~~~0 E 60gn 60s ' 40 0 /I i 20a e 20 cen.i sc Time in minutes FIG. 1. (a) 02 conisumption per 1-mmi intervals and total no acid NHs-N wa production, ode,tewrur and (b) C"I-distribution lsswr lce (suspensiones acidified) with time in 2.2 ml of a washed suspension of Bacillus subtilis. An amount of 8 jamoles (183 mac) ofuniformly labeled glutamic acid was added for exogenous system. no acid was added, the Warburg flasks were placed in an ice bath before samples were taken. In some experiments, respiration was halted by mercuric chloride (0.2 ml of 1.0% HgCl2) with essentially the same results as obtained with the chilling procedure. The C14 content of cells (or fractions thereof), supernatant fluids, and the KOH solutions was determined in the Dynacon system (Nuclear-Chicago Corp., Des Plaines, Ill.) as previously described (3). Ammonia in the suspensions or in Kjeldahl digests was determined by titration with standard acid of steam distillates collected in 2% boric acid solution. In some experiments, ammonia was determined colorimetrically after the addition of Nessler's reagent to boric acid employed as the absorbent in Conway vessels. A 0.2-ml amount of a 50% solution of Rochelle salts was added per colorimeter tube to stabilize the color. Amino acids were determined by the cuprizone colorimetric procedure of Borchers (2). Radioactive areas in paper chromatograms [which had been developed in phenol saturated with m phosphate buffer (ph 12) or in a 6:6:4:1 mixture of methanol, pyridine, water, and acetic acid] were detected with the aid of a 4 pi Actigraph (Nuclear-Chicago Corp.). Pentose was determined by the orcinol method, and pentose nucleic acid (PNA) was estimated from the absorbancy at 260 mjai, the results being expressed in terms of the sodium salt of nucleic acid (General Biochemicals, Chagrin Falls, Ohio).- RESULTS AND DISCUSSION Endogenous substrate. It is apparent from the values recorded in Fig. 1 for ammonia production by cells respiring endogenously that nitrogenous matter serves as a substrate for the endogenous Time Amt of NH-N Ratio of Cell N* PNA increase interval 02 NH- 02-NH3 decrease in supernatant fluid hr palilers lug pg Ag * Initial value of 915,ug of cellular nitrogen per 2 ml. respiration of B. subtilis. This organism does not form poly-,8-hydroxybutyrate, and relatively constant carbohydrate values (anthrone) indicated that polysaccharide is not a major endogenous substrate for cells grown on nutrient agar. When the cells were starved, marked increases with time were noted in absorbancy at 260 m,u of supernatant fluids from nonacidified suspensions. The adsorption spectrum over the range of 220 to 320 m,u was similar to that for ribonucleic acid (RNA). Substances reacting with orcinol, but not with diphenylamine, were released from the cells. These observations suggest release of nucleic acids from the cells. The increases in absorbancy at 260 m,u are reported in terms of PNA in Table 1, but may, at least in part, be caused by other substances. When the suspensions were shaken for longer than 2 hr, the amount of orcinolpositive material in the supernatant fluids tended to decrease, although the absorbancy at 260 m,u continued to increase. Marked decreases (Table 1) in cellular nitrogen also were observed during the course of endogenous respiration, together with corresponding increases in supernatant values. During the 1st hr of endogenous respiration (Table 1), the cellular nitrogen decreased by 12%, but only about 3 % of the cellular nitrogen was converted to NH3-N. NH3-N production per hour decreased to about 1.6% of the cellular nitrogen values for the next 2 hr, and the cellular nitrogen itself decreased by about 5.5% per hr. The most marked change with time in nitrogen distribution in the cells occurred in the cellular fraction soluble in cold 5% trichloroacetic acid. In a typical experiment, the nitrogen content of this fraction decreased from 320,ug per 2 ml of suspension to 228,ug at 60 min and to 140,ug at 120 min. This suggested that the loss occurred from an amino acid pool, which was confirmed by amino

3 548 CLIFTON AND CHERRY J. BAcrERioL Time in minutes FIG. 2. Endogenous 02 consumption per 20-min intervals (() and micromoles ofamino nitrogen (cuprizone test) in supernatant fluids from I ml of acidified (0) and nonacidified (0) suspensions of Bacillus subtilis. acid determinations (cuprizone) on supernatant fluids from acidified and nonacidified suspensions (Fig. 2). Molar ratios of 02 to NH,3 also indicated that amino acids serve as the major, initial endogenous substrate. Gronlund and Campbell (8) reported a ratio of 02 to NH3 of 3.36 for their strain of B. subtilis. A ratio of about 3.0 was noted for the experiment recorded in Fig. 1, and an overall value of 3.6 for the experiment reported in Table 1. It is apparent, however, from the results in Table 1, that the ratio decreased with time, a behavior which suggests utilization of different amino acids or incomplete oxidation. Utilization of glutamate. Typical results for endogenous and exogenous 02 consumption and NHs-N production, together with those for the distribution of C" from 8,umoles of uniformly labeled glutamic acid (183 m,uc), are presented in Fig. 1. Values for 02 consumption per 10-min intervals, rather than cumulative amounts, are plotted against time, since this type of plot illustrates more clearly the observed behavior. Acid was tipped at various times into the main compartment of the Warburg flasks to halt respiration. The results on glutamate utilization recorded in Fig. 1 show a high rate of metabolic activity during the first 40 min after the addition of glutamate to the suspension. By the end of 40 min, the rates of 02 consumption and CO2 or NH3 production were decreasing, unless more than 8,moles of glutamate were added at zerotime. Unpublished results indicate that the initial concentration of glutamate had a marked influence on the metabolic rates, since these rates were much lower when the initial concentration of glutamate was below 0.6,mole per mg (dry weight) of cells. Since acidification releases metabolic pool compounds from the cells, the cell-c'4 values are a measure of the glutamate-c"4 firmly bound by the cells, and not of the total intracellular amounts. This is illustrated by results of a similar experiment wherein a total of 86 m,uc of glutamate-c'4 existed in the cells at the end of 50 min; this decreased to 20 m,uc on acidification of a duplicate sample of the suspension, thus indicating a pool of 66 m,uc. Chromatographic tests indicated that the pool was primarily glutamic acid. Influence of glutamate on endogenous respiration. The results of an experiment designed to determine the extent of endogenous respiration according to the procedure employed by Blumenthal (1) are recorded in Table 2. As is common practice, the suspension was shaken for some time to reduce the endogenous respiration before addition of the glutamate. The initial specific activity of the glutamate was 6.0 muc/,umole of carbon. An amount of 51 m,uc (or 8.5 smoles) of C1402 was produced in 30 min. A total of 15.4,umoles of C02-C was produced; hence, = 6.9,umoles of endogenous C02-C. In the endogenous system, 9.0,umoles of CO2-C was produced during the 30-min test period. The extent of inhibition of endogenous respiration in the presence of glutamate, therefore, was 23% ( x 100/9.0). Similar calculations for 60 and 120 min yielded values of 24 and 9%, respectively. In one experiment with labeled cells (cells grown on nutrient agar plus algal hydrolysate, and not uniformly labeled), there was somewhat less inhibition (ca. 19%) of endogenous respiration during the first 60 min in the presence of glutamate. In some experiments, however, the amounts of Time TABLE 2. Endogenous and exogenous CO2 production by Bacillus subtilis* Exogenous C02 (umoles) Total C402 Difference Endogenous production min * An amount of 8,umoles of uniformly labeled glutamic acid of specific activity of 6.0 mpuc per,umole of C was added to the exogenous system.

4 VOL. 9 1, ENDOGENOUS RESPIRATION OF B. SUBTILIS 549 CO2 not derived from glutamate (Blumenthal calculations) were greater for the exogenous than for the endogenous system. For example, in one experiment, 5.7,umoles of nonglutamate CO2 were produced in 30 min in the exogenous system, and 5.3 in the endogenous system. The variations did not appear to be the result of experimental errors, since results of duplicate experiments agreed closely. Because of these variations, a study was made of the effect of reducing the endogenous rate by shaking the suspensions for various periods of time before the addition of the glutamate. Both the endogenous and exogenous rates of 02 consumption decreased fairly rapidly, but at different rates. This makes it difficult to select any end point for comparative purposes. Tests, therefore, were conducted either for fixed periods of time or of similar amounts of 02 consumption in the exogenous systems. Influence of age of cells on respiration. If a fixed period of time was employed, the amounts of 02 consumed, or of CO2 produced endogenously and exogenously, decreased markedly as the cells aged. For example, 368,uliters of 02 was consumed in 60 min if the cells were harvested and washed as rapidly as possible, and if glutamate was added to the washed cells after 10 min of equilibration. The amount of 02 consumed in 60 min decreased to 53% of the initial value for cells aged 3 hr at 30 C and to 33% for cells shaken for 6 hr. Corresponding values for C'402 production by the aged cells during 60 min were reduced to 71 and 41% of the 65 m,uc liberated by the freshly harvested cells. The differences between 02 and C1402 values reflect the effect of variation in endogenous 02 consumption with time. Production of nonradioactive CO2 during 60 min by the freshly harvested cells in the presence of glutamate was 1.1 times that observed in the endogenous control, indicating slight stimulation of endogenous respiration. The calculated endogenous respiration of the aged cells in the presence of glutamate was reduced to 0.71 and 0.58 of the corresponding endogenous values for cells starved for 3 and 6 hr, respectively. In other experiments, respiration in both endogenous and exogenous systems was allowed to continue until approximately the same total amounts of 02 had been consumed in the exogenous systems. About 1.6 times as long was required for the same amount of respiration after the addition of glutamate to cells aged for 4 hr as for cells tested at zero-time. During this longer time interval, the endogenous 02 and CO2 values in the absence of glutamate were about one-half of those observed in the zero-time tests. A value of 4.3,rmoles was noted for the endogenous CO2 produced by freshly harvested cells, and 4.4,moles in the presence of glutamate. Corresponding figures for cells aged 4 hr were 1.8 and 2.2. As an additional check, similar tests were conducted with 8,moles of non-nitrogenous substrates, uniformly labeled glycerol, or glucose. In 30 min, freshly harvested cells produced 12.2,moles of CO2 in the presence of glycerol, 7.3,umoles of which was derived from glycerol and 4.9 from endogenous sources. During the same time, 3.8,umoles of CO2 was produced in the endogenous system. Cells aged 4 hr required twice as long (60 min) to consume the same amount of 02, and they produced 10.7 jamoles of CO2 from glycerol and 1.0 from endogenous sources, whereas 2.3,moles was produced in the endogenous system. A similar behavior was noted with glucose as the substrate, but the results were complicated by apparent fermentative activities which were more pronounced with glucose as the substrate. In either type of experiment described above, if the cells were aged for 1 hr or longer, the endogenous respiration was reduced after the addition of glutamate, glycerol, or glucose. The addition of substrate to suspensions shaken for shorter periods of time had lesser effect on endogenous respiration, or even had some stimulatory effect. This latter behavior reflects the influence of the high initial rate of endogenous respiration on the effect of an exogenous substrate. Blumenthal (1) reported that the degree of inhibition of the endogenous respiration of Neurospora crassa is influenced by many factors, including starving the cells or nature of the substrate. In contrast to results with B. subtilis, we observed that shaking suspensions of Escherichia coli for various periods of time did not introduce marked variations in the observed behaviors (unpublished data), since the rate of endogenous respiration tended to be low initially and to decrease slowly with time. Also, the exogenous rate of 02 consumption noted after the addition of the substrate was about the same for cells aged as long as 24 hr as for freshly harvested cells. The observed variations in the influence of an exogenous substrate on endogenous respiration also reflect the instability of B. subtilis as reported by Hadjipetrou and Stouthamer (9) and as indicated by rapid decreases in turbidity of our suspensions on shaking, e.g., from a Klett reading of 113 to 100 on shaking for 30 min and to 95 at 120 min. Variations also may be introduced as a result of subsequent utilization of material lost by the cells on shaking the suspensions. Demain, Burg, and Hendlin (7), for example, reported excretion of RNA of high molecular

5 550 CLIFTON AND CHERRY J. BACrERIOL. weight and subsequent extracellular breakdown into smaller utilizable derivatives during growth in broth cultures. We observed that marked and possibly variable losses of cellular materials (unpublished data) also occur on harvesting and washing the cells. ACKNOWLEDGMENTS This study was supported by a grant from the Army Research Office, and by Public Health Service training grant AI 82 to J.C. from the National Institute of Allergy and Infectious Diseases. The technical assistance of Judith Ogrodnik, Laird Bennett, and Margaret Richards is gratefully acknowledged. LITERATURE CITED 1. BLUMENTHAL, H. J Endogenous metabolism of filamentous fungi. Ann. N.Y. Acad. Sci. 102: BORCHERS, R Spectrophotometric determination of amino acids. Anal. Chem. 31: CLIFTON, C. E Endogenous metabolism and oxidative assimilation of typical bacterial species. Ann. N.Y. Acad. Sci. 102: CLFroN, C. E Oxidative assimilation by Bacillus megaterium. J. Bacteriol. 85: DAwEs, E. A., AND D. W. RIBBONS The endogenous metabolism of microorganisms. Ann. Rev. Microbiol. 16: DAWES, E. A., AND D. W. RIBBoNs Some aspects of the endogenous metabolism of bacteria. Bacteriol. Rev. 28: DEMAIN, A. L., R. W. BURG, AND D. HENDLIN Excretion and degradation of ribonucleic acid by Bacillus subtilis. J. Bacteriol. 89: GRONLUND, A. F., AND J. J. R. CAMPBELL Nitrogenous compounds as substrates for endogenous respiration in microorganisms. J. Bacteriol. 81: HADJIPETROU, L. P., AND A. H. STOUTHAMER Autolysis of Bacillus subtilis by glucose depletion. Antonie van Leeuwenhoek J. Microbiol. Serol. 29: MEIKLEJOHN, J The isolation of Nitrosomonas europa in pure culture. J. Gen. Microbiol. 4: Downloaded from on September 11, 2018 by guest