Effects of Selected Inhibitors on Electron Transport in Neisseria gonorrhoeae

Transcription

1 JOURNAL OF BACTERIOLOGY, May 1978, P /78/ $02.00/0 Copyright i 1978 American Society for Microbiology Vol. 134, No. 2 Printed in U.S.A. Effects of Selected Inhibitors on Electron Transport in Neisseria gonorrhoeae ELIZABETH A. KENIMER AND DAVID F. LAPP* Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia Received for publication 2 November 1977 The electron transport system of Neisseria gonorrhoeae was partially characterized by using spectrophotometric, spectroscopic, and oxygen consumption measurements. The effects of selected electron transport inhibitors (amytal, rotenone, 2-heptyl-4-hydroxyquinoline, antimycin A1, and potassium cyanide [KCN]) on electron transfer in whole-cell and sonically treated whole-cell preparations of N. gonorrhoeae were examined. The oxidation ofreduced nicotinamide adenine dinucleotide, measured as a decrease in absorbance at 340, was inhibited by each of the compounds tested. Oxygen consumption stimulated by reduced nicotinamide adenine dinucleotide was also inhibited, whereas oxygen uptake stimulated by succinate and malate was inhibited by KCN alone, suggesting the presence of a KCN-sensitive terminal oxidase. Room temperature optical difference spectra indicate an operational electron bypass around the amytalrotenone-binding site. Difference spectra in the presence of 2-heptyl-4-hydroxyquinoline suggest a possible site of interaction of this compound at the substrate side of cytochrome b. Reduced-minus-oxidized spectra of ascorbatetetramethyl-p-phenylenediamine suggest the participation of b-,' a-, and d-type cytochromes in terminal oxidase activity. Hence, N. gonorrhoeae appears to have an electron transport chain containing cytochrome c, two b-type cytochromes (one of which has an oxidase function), and possibly a- and d- type cytochromes. An abbreviated chain exists through which succinate and malate can be oxidized directly by a KCN-sensitive component. As early as 1934, Fujita and Kodama reported the existence of cytochromes a, b, c, di, and d2 in Neisseria gonorrhoeae (2). Cytochromes c and d2 were found in the largest quantities. No further work was done in this area until Jurtshuk and Milligan reported a method for quantitation of the tetramethyl-p-phenylenediamine (TMPD) oxidase reaction characteristic of the genus Neisseria (3). These investigators found that all true Neisseria have high TMPD oxidase activities in common with Azotobacter, Rhizobium, and Pseudomonas. Later, Winter and Morse observed cytochrome c, two b-type cytochromes, cytochrome o, and, possibly, cytochrome a in membrane preparations of N. gonorrhoeae (10). However, thf sequence of electron transfer through these cytochromes and the participation of these cypchromes in oxidative respiration have not b46n previously reported. The purpose of this study is to describe some of the functions of the respiratory components of N. gonorrhoeae and to compare these functions with those of the respiratory components of other aerobic microorganisms and mitochondra. We report data on reduced nicotinamide 537 adenine dinucleotide (NADH) oxidase activity, optical difference spectroscopy, and oxygen consumption in the presence and absence of electron transport inhibitors. MATERIALS AND METHODS Organism. A clinical isolate of N. gonorrhoeae was used in this study. The strain was identified and checked at regular intervals for classical fermentation and fluorescent-antibody reactions. Cultures were incubated in a candle jar at 370C on single-strength GC medium base (Difco Laboratories) plates supplemented with glucose or acetate (5 g/liter). Several colonies, after 18 to 20 h of growth, were inoculated into screw-capped flasks with 1 liter of medium containing proteose peptone no. 3 (4 g; Difco), NaCl (5 g), K2HPO4 (4 g), KH2PO4 (1 g), glucose or acetate (5 g), and sodium bicarbonate (42 mg). At the early stationary phase (after 18 to 20 h of incubation at 37 C), cultures were harvested and washed once with 0.02 M potassium phosphate buffer (ph 7.4). Although it has been reported that acetate cannot support the growth of N. gonorrhoeae (5), our clinical isolate was found to increase in cell number, utilizing acetate as the sole carbohydrate source, but at a much slower rate than cells utilizing glucose. Cells inoculated into basal medium containing glucose were found to increase in

2 538 KENIMER AND LAPP optical density after a lag of only 2 to 3 h. Cells inoculated into basal medium containing acetate were found to increase in number only after a lag of 6 to 7 h. Whole-cell and sonically treated cell preparations. For whole-cell preparations, washed cells were suspended in 0.02 M K2HPO4 buffer (ph 7.4), and buffer was added until a 10-2 dilution of the suspension had an absorbance at 620 of 1.0 (0.1 to 0.2 mg [dry weight] per ml). For experiments requiring a cell sonic extract, whole-cell preparations were broken in a Sonic 300 Dismembrator (Fisher Scientific Co.) at 80 cps with a Larex tip. Cells were sonically treated for a total of 0.5 min in 6-s bursts. Preparations were swirled in an ice bath for 30 s between bursts. Spectrophotometric determination of NADH oxidation. NADH oxidation was measured as a decrease in optical density at 340, using a Gilford 2000 spectrophotometer. The assay mixture consisted of 1.0 to 2.5,ug of protein in a total volume of 2.5 ml of 0.02 M K2HPO4 buffer. To this mixture, 0.3 umol of NADH was added and measurement of the decrease in optical density was begun immediately. The assay was linear for 1.5 min. Inhibition of NADH oxidation was measured by preincubation of the inhibitor with the assay mixture for 1 min before the addition of NADH. Percent inhibition was derived from a comparison of nanomoles of NADH oxidized per milligram of protein per minute in inhibited and uninhibited samples. Fifty percent inhibitory concentrations were calculated from plots of [I] versus V0/V1. The correlation coefficients for these plots were greater than Optical difference spectroscopy. Room temperature spectra of whole-cell and sonically treated cell preparations were recorded on a Cary model 14 spectrophotometer equipped with a model 1462 scatteredtransmission attachment. The spectrophotometer was calibrated with diluted whole cells or with sonic extract immediately before each scan. Assay mixtures consisted of whole-cell or sonically treated cell preparations diluted with 0.02 M K2HPO4 buffer. Base-line spectra were always established before experimental spectra were measured. Reference samples were oxidized by gently bubbling air through the reference cuvette. Inhibitors were added to samples 1 min before scanning. Determination of oxygen uptake. An oxygen electrode (model 53; Yellow Springs Instrument Co., Inc.) was used to measure oxygen consumption in whole-cell and sonically treated preparations of N. gonorrhoeae. The reaction reservoir contained: substrate (1.0 to umol), whole-cell or sonically treated cell preparation (1.5 to 4.5 mg of protein), inhibitors (0.01 to 10.0 ymol when added), and 0.02 M K2HPO4 buffer (3.0 ml; ph 7.4). Reactions were run at 37 C. Whole-cell or sonically treated cell preparations were introduced into a reservoir containing buffer and allowed to incubate for 3 min before the introduction of the electrode. A base-line endogenous respiration rate was followed for 2 to 3 min before other additions were made. Inhibitors were added 1 min before the addition of substrate. The percent inhibition was derived from a comparison of nanomoles of 02 consumed per milligram of protein per minute in inhibited and uninhibited samples. The 50% inhibitory concentration for each inhibitor and each substrate was calculated from plots of [1] versus V0/V1 with correlation coefficients greater than It was assumed that 0.217,umol of dissolved 02 was present per ml of reaction mixture (1). Miscellaneous measurements. Protein was determined by the phenol method (7), with bovine serum albumin as a standard. Chemicals. The following compounds were ob- *tained from the sources indicated: antimycin Al, 2- heptyl-4-hydroxyquinoline oxide (HQNO), NADH, succinate, and malate from Sigma Chemical Co. Sodium amytal and rotenone were kindly supplied by Sam A. Singal. Other chemicals were obtained from other reliable chemical sources. RESULTS J. BACTERIOL. NADH oxidation studies. Inhibition of NADH oxidation by class I (amytal and rotenone, which block between NADH dehydrogenase and cytochrome b), class II (antimycin A1 and HQNO, which block between cytochromes b and c), and class III (potassium cyanide [KCN], which prevents the reduction of molecular oxygen) inhibitors was studied in sonically treated cell suspensions prepared from cells grown with either glucose or acetate as the major carbohydrate source. Slight differences in the sensitivity to inhibition of glucose-grown and acetate-grown sonically treated cell preparations were found only in the cases of rotenone and KCN (Fig. 1). Table 1 illustrates the 50% and the maximum inhibitory concentrations for the compounds listed above. The level of KCN giving maximum inhibition (40 um) was in agreement with reports of terminal oxidase inhibition in N. catarrhalis by Jurtshuk and Milligan (4). The concentration of amytal (0.8 mm) required to produce 50% inhibition in the gonococcus was 10% that required (10 mm) in the staphylococci (8). Antimycin has been reported to inhibit mitochondrial systems at concentrations of about 5 x 10' M (9). Antimycin A1 at these concentrations was not inhibitory. The concentration of rotenone producing 50% inhibition was 10' M, which corresponds to inhibitory concentrations in mitochondria (9). Optical difference spectroscopy. (i) Spectrscopy studies with sodium dithionite, nitrogen, and KCN. Each of the cytochrome and nonheme iron components of N. gonorrhoeae was totally reduced with sodium dithionite crystals (Fig. 2). The a absorbance at 552 to 553 and the absorbance in the fi region at 524 were indicative of a c-type cytochrome. The broad 562- absorbance and the,8 maxima at

3 VOL. 134, 1978 A 80 EFFECTS OF INHIBITORS ON N. GONORRHOEAE B C 539 NCTENONE (,um) ;t ci 4f It I.. w.j lk IWX D AMYTAL (mm) E MONO(IsM) O :0 4C ANTIMYCIN (M)MKCN (# M) FIG. 1. Effects of inhibitors on NADH oxidation by sonic extracts prepared from cells grown with acetate or glucose as major carbohydrate source. Glucose-grown sonic extract (0) and acetate-grown sonic extract (0) were assayed for their ability to oxidize NADH (measured spectrophotometrically) in the presence of (A) rotenone, (B) amytal, (C) HQNO, (D) antimycin A,, and (E) KCN. TABLE 1. Concentrations ofinhibitorsproducing 50% or maximum inhibition of NADH oxidation measured spectrophotometrically Concn (um) of inhibitor pro- Maducing: mum in- Inhibitor hibitio 50% Inhibi- Maximum in- hibition tion hibition Amytal 832 1, Rotenone HQNO -a Antimycin A, KCN a-, Maximum inhibition below 50%. 528 to 530 were within the range of b-type cytochromes. The observation of a broad absorption band centered around 600 with a 440- Soret shoulder may indicate the presence of an a-type cytochrome. In addition, low extinction of this absorbance and of the Soret 440- absorbance would suggest a relatively small concentration of this component. After observation of similar absorbances, Winter and Morse could not eliminate the possibility that an a-type cytochrome exists in this organism (10). N. cartarrhalis exhibits an oxidized-minus-reduced spectrum similar to that of N. gonorrhoeae, and Jurtshuk and Milligan have suggested that it contains cytochrome a, (4). Fujita and Kodama proposed that the gonococcus contains cytochrome d in addition to cytochrome a (2). Because cytochrome d has an a absorbance at 620 to 630 (,8 absorbance at 518 to 522 ), the combined a region absorbances of cytochromes d and a could account for the broad absorption in the 600- region. This possibility will be considered in more detail below. The difference spectrum obtained after inhibition with KCN is shown in Fig. 3. Absorbances in the a, /i, and -y regions can be seen, although they are much less well defined than those produced by dithionite reduction. There in no bleaching in the 450- region, indicating oxidation of flavoprotein in the presence of KCN.

4 540 KENIMER AND LAPP J. BACTERIOL :f A 0Q ' FIG. 2. Optical difference spectra of acetate-grown whole-cell sonic extract after reduction with sodium dithionite. Solid sodium dithionite crystals were added to the sample cuvette containing a total of 2.5 ml of assay mixture. (-) Spectrum obtained after reduction of 2.5 mg of sonicate protein; (----) reduction spectrum of 10.0 mg ofsonicate protein; and (-----) base line. The reference cuvettes were bubbled gently with air for 1 min immediately before scanning. AA, Change in absorbance. 425 \3}40 A U IAzA001 r _ 4 i _ FIG. 3. Oxidized-minus-reduced spectrum ofsonic extract obtained after KCN inhibition. ( ) Difference spectrum obtained after addition of 1.0 umol ofkcn to the sample cuvette containing 2.98 mg ofprotein in a total volume of 2.5 ml of KH2PO4 buffer (ph 7.4); (-----) base line. Assay mixture and inhibitor were preincubated for I min. The reference cuvette was gently bubbled with air for I min immediately before scanning. No substrate was added. AA, Change in absorbance. However, an absorbance appears at 340 mm due to NADH. The low, broad absorbance in the 600- to 620- region is not seen in the presence of KCN. Cytochromes of the d-type have been reported by several investigators to be CN- insensitive. A CN--insensitive terminal oxidase would perhaps explain the oxidation of flavoprotein in the presence of this inhibitor.

5 VOL. 134, 1978 The difference spectrum induced by nitrogen bubbling for 1 min can be seen in Fig. 4. The absorbances are qualitatively similar to but lower than those observed after sodium dithionite reduction. However, the extent of steadystate reduction after nitrogen exposure was much less than the total reduction by dithionite. The appearance of the 450- trough (reduced flavin) and the broad absorbance in the 600- region indicate a more complete blockage of terminal oxidase activity with nitrogen than with KCN. Identical results were obtained when the sample was gassed with nitrogen for 2 or 3 min. (ii) Spectroscopy with class I inhibitors. The effects of sodium amytal and rotenone, class I inhibitors, were studied spectroscopically in whole-cell and sonically treated cell preparations of N. gonorrhoeae. After introduction of concentrations of amytal (1.12 mm) or rotenone (7.5,uM) producing maximal inhibition of NADH oxidation (Fig. 1), no reduction peaks were observed (data not shown). This suggests the existence of a possible by-pass for NADH reducing equivalents around the amytal-rotenone-binding site. (iii) Spectroscopy with class H inhibitor HQNO. No difference in the effect of HQNO was found in spectra generated from whole-cell and sonically treated cell preparations. In our system, a reduction peak was observed only at 340 (NADH) after 2.0,tg of HQNO was introduced (Fig. 5). After addition of succinate to a sample inhibited with HQNO, a broad absorbance centered at 437 to 438 was observed in addition to the 340- absorbance. This new absorbance, possibly a Soret band of b-type cytochromes, may indicate the steady-state reduc- EFFECTS OF INHIBITORS ON N. GONORRHOEAE 425 i AA tion of a b-type cytochrome in the presence of HQNO. These data suggest a binding site(s) for the HQNO substrate side of the ubiquinonecytochrome b level. (iv) Ascorbate-TMPD partial reduction. Ascorbate, in the presence of catalytic amounts of TMPD, is assumed to reduce cytochrome c and, indirectly through cytochrome c, all electron transport components on the oxygen side of it. Because a CN--cytochrome a complex could not be detected, but an absorbance in the 600- region could be detected with sodium dithionite, the electron donors ascorbate and TMPD were employed to study the reduction of cytochrome c. The ascorbate-tmpd difference spectrum (reduced minus oxidized) is shown in Fig. 6. Absorbances were found in the 600- to 630- region and at 550 (cytochrome c). Absorbances in the I8 region were similar to those observed after dithionite reduction (518 to 520, 523 to 524, and 530 ). The combined absorbances of an a-type cytochrome (a, 598 to 604 ; Soret 440 ) and a d-type cytochrome (a, 620 to 630 ; fl, 518 to 522 ) along with a,8 absorbance of a b-type cytochrome (530 ) could account for the observed maxima. The presence of the /3 absorbance at 530 in both ascorbate-tmpd and dithionite spectra suggests a possible terminal function for b-type cytochrome. Oxygen electrode studies. Oxygen uptake was studied as a function of substrate in sonically treated and whole-cell preparations. Rates of oxygen consumption with NADH, succinate, and malate are given in Table 2. No endogenous respiration was observed over the 2- to 3-min interval before the addition of substrate in the sonically treated preparation. Glucose and ace ' 600 i, 4 A, FIG. 4. Difference spectrum ofnitrogen-reduced sonic extract. Reference and sample cuvettes each containing 3.18 mg ofprotein in 2.5 ml ofkh2p04 buffer (ph 7.4). The reference cuvette was bubbled with air and the sample cuvette was bubbled with nitrogen (99% pure) for 1 min immediately before scanning. (-----) Base line. No substrate was added. AA, Change in absorbance.

6 542 KENIMER AND LAPP 340 J. BACTERIOI I AA SO.0I 43? FIG. 5. Difference spectra obtained after HQNO inhibition in the presence and absence of succinate. Sonic extract (3.95 mg of protein in a total volume of 2.5 ml) was reduced after inhibition with 3.0 glm HQNO (-). Assay mixture and HQNO werepreincubated for min before scanning. Reference cuvette was bubbled with air for I min immediately before scanning. (-----), Base line; (---) spectrum obtained after addition of 50 umol of succinate to the HQNO-inhibited sample; ( ) 3.0 M HQNO. AA, Change in absorbance. tate stimulated oxygen uptake only very slightly in whole cells. The rate of NADH-stimulated 02 uptake decreased by 50% with sonic oscillation. All determinations of malate-stimulated oxygen uptake were made in the presence of 33 mm malonate, which inhibited succinate-stimulated oxygen consumption by 88%. The effects of inhibitors on the oxygen consumption stimulated by NADH, succinate, and malate are given in Table 3. Antimycin A1 and KCN are the only inhibitors that affected oxygen consumption stimulated by all three substrates. Antimycin A1 maximally inhibited oxygen uptake stimulated by NADH, succinate, and malate 53, 55, and 44% respectively. The maximum inhibition attained with KCN was approximately the same with all three substrates. However, the 50% and maximal inhibitory concentrations differed. The amount of KCN required to inhibit succinate-stimulated oxygen consumption was approximately 50 and 25% of the amount needed to inhibit malate- and NADHstimulated oxygen uptake, respectively. This suggests the sharing of malate-reducing equivalents between a pathway favoring succinate oxidation (KCN sensitive) and a pathway favoring NADH oxidation (KCN sensitive). DISCUSSION Morse et al. (5) reported that N. gonorrhoeae preferentially ferments glucose, resulting in the excretion of acetate into the medium, which is oxidized only after glucose depletion. To examine the possibility of cytochrome induction in the gonococci, rates of NADH oxidation in sonic extracts prepared from cultures grown in excess glucose or acetate were compared. Rates (measured spectrophotometrically) did not differ significantly in the two types of preparations and were affected in a imilar manner by amytal, antimycin A1, and HQNO. It can be concluded that all components necessary for electron transport were present in sonic extracts prepared from both types of culture. However, branches of the electron transport pathway could be turned off in cells growing in excess glucose

7 VOL. 134, EFFECTS OF INHIBITORS ON N. GONORRHOEAE 543 IAA % 4 I2 '^, ; ; 55. * o., FIG. 6. Difence spectrum of ascorbate-tmpd reduction of whole-ceu preparation. The sample cuvette contained 5.21 mng ofprotein in a total volume of 2.5 ml of KH2PO4 buffer (ph 7.4). (-) Pigments reduced by 18 mm ascorbate ph 7.4) and 80 mm TMPD; (-----) base line. AA, Change in absorbance. TABLE 2. Rate of substrate-stimulated oxygen consumption in whole-ceu and sonically treated ceu preparations 02 consumed' (nol/mg of protein per min) Substate Subsotmt Whole celis Sonic extract -Substrate +Substrate -Substrate +Substrate Glucoe ± ±i 1.2()b 0 0 Acetate ± :± 1.0 (7) 0 0 Malate ± ± 2.4 (5) ± 1.8 (10) Succinate ± ± 4.4 (8) ± 3.8 (7) NADH ± ± 1.2 (4) ± 4.6 (16) Values given are the average ± standard deviation. b NUmbers in parentheses indicate number of experiments per determination. because quantitative differences in the inhibition by rotenone and KCN do exist in the two types of preparations. Amytal and rotenone have been shown to block electron transport on the oxygen side of NADH dehydrogenase. Thus, in mitochondria, treatment of electron transport particles with the compounds listed above results in spectroscopic observation of an NADH peak and reduced flavoprotein absorbance (9). Under the conditions specified, treatment of sonic extract with class I inhibitors did not result in the reduction of these components. However, amytal and rotenone blocked both NADH oxidation and NADH-stimulated oxygen uptake. The site of interaction of HQNO with the respiratory chain was variable. HQNO inhibited reoxidation of b-type cytochromes in classical mitochondrial systems and in some bacterial systems. In Escherichia coli and Proteus vulgariw systems, HQNO has been reported to prevent the reduction of cytochrome b (6). An ab-

8 544 KENIMER AND LAPP J. BACTrERIOL. TABLE 3. Effect of inhibitors on substrate-stimulated rates of oxygen consumption Concn (pm) inhibiting O2 uptake: Maximum in- Substrate Inhibitor hibitio 50% Maximum hibition (%) NADH Amytal -a Rotenone HQNO Antimycin KCN Succinate Amytal, HQNO, and rotenone Antimycin KCN Malate Amytal, HQNO, and rotenone Antimycin KCN a _, Maximum inhibition less than 50%. sorbance of NADH is the only reduction peak observed in oxidized-minus-reduced spectra after HQNO treatment of sonic extract. Adding succinate to a sample inhibited with HQNO resulted in the appearance of an additional absorbance at 430 to 432. This band, in the Soret region of b-type cytochromes, was probably due to steady-state reduction of cytochrome b and/or cytochrome o. Cytochrome o has been reported to be insensitive to HQNO. Under the conditions specified, HQNO apparently blocked electron transfer on the NADH dehydrogenase side of cytochrome b. KCN and nitrogen were employed to inhibit terminal oxidase activity. Nitrogen and sodium dithionite reduced-minus-oxidized spectra resulted in similar reduction peaks, although dithionite treatment more completely reduced the system. In KCN inhibited-minus-oxidized spectra, no reduced flavoprotein absorbance was observed. Also, a CN--cytochrome a complex absorbance could not be detected. These data suggest a possible electron leak from a CN--sensitive terminal oxidase to a CN--insensitive terminal oxidase. Thus, flavoprotein could be reoxidized in the presence of KCN. The existence of a CN--insensitive terminal oxidase was also suggested by oxygen electrode studies. Succinate-stimulated oxygen consumption in sonically treated cell preparations was found to be four to five times more sensitive to KCN inhibition than was NADH-stimulated oxygen uptake. These data indicate the presence in N. gonorrhoeae of a CN--insensitive pathway favoring NADH oxidation and a CN--sensitive pathway favoring succinate oxidation. However, since inhibition was not complete with either substrate, both substrates must have access to both pathways. The electron donor pair, ascorbate-tmpd, was employed to determine the site of convergence of the NADH-oxidizing and the succinateoxidizing pathways. Succinate- and malate-stimulated oxygen consumption was found to be sensitive to inhibition by KCN only. Therefore, reducing equivalents from these substrates must enter the electron transport chain at a site below the HQNO block. Ascorbate, in the presence of catalytic amounts of TMPD, reduces cytochrome c and, indirectly through cytochrome c, all components on the oxygen side of it. Ascorbate-TMPD reduced-minus-oxidized spectra indicate the presence of a-type, d-type, and b-type (probably cytochrome o) cytochromes. Cytochromes of the d-type have been reported to be CN--insensitive in some bacterial systems. Fujita and Kodama reported the presence of d1- and d2-type cytochromes in N. gonorrhoeae (2). However, Winter and Morse suggested the possibility that the broad, low absorbance in the 600- region is due to nonheme iron components (10). Because it has been established that the absorbance in the 600- region is due to a component participating in electron transfer between cytochrome c and oxygen, this component is probably a cytochrome of the d-type. In the mammalian respiratory chain, antimycin A1 reacts with an unidentified factor (presumably protein) to inhibit electron transport. In bacteria, this factor seems to be absent. Antimycin A1 was found to be inhibitory only at concentrations 1,000-fold greater than those required to inhibit mammalian mitochondrial electron transport. Complete inhibition of electron transfer was not attained with any of the inhibitors tested under the specified conditions. However, significant inhibition of NADH oxidation and NADH-

9 VOL. 134, 1978 stimulated oxygen uptake was achieved with concentrations of amytal, rotenone, HQNO, and KCN well within concentration ranges reported for mitochondria and other bacteria. More detailed studies are required to determine the exact site and extent of inhibitor interaction. ACKNOWLEDGMENTS The advice and interest of S. A. Singal throughout the duration of this project are gratefully acknowledged. LITERATURE CITED 1. Barnes, F. M., and H. R. Kaback Mechanisms of active transport in isolated membrane vesicles. I. The site of energy coupling between D-lactic dehydrogenase and fi-galactoside tranport in Eschericia coli membrane vesicles. J. Biol. Chem. 246: Fujita, A., and T. Kodma Untersuchunzen wher atmung und garung pathogener bakterien. Biochem. J. 273: Jurtshuk, P., and T. W. MiNlgan Quantitation of the tetramethyl-p-phenylenediamine oxidase reaction EFFECTS OF INHIBITORS ON.N. GONORRHOEAE 545 in Neisseria species. Appl. Microbiol. 28: Jurtshuk, P., and T. W. Milligan Preliminary characterization studies on the Neisseria catarrhalis respiratory electron transport chain. J. Bacteriol. 120: Morse, S. A., S. Stein, and J. Hines Glucose metabolism in Neisseria gonorrhoeae. J. Bacteriol. 120: Smith, L The respiratory chain of bacteria, p In T. P. Singer (ed.), Biological oxidations. Interscience Publishers, New York. 7. Sutherland, E. W., C. F. Cori, R. Haynes, and N. S. Olsen Purification of the hyperglycemic-glycogenolytic factor from insulin and from gastric mucosa. J. Biol. Chem. 180: Taber, H. W., and AL Morrison Electron transport in staphylococci. Properties of a particle preparation from exponential phase Staphylococcus aureus. Arch. Biochem. Biophys. 106: Waines, W. W The mammalian mitochondrial respiratory chain. Academic Press Inc., New York. 10. Winter, D. B., and S. A. Morse Physiology and metabolism of pathogenic Neisseria: partial characterization of the respiratory chain of Neisseria gonorrhoeae. J. Bacteriol. 123: Downloaded from on May 14, 2018 by guest