Nitrate Reductase and Respiratory Adaptation in Bacillus Stearothermophilus R

JOURNAL OF BACTERIOLOGY, Feb., 1966 Vol. 91, No. 2 Copyright © 1966 American Society for Microbiology Prined fn U.S.A. Nitrate Reductase and Respiratory Adaptation in Bacillus stearothermophilus R. J. DOWNEY Lobund Laboratory, Department ofBiology, University ofNotre Dame, Notre Dame, Indiana Received for publication 13 September 1965

ABSTRACT DOWNEY, R. J. (University of Notre Dame, Notre Dame, Ind.). Nitrate reduc- tase and respiratory adaptation in Bacillus stearothermophilus. J. Bacteriol. 91:634- 641. 1966.-Bacillus stearothermophilus 2184 required nitrate to grow in the ab- sence of oxygen. Like many facultative microorganisms, the growth obtained anaerobically was considerably less than that obtained aerobically, even though the dissimilatory reduction of nitrate is, in effect, anaerobic respiration. The ability to re- duce nitrate depended on the induction of nitrate reductase. Although oxygen at low levels did not retard induction of the enzyme, enzyme synthesis was consider- ably lessened by aeration. A semisynthetic medium containing nitrate supported aerobic growth of the thermophile but did not support anaerobic growth. The adaptation to nitrate resulted in a decrease in the level of cytochrome oxidase nor- mally present in aerobically grown cells. Although the aerobic oxidation of suc- cinate by the respiratory enzymes from aerobically grown cells was inhibited by 2-N-heptyl-4-hydroxyquinoline-N-oxide, the anaerobic oxidation of succinate by nitrate in a similar preparation from nitrate-adapted cells was not. The nitrate reductase in the bacillus was strongly inhibited by cyanide and azide but not by carbon monoxide. The nitrate reductase catalyzed the anaerobic oxidation of re- duced nicotinamide adenine dinucleotide, and appeared to transfer electrons from cytochrome bi to nitrate. Cytochrome cl did not appear to be involved in the trans- fer.

Bacillus stearothermophilus, like most aerobic dant serve to limit growth on a complex spore formers, utilizes an aerobic respiration medium. whenever the supply of oxygen permits. As a deni- In addition to describing the changes noted in trifler, the organism possesses the ability to respire the constitutive oxidative components of the anaerobically with nitrate as an oxidant. To do bacillus after adaptation to nitrate, this report so, an adaptive enzyme, nitrate reductase, must be includes some observations on the relationship synthesized. In addition to the presence of nitrate, of nitrate reductase to the respiratory chain, its one of the conditions necessary for induction of role in anaerobic oxidation of reduced nicotin- nitrate reductase appears to be a low oxygen amide adenine dinucleotide (NADH2), and its tension. An organism possessing both means of behavior in the presence of conventional respira- respiration presumably alters its oxidative tory inhibitors. mechanisms to suit the availability of the oxidant. Although the free energy available from nitrate MATERLALS AND METHODS reduction is less than that available via oxygen Organism, growth medium, and method of cultiva- respiration, it is conceivable that more extensive tion. B. stearothermophilus 2184 was grown on a use of the former could serve as a compensatory medium containing Trypticase (BBL), 20 g; yeast mechanism in meeting the biosynthetic require- extract, 10 g; FeCl136H20, 7 mg; MnCl2.4H20, 15 ments of growth. mg; MgSO4-7H2O, 15 mg; and sucrose, 5 g; in 1,000 Once a culture has adapted to nitrate, the ml of water. Potassium nitrate (1 g per liter) was used where indicated for anaerobic growth. After availability (solubility) of this oxidant, being autoclaving, the medium was adjusted to pH 7.4 considerably greater than oxygen, might permit with sterile KOH. Mass culture of aerobic cells was growth rates comparable to those obtained with achieved in shake flasks rotating at 200 rev/mnn in a oxygen. The experiments reported here show that New Brunswick gyrotary incubator describing a 1- factors other than the availability of an oxi- inch (2.5-cm) circle. Each flask contained 1 liter of 634 VOL. 91, 1966 NITRATE RESPIRATION IN B. STEAROTHERMOPHILUS 635 medium and received approximately 2.8 X 108 mid The NADH2 oxidase was assayed by a method de- log-phase cells as inoculum. After 5 hr of growth at scribed previously (4). The oxidation of NADH2 60 C, the cells were harvested and washed twice with nitrate as sole hydrogen acceptor was observed in phosphate buffer (0.067 M, pH 7.4) and 0.87% with Thunberg cuvettes. The reaction mixture con- KCl (1:1, v/v). tained 0.15 M phosphate buffer (pH 7.6), enzyme (300 Cultivation under anaerobic conditions was ,ug of protein), 20 mm KNO3, 0.72 mm NADH2, accomplished in large carboys (14 liters), filled to the and water to a final volume of 2 ml. When used, in- neck with sterile medium and sparged with purified hibitors were added to the final concentrations indi- nitrogen (300 ml/min) for 90 min prior to receiving cated in the text. After 10 min of preincubation at 1 liter of mid log-phase nitrate-adapted cells. After 6 60 C, the NADH2 was tipped in, and the change in hr of growth at 60 C, the cells were harvested and absorbance at 340 mMu was recorded for 3 to 5 min washed as above. in a Beckman DB spectrophotometer. The activity Growth curves were determined with the use of is expressed as micromoles of NADH2 oxidized per nephelometer flasks containing 100 ml of complex minute per milligram of protein. medium (TY), complex medium with sucrose (TYS), The oxygen consumption of aerobic cells and elec- complex medium with nitrate (TY-NO3), or complex tron-transport particles from such cells was deter- medium with sucrose and nitrate (TYS-NO3). For mined in a Gilson differential respirometer at 125 anaerobic growth, the screw-cap nephelometer flasks oscillations per min with air as the gas phase. The contained medium up to the neck. This was bubbled temperature used in these experiments was 55 C, the with nitrogen prior to addition of inoculum. The maximum attainable with the above instrument. flasks were not shaken. A minimal medium with or For the induction experiments, cells were grown without casein hydrolysate (17) was used in the study aerobically for 3 hr on TYS medium, harvested, of the induction of nitrate reductase. With either the and washed two times with sterile buffer-salts solution complex or minimal medium, the flasks were inocu- containing: Na2HPO4, 2.5 g; KH2PO4, 1.0 g; KCI, lated to an absorbancy of 0.1 to 0.15 and incubated 1.0 g; (NH4)2SO4, 1.0 g; FeCl3-6H20, 5 mg; MgCl2* at 60 C in a rotary shaker. The growth rate is expressed 6H20, 5 mg; and CaCl2-2H20, 5 mg; per liter of dis- by the constant (k) from the relationship dA/dt= tilled water. The washed cells were suspended in 100 kA. A is the absorbancy of the culture and t is the ml of the above buffer solution and added to 300 time in hours. The linear portion of semilogarithmic ml of the minimal medium supplemented with 0.1% plots of growth provided the values for k. casein hydrolysate. The flasks were inoculated to an Preparation of the particulate nitrate reductase. absorbancy of 0.10 to 0.15 and incubated at 60 C The electron transfer particles (ETP) were prepared for 5 to 6 hr. The medium was bubbled with nitrogen from washed sonically disrupted protoplast mem- for 30 min at 60 C prior to adding KNO3 (0.1%). branes of B. stearothermophilus (4). The bulk of Every 30 min during the 5-hr period of adaptation, nitrate reductase activity resided in a particulate samples (2.0 ml) were removed for assay of nitrate fraction obtained after a centrifugation at 140,000 X reductase and dry-weight determination. When in- g for 90 min. The pellet was resuspended in a solution dicated, chloramphenicol (50,ug/ml) was added to the containing 67 mm tris(hydroxymethyl)aminomethane medium. (Tris)-chloride buffer (pH 7.6), 5 mm MgCl2, and Determination of cytochromes. The cytochrome 30 mm KCI, and was sedimented at 60,000 X g for patterns in the ETP from aerobically and anaerobi- 60 min. The pellet, containing 87% of the total nitrate cally grown bacilli were determined by the difference reductase activity of the parent fraction, was used in spectra methods of Chance (2). The spectrum repre- the experiments reported here. sents the difference between oxidized and substrate Enzyme assay procedures. Nitrate reductase was (malate)-reduced particles. A few crystals of potas- assayed anaerobically in Thunberg tubes by the Diazo sium ferricyanide were added to each cuvette to oxi- coupling reaction for nitrate (10). The reaction mix- dize the components of the respiratory chain. Sub- ture contained 0.15 M phosphate buffer (pH 7.6), sequently, the cytochromes in the ETP were reduced enzyme (300 jig of protein), and water in the main by the addition of 0.1 ml of 0.25 M sodium malate to body, and 20 mM KNO3 and 15 mm sodium malate one of a pair of cuvettes. in the side arm, in a final volume of 2.0 ml. The vessel Determination of oxygen concentration during was evacuated and preincubated for 10 min at 60 C growth. The concentration of oxygen during growth prior to tipping in substrate. After 30 min, the tubes was monitored by use of a Clark electrode (Beckman were plunged into crushed ice, and 0.5 ml of 1.0% physiological gas analyzer, model 160). The macro- sulfanilamide was added immediately. After addition electrode was calibrated against 0.1 M phosphate buf- of 0.5 ml of 0.02% N-(l-naphthyl)-ethylenediamine fer (pH 7.4) saturated with oxygen at 60 C (700 ,AM). dihydrochloride to develop the color, the tube con- The electrode was inserted in the side port of a special tents were cleared of precipitate by sedimentation, the of the shake flask. The rotary motion of the medium served and absorbancy supernatant liquid was renew the surface of the electrode. determined at 540 mM4. A standard containing a known to continually The concentration of nitrite was constructed in a mixture proportions of gases (N2:02:C02) in the mixture similar to the above but lacking substrate and ni- admitted to the incubator were adjusted periodically trate. A blank mixture, lacking substrate, was used to achieve the desired P02 in the growth medium. to correct for any endogenous activity that prevailed. Protein determinations. The method of Lowry et al. The activity is expressed as micromoles of nitrite (7) was used for protein. produced per 10 min per milligram of protein. Inhibitors. The inhibitors used in this study were 636 DOWNEY J. -BAC"MIL.' obtained as follows: antimycin A, 2-N-heptyl-4- hydroxy-quinoline-N-oxide (HOQNO), 2-N-nonyl- 4-hydroxy-quinoline-N-oxide, Sigma Chemical Co., St. Louis, Mo.; quinacrine, Nutritional Biochemicals Corp., Cleveland, Ohio; p-chloromercuribenzoate, Mann Research Biochemicals, New York, N.Y.; sodium-amytal, Eli Lilly & Co., Indianapolis, Ind.; potassium cyanide, sodium azide, ethylenediamine- k tetraacetic acid, Fisher Chemicals, Chicago, Ill.; and 8-hydroxy-quinoline, Mallinckrodt Chemical Works, St. Louis, Mo. RESULTS Influence of oxygen tension on the growth of B. stearothermophilus. At a pressure of one atmos- phere, assuming saturation of growth medium with air, the maximal oxygen concentration available to a culture at 60 C was calculated to be U 10 20 30 40 approximately 140 ,Mm or 0.28 ,uatom per ml. FKN03] mM Measurement via a Clark electrode indicated that the level of oxygen in the growth medium at 60 FIG. 2. Effect ofnitrate concentration on the anaero- C was 121 mm of Hg or 139 ,UM, a value which is bic growth rate of Bacillus stearothermophilus l com.- in good agreement with that expected. It can be plex and supplemented minimal medium. Growth rates seen in Fig. 1 that the yield in cell mass of bacilli, were determined as described in Materials and-Methods. In each experiment, the inoculum consisted oflate log- after a prescribed period of incubation on TYS cells. medium, was a function of the phase nitrate-adapted Temperature, 60 C. Sym- p02 during growth. bols: *, complex medium (TYS); 0, minimal me- The growth yield was optimal at or near the oxy- dium (MCHS) with casein hydrolysate, sucrose (0.5%); gen concentration normally available in the broken line, minimal medium (0.5% sucrose). mesophilic range of temperatures, viz., 137 to 147 min of Hg (143 to 240 ,uM). Increasing the Influence of nitrate on the growth rate of baCil- oxygein concentration beyond this point retarded lus. The anaerobic growth rate of B. stearother- the griowth of the thermophile (Fig. 1). mophilus increased in proportion to the nitrate provided (Fig. 2). At a concentration otappri - 10 mately 20 mm nitrate, the k value was maximal. Higher concentrations of nitrate had no effect on 0 the growth. Assuming the above concentration of oxygen (140 ,uM) to be the maximum attainable at 60 C, under the conditions specified, the initial 0 level of nitrate available in the medium (20 mM) was approximately 143 OEA0.E times this. Nitrate, as a a a O 0 / \ nonreplenishable oxidant, conceivably declined 0 _ \ as a function of growth and eventually became ): rate-limiting unless it was provided at an initial concentration of 20 mM. At a. O / \ this concentration, factors other than nitrate seem to limit the ability w to grow anaerobically. E D 9t \ In an attempt to assess the bis quirements of the organism during a ______._._.___. to nitrate, a minimal medium supplen it 0 60 120 180 240 300 casein hydrolysate was employed. Thib,iu supported aerobic growth of B. stearotheroph- PO2 (mm Hg) ilus. The requirements for synthesis of mtr reductase FIG. 1. Effect ofoxygen concentration on the growth obictgotcouldwasnotnotbeberveddetermined,o sinpeiniaar-.mer rate ofJBacillus stearothermophilus on complex medium. contai notrabsewved onwh sein The oxjygen concentration during growth was monitored dium contining nirate with or withou .cei- as desc-ribed in Materials and Methods. The total hydrolysate (Fig. 2). period (of growth for each experiment at a given PO2 The addition of a carbon sourcem(( was 4 hi r. Each point on the curve represents the average increased the aerobic growth rate of B. `-Shar- ofat lec1st three experiments. thermophilus, but produced a negligible effect on VOL. 91, 1966 NITRATE RESPIRATION IN B. STEAROTHERMOPHILUS 637

TABLE 1. Influence ofaerobiosis and anaerobiosis on the growth rate of Bacillus stearothermophilus on complex and minimal media with and without added nitrate o50

k* - Medium Aerobic Anaerobic 30 TY ...... 0.76 0.01 0 0 ...... 0.03 ML-20

0.05 ~~~~~~0.04 0 0O.04 \o__- / A )003WAVELENGTH ( 0*02* . - 0.08 0.02 420 460 500 5 10 15 20 Z - 0.06 I ~~~~SECONDSw2 ' '- 0.0I . I Z w 0.04 I. ~~~~~z 0.00 9 w 0+0.02 z 520 540 560 580 600 620 * 0.00 WAVELENT(mu)cvtWAVELENGTH (mY) FiG. 5. Influence of nitrate on the cytochromes in -0.02 the ETP from nitrate-adapted cells. Spectra represent 4.0 mg/mi ofparticle protein in 0.1 ms phosphate buffer 420 460 500 540 580 620 ~~~500(pH 7.6). A = malate reduced vs. ~~~~~~~~ oxidized, reference cuvetle shaken to admit air. B = malate reduced vs. WAVELENGTH (mM) nitrate oxidized, reference cuvette maintained in FiG. 4. Difference spectra of ETP from aerobic vacuo; the KNO, (40 iAmoles) was added from. the and anaerc)bic (nitrate-adapted) cells. Spectra repre- side arm after evacuation of the cuvette. C = oxidized sent 4.5 mg?/ml ofparticle protein in 0.10 mf phosphate vs. oxidized, both cuvettes shaken to admit air. oufer (pR 1.0). A = malate reaucea vs. oxiaizea in ETP from aerobically grown cells. B = malate re- duced vs. nitrate oxidized in ETP from nitrate-adapted cells. Inset: Reoxidation of malate-reduced cyto- chrome b by nitrate. System contained: phosphate buffer, 0.10 M, pH 7.6; ETP, 6.0 mg/ml of protein; malate, 15 mm; nitrate, 10 mm; HOQNO, 2.0 mm. m = malate added at zero-time; n = nitrate added C. at 15 sec; solid line, ETP; dashed line, ETP + 0 HQNO; dotted line, ETP, no nitrate added; dash- -I dot line, ETP, no malate added. Temperature, 60 C. of reduced cytochrome b and ultimate reoxida- tion of the remaining cytochromes. The oxidation of cytochrome b from nitrate- adapted Bacillus cells was not inhibited by HOQNO (Fig. 4, inset). The introduction of substrate (m in figure) quickly reduced the MINUTES cytochrome b, whereupon the addition of nitrate FIG. 6. Inhibition of oxygen consumption in whole (n in figure) oxidized it more rapidly. The nitrate cells and ETP from Bacillus stearothermophilus in the if it represents the terminal of presence of HOQNO. Reaction vessels contained: reductase, segment phosphate buffer, 100 mm, pH 7.4; cells, 2.0 pig (dry an adaptive pathway in this preparation, did not weight) or ETP, 0.54 mg of protein; sueclnate, 20 appear to be rate-limiting. The data presented as mm; HOQNO, 50 ,g/ml; and water to a total-v"olme nitrate reductase actively in the ETP should be of 2.0 ml. Cells were grown aeroblcallyr on 'dhw0e interpreted accordingly. medium. Symbols: *, ETP; 0, cells; *, HOQAVQ The effects of several classical inhibitors are added at zero-time; 0, HOQNO add at 15 min. indicated in Table 2. From these data, it can be Temperature, 55 C. Values have been corrected for seen that metal-binding agents were the most the endogenous oxygen uptake. effective against the action of nitrate reductase. With regard to the effect of inhibitors, there was, the particle-bound enzyme more soluble WeMot in general, good correlation between the behavior successful. The distribution of nitrate reductase of a natural substrate (malate) and an artificial activity from a sonically disrupted cell mem e substrate (benzyl viologen). Several attempts to fraction was such that 80% of the total activity alter the susceptibility to inhibitors by rendering was retrieved in the supernatant liquid after VOL. 91, 1966 NITRATE RESPIRATION IN B. STEAROTHERMOPHILUS 639

TABLE,2.T Effect of inhibitors on nitrate reductase* from Bacillus stearothermophilus

Inhibition Inhibitor Concn Substrate A Substrate B Substrate C

KCN ...... 1.5 X 10-3 M 95 97 93 NaN3 ...... 2.0 X 10-3 M 76 65 87 Quinacrine...... 1.5 X 10-5 M 69 0 73 2-N-heptyl-4-hydroxyquinoline-N-oxide. 50 ,ug/ml 13 0 0 2-N-nonyl-4-hydroxyquinoline-N-oxide ...... 30 ,Ag/ ml 10 0 0 Antimycin A...... 5.0 X 10-4 M 0 0 0 Carbon monoxide...... 5-min bubbl- 0 0 0 ing 8-Hydroxyquinoline ...... 5.0 X 10-2 M 71 95 100 p-Chloromercuribenzoate ...... 4.0 X 10-3 M 0 0 0

Sodium amytal ...... 3.0 X 10 3 M 0 0 0

Ethylenediaminetetraacetate ...... 2.0 X 10-2 M 89 93 100

* The standard assay was performed as described in Materials and Methods. Each tube contained: phosphate buffer, 0.15 M, pH 7.6; KNO3, 20 mM; protein, 300 MAg; malate (substrate A), 15 mM; reduced benzyl viologen (substrate B), 3.0 mM; or succinate (substrate C), 15 mM; and inhibitor in the concentration indicated. Temperature, 60 C. sedimentation at 20,000 X g for 30 min. Sub- TABLE 3. Aerobic and anaerobic oxidation of re- sequently, 87% of the nitrate reductase activity duced nicotinamide adenine dinucleotide by the in this fraction resided in the pellet obtained after nitrate reductase system from anaerobically a centrifugation at 144,000 X g for 90 min. The grown Bacillus stearothermophilus* use of detergents like deoxycholate, Tween 80, Activity or Triton-X-100 resulted in a sizable loss of System (donor-acceptor) condition moles nitrate reductase activity when they were used on mg-i) the particulate fraction. Oxidation of NADH2 by the nitrate reductase 1. NADH2-02 Aerobic 1.66 system. Since aeration retarded synthesis of 2. NADH2-NO3- Anaerobic 3.86 nitrate reductase in B. stearothermophilus, it was 3. NADH2-02 + N03- Aerobic 4.83 of interest to test the ability of ETP from 4. NADH2-NO3- + HOQNO Anaerobic 3.50 anaerobically grown cells to oxidize NADH2 with (1.8 X 103 M) oxygen in the presence and absence of nitrate as 5. NADH2-NO- + 02 + Aerobic 3.03 an electron acceptor. The ETP from nitrate- HOQNO (1.8 X 10-3 M) 6. NADH2-02 + HOQNO Aerobic 1.57 adapted cells catalyzed the aerobic oxidation of (1.8 X 10-3 M) NADH2 at approximately one-third the rate of 7. NADH2-NO3- + KCN Anaerobic 0.11 the anaerobic oxidation with nitrate as the (2.0 X 10-3 M) electron acceptor (Table 3). The enzyme was 8. NADH2-NO3- + CO (5- Anaerobic 3.75 inactive with reduced nicotinamide adenine min bubbling) dinucleotide phosphate as substrate. Addition 9. NADH2-NO3- + 02 + Aerobic 3.56 of nitrate to the aerobic system (No. 3 in Table CO (5-min bubbling)

3) appeared to be additive. As with the aerobic * oxidation of malate (Fig. 5), HOQNO had a Reaction mixture contained: phosphate buffer, 0.15 M, pH 7.6; KNO5, 20 mM; NADH2, notable effect on the aerobic oxidation of NADH2 0.72 mM; protein, 300,g; and inhibitor in the con- but no appreciable effect on the anerobic oxida- centration indicated. The system was incubated tion with nitrate as electron acceptor (Table 3). (60 C) for 15 min in presence of inhibitors before Whereas carbon monoxide produced little inhibi- addition of substrate. Temperature, 60 C. tion, cyanide proved to be quite effective against the oxidation of NADH2 by the ETP from aerobic and anaerobic pathways. Nitrate did not nitrate-adapted cells. prove to be a complete substitute for oxygen in the growth of B. stearothermophilus on a semi- DiscussioN synthetic medium. Under aerobic conditions, the The reduction of nitrate appears to be an presence of nitrate made little difference in the adaptive type of respiration employing certain growth rate. This indifference correlates with the catalytic components which are common to both repression effect that oxygen exerted on the in- 640 DOWNEY J. BACTERtOL. duction of nitrate reductase in this organism. electron transfer only from cytochrome b1, then Under conditions of suitable aeration, the nitrate the participation of cytochrome cl in nitrate res- reductase was not detectable to any appreciable piration of B. stearothermophilus appears ques- extent, whereas in the anaerobic state the enzyme tionable at the present time. was induced and growth was proportional to the Further evidence that cytochrome cl is not a concentration of nitrate. functional intermediate in the reduction of nitrate In addition to the sizable difference in response in the thermophile is suggested in the insensitivity to nitrate versus oxygen as a terminal oxidant, the of the nitrate reductase to the 2-N-alkyl-hydroxy- thermophile exhibited a rather sharp decline in its quinoline-N-oxides, which are currently thought ability to grow at elevated oxygen levels (Fig. 1). to be specific in the inhibition of the transfer of It thus seems that certain metabolic mechanisms electrons from cytochromes b1 to cl. The aerobic are attuned to a concentration of oxygen that is oxidation of succinate appeared to involve the attainable at the optimal growth temperature of HOQNO-sensitive site, but the anaerobic oxida- many mesophilic organisms, and is quite intol- tion via nitrate did not (Table 2). The indifference erant of oxygen levels in excess of this. of the anaerobic oxidation of NADH2 to HOQNO That nitrate serves its role as an oxidant by a (No. 4, Table 3) would seem to support further mechanism which is distinct and possibly only the notion that the cytochrome b to nitrate indirectly related to that of oxygen is suggested transfer does, in effect, circumvent the HOQNO- by the fact that optimal growth in nitrate-adapted sensitive site. However, since the aerobic oxida- cultures required nitrate levels over 100-fold tion by this same preparation was insensitiv to greater than the oxygen levels which provided HOQNO (No. 6, Table 3), the aerobic oxidation optimal growth in aerobic cultures. In support of NADH2 could involve a separate pathway. of a direct relationship, however, is the fact that The oxidation of NADH2 by the ETP from oxygen not only suppressed formation of nitrate nitrate-adapted cells occurred with either nitrate reductase but also appeared to influence the level or oxygen, or both, as electron acceptor. The of cytochrome a3, which is a constitutive enzyme effects were additive in the case where both in the thermophile. acceptors were present. Just which one of the Instead of an outright anaerobic condition, a pathways mediates this oxidation preferentially lowered oxygen concentration in the growth will be difficult to discern unless the rate forma- medium apparently was sufficient to induce the tion of nitrite and the rate consumption of synthesis of nitrate reductase. Vigorous aeration oxygen are measured and compared in a reac- retarded synthesis of the enzyme, but synthesis tion in which NADH2 is provided in substrate began rather quickly after cessation of aeration amounts. (Fig. 3). Although there was practically no lag In general, the effects of inhibitots on the in the synthesis after the termination of aeration, nitrate reductase from B. stearothermophilus are there was a characteristic lag after the start of the in good agreement with those reported for other induction experiment. The lag after the aeration systems. A closer scrutiny of the effects of these period may be shortened because most of the inhibitors will doubtless require an enzyme of adaptation to the medium, etc., occurred during substantially greater purity. the initial lag period. Also, the aerobically grown The soluble nitrate reductases from Neurospora cells used in the induction experiment would, (9), Achromobacterfisheri (12), and Pseudomonas by virtue of their respiration, consume the oxygen aeruginosa (5) have been characterized relative to in the medium to the point where earlier induc- electron donors, metal ion dependence, and sen- tion and synthesis could proceed. Although the sitivity to inhibitors. Other bacterial nitrate minimal level of oxygen that just represses reductases, from Escherichia coli (16), Rizobium nitrate reductase formation in growing or resting japonicum (3), and Micrococcus (1), have been cells of B. stearothermophilus has not been as- membrane-bound in their original physical state, certained, it should be possible to do so by the and in both soluble and particulate systems hrt techniques described here. these organisms cytochrome b is believed to The alterations in the respiratory chain. which mediate the transfer of electrons from flavin to accompanied adaptation to nitrate appeared to nitrate reductase (8). involve only the terminal oxidase (cytochrome In view of the evidence presented in this report, a3). It is interesting that nitrate did not reoxidize the nitrate reductase system from B. sterother- malate-reduced cytochrome cl in the ETP from mophilus is not appreciably different fromt anaerobically grown cells (Fig. 5). Reoxidation systems mentioned above. However, there are a did occur, although quite slowly, with aeration. few features which indicate that the system in the If the anaerobic state suppresses formation of thermophile may be somewhat unique.' cytochrome as and if nitrate reductase catalyzes In many of the systems mentioned above, thie VOL. 91, 1966 NITRATE RESPIRATION IN B. STEAROTHERMOPHILUS 641 reduction of nitrate was inhibited by HOQNO. diate in the respiration of Bacillus stearother- The indfference of the enzyme in the thermophile mophilus. J. Bacteriol. 84:953-960. would seem to set it apart from these in its 5. FEWSON, C. A., AND D. J. D. NICHOLAS. 1961. Nitrate reductase from Pseudomonas aerugi- relation to the established HOQNO-sensitive nosa. Biochim. Biophys. Acta 49:335-349. segment (cytochrome bi to cytochrome cl) in the 6. LIGHTBOWN, J. W., AND F. L. JACKSON. 1956. electron transfer sequence derived from aerobi- Inhibition of cytochrome systems of heart cally grown cells. In contrast to the report of muscle and certain bacteria by antagonists of Sato (13), the reduced steady state of the cyto- dihydrostreptomycin: 2-alkyl-4-hydroxyquino- chromes in B. stearothermophilus is affected by line-N-oxides. Biochem. J. 63:130-137. the nitrate reduction process. 7. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, Regardless of the difference in reaction with AND R. J. RANDALL. 1951. Protein measure- inhibitors, the reduction of nitrate seems to ment with the Folin phenol reagent. J. Biol. Chem. 193:265-275. involve a heme protein, probably cytochrome 8. NASON, A. 1962. Symposium on metabolism of bi. The nitrate reductase from Nitrobacter agilis inorganic compounds. II. Enzymatic pathways (15) contains cytochrome b, c, a, and a,, in of nitrate, nitrite, and hydroxylamine metabo- addition to cytochrome oxidase. The partially lisms. Bacteriol. Rev. 26:16-41. purified enzyme is a heat-labile particulate 9. NASON, A., AND H. J. EVANS. 1953. Triphos- enzyme which uses cytochrome c as electron phopyridine nucleotide-nitrate reductase in donor. It is insensitive to carbon monoxide and Neurospora. J. Biol. Chem. 202:655-673. sensitive to cyanide and azide. 10. NICHOLAS, D. J. D., AND A. NASON. 1957. De- A good deal more characterization of the termination of nitrate and nitrite, p. 983-984. here will be essential In S. P. Colowick and N. 0. Kaplan [ed.], nitrate reductase described Methods in enzymology, vol. 3. Academic to an adequate understanding of the respiratory Press, Inc., New York. adaptation which occurs in Bacillus. To properly 11. PICHINOTY, F., AND L. D'ORNANO. 1961. In- assess the energetics of growth of Bacillus during hibition by oxygen of biosynthesis and activity nitrate adaptation, experiments of the type of nitrate-reductase in Aerobacter aerogenes. described by Senez (14) might be indicated. The Nature 191:879-881. role of nitrite reductase in the enzymatic transfer 12. SADANA, J. C., AND W. D. McELROY. 1957. of electrons from nitrite to molecular oxygen, the Nitrate reductase from Achromobacter fischeri. extent to which phosphorylation is coupled, and Purification and properties: function of flavins Arch. the influence of oxygen on the terminal oxidases and cytochrome. Biochem. Biophys. 67:16-34. of this facultative microorganism are but a few of 13. SATO, R. 1956. The cytochrome system and many problems which require intensive study. microbial reduction of nitrate, p. 163-175. In W. D. McElroy and B. H. Glass [ed.], ACKNOWLEDGMENTS Symposium on inorganic nitrogen metabolism. Baltimore. This investigation was supported by Public Health Johns Hopkins Press, Service grant Al 05820 from the National Institute of 14. SENEZ, J. C. 1962. Some considerations on the Allergy and Infectious Diseases. energetics of bacterial growth. Bacteriol. Rev. 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