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Cytochrome bd-typQ quinol oxidase in a mutant of Bacillus stearothermophilus deficient in caa3-type cytochrome c oxidase
MICROBIOLOGY LETTERS
ELSEVIER
FEMS Microbiology Letters 143 (1996) 151-158
Cytochrome M-type quinol oxidase in a mutant of Bacillus stearothermophilus deficient in caas-type cytochrome c oxidase Junshi Sakamoto *, Akira Matsumoto,
Kenji Oobuchi, Nobuhito
Sone
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Kawazu 680-4. Iizuka. Fukuoka-ken 820. Japan
Received 1 July 1996; revised 28 July 1996; accepted 28 July 1996
Abstract
Gram-positive thermophilic Bacillus species contain cytochrome caas-type cytochrome c oxidase as a terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated B. stearothermophilus mutants defective in the cuus-type oxidase activity. One mutant contained little cytochrome a and had low cytochrome c oxidase activity. However, growth and the respiratory activity of membranes in the presence of NADH were close to normal, suggesting that the mutant contains an alternative electron transfer pathway. A novel oxidase was isolated from the membrane fraction of the mutant. The enzyme is a cytochrome bd-type qulnol oxidase composed of two subunits of 52 and 40 kDa, whose N-terminal regions show sequence similarity to polypeptides of the bd-type oxidase from Escherichiu colt’ and Azotobucter vinelundii. This is the first report of a bd-type terminal oxidase purified from a Gram-positive bacterium. Keywords: Thermophilic
Bacillus stearothermophilus; bd-type quinol oxidase;
bacteria;
1. Introduction Gram-positive spore-forming thermophilic bacilli such as Bacillus PS3 and B. stearothermophilus contain cytochrome caas-type cytochrome c oxidases as the terminal oxidases in the respiratory chain [ 1,2]. Based on the prosthetic groups present, peptide se-
* Corresponding author. Tel.: +81 (948) 297823; Fax: +81 (948) 297801; E-mail: [email protected] Abbreviations: MEGA 9+10, 1:l mixture of n-nonanoyl Nmethylglucamide and n-decanoyl N-methylglueamide; NTG, lmethyl-3-nitro-1-nitrosoguanidine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel N, N, N’, N’-tetramethyl-p-phenylene 03781097/96/$12.00 Copyright PIISO378-1097(96)00312-6
electrophoresis; diamine
TMPD,
0 1996 Federation
of European
Cytochrome
d
quence data and function, the enzyme is regarded as a member of the heme-copper respiratory oxidase family [3,4]. This family includes many members such as cbba-type cytochrome c oxidase of Bradyrhizobium japonicum, aas-type cytochrome c oxidase of Paracoccus denitrljicans and bo-type quinol oxidase of Escherichia coli. In addition to the main terminal oxidases, which function under regular growth conditions, alternative oxidases have been found in many species. For example, E. coli expresses a bdtype oxidase in stationary phase [5,6] in addition to the bo-type oxidase expressed in exponential growth phase. The former enzyme does not have copper in its active site and shows no appreciable sequence similarity to the oxidases of the heme-copper terminal oxidase family. In the case of thermophilic bacilli, a caos-type cyMicrobiological
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J. Sukumoto
CI ul. IFEMS
Microbiology
tochrome c oxidase has been identified in cells grown under air-limited conditions. Its four polypeptide subunits are the same as those of the caas-type enzyme, but high spin heme 0 is replacing the heme A at the 02-reducing binuclear site [7]. The presence of additional oxidases in thermophilic bacilli has been suggested but not identified yet. In order to identify alternative oxidases in thermophilic bacilli, we isolated B. stearothermophilus mutants defective in oxidase activity. In one of the mutants, named K17, the content of heme A is low while the respiratory activity of its membrane fraction is comparable to that of the wild strain when NADH was used as the substrate, suggesting that an alternative electron transfer pathway operates in the mutant. Here, we report that a cytochrome bd-type quinol oxidase exists in the mutant and that the purified enzyme shows clear sequence similarity to the hd-type oxidases of E. coli [lO,l l] and Azotobucter vinelandii [12]. To our best knowledge, this is the first bd-type terminal oxidase purified from a bacterium outside the gamma subclass of proteobacteria.
2. Materials and methods 2.1. Mutagenesis und screening jtir mutants B. steurothermophilus K1041 [l l] was aerobically cultured at 60°C in 5 ml of medium containing 0.8% polypeptone, 0.4% yeast extract, 0.3% NaCl, and 0.1% dipotassium phosphate with pH adjusted to 7.0 (medium A). At an optical density of 1.5 at 650 nm, the cells were sedimented, washed with medium A, and gently suspended in medium A containing 50 or 100 ug ml-’ 1-methyl-3-nitro- 1-nitrosoguanidine (NTG) with or without streptomycin (0.1 or 0.3 pg ml-‘), followed by incubation with shaking at 30°C for 20 min. After being washed three times, the cells were again suspended in medium A and cultured at 50°C for 3 h. The cells were then diluted, spread on 2.0% agar plates containing medium A and incubated overnight at 60°C. The TMPD plate assay described by Mueller and Taber [12] was employed with some modifications as a screening method for mutants with low TMPD oxidase activity. Soft agar at 0.6% containing 0.5% NaCl, 20% glycerol, and 10 mM N-2-hydroxyethylpiperazine-N’-2-ethane sulfo-
Letrm
143 11996)
151-158
nit acid-HCl, pH 7.0 was autoclaved, and cooled to about 60°C then supplemented with N,N,N’,N’tetramethyl-p-phenylene diamine (TMPD) to a final concentration at 1.9 mM, and immediately poured over the culture plate as evenly as possible. Most of the colonies quickly turned purple while some of them stayed white for a while. The latter colonies were picked (1st screening). Each colony was streaked on another plate and screened for TMPD oxidation as above (2nd screening). Mutant clones were cultured in 200 ml of medium A, and cells were harvested to prepare the membrane fraction as described previously [13]. The isolates whose membrane preparations showed low TMPD oxidase activity and modified cytochrome content were selected for further study (3rd screening). 2.2. Enzyme preparation Growth and membrane preparation of B. steurothermophilus were performed as previously described [13]. The membranes (1 g of protein) were washed twice with 2% (w/v) Na-cholate at 10 mg protein ml-‘, and then solubilized with 1% (w/v) of a 1 : 1 mixture of n-nonanoyl N-metylglucamide (MEGA 9) and n-decanoyl N-methylglucamide (MEGA 10) in 100 mM NaCl, 1 mM EDTA, and 20 mM sodium phosphate buffer, pH 6.0. The mixture was centrifuged at 140 000 Xg for 40 min, and the supernatant was dialyzed against 20 mM sodium phosphate buffer, pH 6.0, supplemented with 1% (w/v) MEGA 9+10, and then applied to a DEAEToyopearl column (1 .OX 6.4 cm) equilibrated with 0.5% (w/v) MEGA 9+10, 1 mM EDTA, 20 mM sodium phosphate, pH 6.0. The column was washed with 300 ml of the same solution and proteins were then eluted using a lOO-ml linear gradient from 0 to 100 mM NaCl in the buffer followed by 30 ml of the buffer containing 500 mM NaCl. Peak fractions were combined and applied to a hydroxylapatite column (0.5 X 1.5 cm) and proteins were stepwise eluted with buffer containing 0.5% (w/v) MEGA 9+10 and increasing concentration of sodium phosphate. The peak fraction was concentrated using a Centriconconcentrator (Amicon Inc., Beverly) and applied to a G3000SW glass gel filtration column (Tosoh Co., Tokyo).
J. Sakamoto et al. IFEMS
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Microbiology Letters 143 (1996) 151-158
550
Wavelength
600
153
650
(nm)
Fig. 1. Redox difference spectra of B. stearothermophilus membranes and the quinol oxidase. Samples were membrane fractions prepared from wild-type cells (A) and mutant K17 (B) at 5 mg protein/ml and the purified bd-type quinol oxidase (C) at 2.1 PM. Spectroscopy was done as described in Section 2.
2.3. Enzymatic assay and optical spectroscopy Oxygen consumption was measured using a Clarktype oxygen electrode at 40°C [13]. TMPD oxidase activity was spectrophotometrically measured at 22°C by monitoring the increase in Asss. The buffer for these two measurements was 100 mM NaCl, 1 mM EDTA (sodium salt), and 50 mM sodium phosphate buffer, pH 6.0. Duroquinol oxidase activity was measured at 40°C in the 20 mM sodium phosphate buffer, pH 6.7 by monitoring the increase in A264.5minus A2ss.s. Enzyme activities were calculated millimolar extinction coefficients using the 10.5 mM_’ cm-’ and A&zs4.5__2ss.5 = 19.5 A&562 = mM_’ cm-‘, respectively. Redox difference spectra were recorded using a Beckman DU-70 spectrophotometer at room tem-
perature. Air-oxidized sample was taken as the baseline and a few grains of solid sodium dithionite were added to the oxidized sample, followed by spectral measurement. Contents of hemes were calculated from redox difference spectra of cytochromes using the millimolar extinction coefficients Aassi--58s = 21 .O M-’ cm-’ for protoheme IX and A~+~ss-s~~= 27.9 M-’ cm-’ for heme D, determined for E. coli bdtype quinol oxidase [14]. 2.4. Electrophoresis
and peptide sequencing
SDS-PAGE was performed according to the method described by Laemmli except that boiling of protein samples was omitted. Molecular masses of polypeptides were estimated by Ferguson plot analysis [ 151 using 10, 12, 14, and 16% (w/v) acrylamide
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J. Sakumoto et 01.I FEMS Microbiology Letters 143 (1996) 151-158
gels. For sequence analysis, proteins were separated by SDS-PAGE and electro-transferred to a polyvinylidene difluoride membrane for 3 h in the presence of 25 mM tris(hydroxymethyl)aminomethane, 192 mM glycine, 0.05% (w/v) SDS and 20% (v/v) methanol. The membrane was washed extensively with water to remove glycine, treated with 0.6 N HCl at room temperature for 24 h to cleave a possible Nterminal formyl group [16], and applied on a pulseliquid peptide sequencer (Applied Biosystems, model 477A). 2.5. Materials N-2-Hydroxyethylpiperazine-N’-2-ethane sulfonic acid, MEGA 9 and MEGA 10 were purchased from Dojin Co. (Kumamoto, Japan). DEAE-Toyopearl anion exchange gel and hydroxylapatite were obtained from Tosoh (Tokyo) and Bio-Rad (Hercules). Proteins used as molecular mass standards. polyvinylidene difluoride membranes and NTG were obtained from Sigma, Millipore (Bedford, MA) and Aldrich Chemical (Milwaukee), respectively. TMPD was purchased from Wako (Kyoto). Other reagents were analytical grade.
3. Results 3.1. Isolation of mutants with low TMPD oxidase activity Since the presence of high oxidase activity due to the cytochrome caas-type complex might affect the synthesis and/or detection of alternative oxidases with much lower activity, it was decided to isolate Table I Properties Strain
wild type K7 K17 K21 K22 K25 K41
of B. stearothermophilus Growth
+++ ++ ++ + ++ + +
rate
mutant
mutants with low or no caa3-type oxidase activity and a low content of cytochrome c or a. Cells treated with NTG were spread on culture plates, which sometimes contained streptomycin since mutants deficient in caas-type oxidase would be more resistant to the antibiotic because its uptake into cells is suggested to be dependent on proton motive force across the cell membrane. The TMPD plate assay as described by Mueller and Taber [12] was employed as a screening method for mutants. Sixteen mutant colonies were isolated at the second screening and grown in liquid culture medium to obtain membrane fractions of the cells. All mutant strains showed less than 50% TMPD oxidase activity compared to that of the wild strain. To focus on mutants substantially different from the wild type, we further screened for those which showed clearly modified redox difference spectra and less than 25% of TMPD oxidase activity. Table 1 summarizes the properties of six mutants selected on those criteria. The overall frequency of mutants obtained in the presence of streptomycin (K7 through K25) was 0.07% (5/6900) and higher than that (O.OOS%, l/19400) of mutants obtained in its absence (K41). Mutant K17 showed no apparent absorption peak of cytochrome a at about 600 nm (Fig. lB), suggesting that it lacks the cytochrome caas-type oxidase. However, it grew aerobically at a near normal rate and isolated membranes consumed oxygen in the presence of NADH at wild-type rates, suggesting that it possesses an alternative pathway of respiration. 3.2. IdentiJication of a cytochrome bd-type quinol oxidase In order to identify the alternative
oxidase operat-
strains Cytochrome
Oxygen uptake rate in membranes
a
h
‘
NADH
++ ++ _
++ + ++ ++ + + _
++ ++ + _
480 7 536 27 46 II 73
+ ++ + +
++ + ++
(200 PM)
TMPD 553 45 115 19 67 9 4
(ng atom mg-’
(100FM)
min-‘)
cyt. c (20 FM) 133 4 44 0 9 3 6
155
J. Sakamoto et al. I FEMS Microbiology Letters 143 (1996) 151-158
0.08 c V
o^ 0.04 V
1.0
I’
100
3 0.06
B 5 0.03 >r .z
h
5
^n V 0.5 T
ti t 2
0.02
g
0.01
50 s & C w f.
a
0
z ,O
0
1
20
30
40
50
Fraction Number
-4 0,
1 1
0.08
I.5 100
E 0.06
B s E :S *t; $ d g
3-
2-
,_
1.0
g 2 $ 2 s 0 i;
a^
”
V
0.04
N
2
50:
w z
a 0.5 0.0; 0
t
0
Fraction Number Fig. 2. Elution profiles of B. stearothermophilus oxidases in DEAE-anion-exchange chromatography. Membranes from mutant K17 (A) and the wild type (B) were solubilized with MEGA 9+10, applied to a DEAE-Toyopearl anion-exchange column and eluted with a linear gradient of NaCl. TMPD (w) and duroquinol oxidase activity (0) were measured as described in Section 2. Absorbance at 412 nm (A ).
ing in mutant K17, isolated membranes were solubilized with non-ionic detergents and subjected to anion-exchange chromatography. Fig. 2 compares the elution patterns of such chromatography of membrane proteins from the wild type and mutant K17. In the case of wild type (Fig. 2B), TMPD oxidase activity was found in the fraction eluted with buffer
containing about 70 mM NaCl, and corresponded to caas-type cytochrome c oxidase. In the case of mutant K17 (Fig. 2A), TMPD oxidase activity in the 70 mM NaCl eluate was less than 1% of that of the wild type. Instead, quinol oxidase activity was found in the 50 mM NaCl eluate. Practically no quinol oxidase activity was detected in the corresponding frac-
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tions of the wild type (Fig. 2B). The apparent duroquinol oxidation activity found in the fractions eluted with 90 mM NaCl might be due to succinate dehydrogenase, since the polypeptide profile after SDS-PAGE was very similar to that of Bacillus subtilis succinate dehydrogenase [17] and the fraction mainly contained cytochrome b,5:,8which was reducible with Na-succinate. Mutant K17 showed duroquino1 oxidation activity due to succinate dehydrogenase at the corresponding fractions but with a lower activity. The quinol oxidase in mutant K17 was further purified using a hydroxylapatite column and a gel filtration column as described in Section 2. Table 2 shows a summary of the purification. The oxidase activity was not very stable after solubilization even though MEGA 9+10 was more suitable than other detergents tested. The purified oxidase in the reduced state showed absorption peaks at 560 and 618 nm (Fig. 1C). The redox difference spectrum of its pyridine hemochrome showed peaks at 557 and 613 nm, indicating that the chromophores are protoheme IX and heme D. The contents of protoheme IX and heme D were 14.5 and 8.5 nmol (mg protein))‘, calculated from redox difference spectra of cytochromes. The turnover number, measured as duroquinol oxidase activity, was 0.39 ss’ based on the assumption that the enzyme contained two protoheme IX and one heme D as purple bacterial bdtype oxidases. Upon SDS-PAGE the oxidase preparation showed three polypeptide bands migrating at 65, 39, and 27 kDa (Fig. 3). The stoichiometry of the 39 and 27 kDa bands was close to 1: 1 as judged by densitometric scanning, while the relative amount of the 65 kDa band to those of the two lower bands was much less than unity and varied from preparation to preparation. Molecular masses of the two main polypeptides were determined to be 52 and 40 Table 2 Purification Experimental
of cytochrome step
Letters 143 (19961 151-158
66 45 36 29
20
Ml
23
4
Fig. 3. SDS-PAGE of the hd-type quinol oxidase isolated from R .rre~lrothrrmophilus. The concentration of acrylamide was 13.5%. The samples are membranes from mutant K17 (lane I). the MEGA 9+10 extract (lane 2), the peak fraction from DEAEToyopedrl chromatography (lane 3) and the peak fraction from hydroxylapatite chromatography (lane 4). The protein amounts in these lanes are 20, 3, 1.5 and 3 pg. respectively. The proteins used as molecular mass standards (lane M) are bovine serum abumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa) and trypsin inhibitor (20 kDa).
kDa by Ferguson plot analysis. This is consistent with the fact that hydrophobic subunits of oxidases generally migrated faster than hydrophilic proteins of similar size used as molecular mass standards. The three polypeptides were subjected to N-terminal protein sequence analyses. The largest polypep-
h&type quinol oxidase from B. .stc,uuothPrmo)/)hllu.s
Total protein (mg)
Total activity (unit)
Recovery (‘%I)
Specific activity
Purification
(X IO-” unit/mg)
Membranes Cholate wash MEGA extract DEAE fraction Purified enzyme
882 280 138 2.51 2.13
0.676 0.563 1.014 0.200 0.074
100 83 150 29.6 IO.9
0.77 2.01 7.34 79.1 34.5
I 2.62 9.58 104 45.0
(-fold)
J. Sakamoto 1
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et al. I FEMS 15
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20
B. s. 52 kDa subunit E. co/i
cydA
E.coli appC A. vinelandii
cydA
Fig. 4. Comparison of the N-terminal amino acid sequences of the subunits of B. stearothermophilus bd-type quinol oxidase with the corresponding polypeptides of E. coli and A. vinelandii bdtype quinol oxidases.
tide gave comparable amounts of two main amino acids per every Edman reaction cycle and they were identical to the combined N-terminal sequences of the two smaller polypeptides, indicating that the band found at 65 kDa is composed of the two true subunits of the oxidase which have not been dissociated in the presence of SDS. In Fig. 4 the N-terminal sequences of the 52 and 40 kDa subunits are compared with those of subunits of the bd-type quino1 oxidases from E. coli [8,9] and A. vinelundii [lo].
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position, molecular mass and tendency to resist dissociation by SDS. The N-terminal sequences of the two subunits show similarity to those of E. coli and A. vinelandii, although the similarity is lower than that between the proteobacterial enzymes (Fig. 4). These oxidases might compose an oxidase family distinct from the heme-copper terminal oxidase family [41. The bd-type oxidase was not detected in the wild strain of B. stearothermophilus K1041 (Fig. 2B), or in other thermophilic bacilli grown under highly aerated or air-limited conditions. Cytochrome d has been found in the mesophilic bacteria Bacillus cereus [22] and B. subtilis [23] in stationary growth phase or at low oxygen tensions. It is thus likely that K17 is a mutant that can grow because repression on the operon for the bd-type oxidase is released in some way and that the consequently synthesized bd-type oxidase complements the deficiency in caas-type cytochrome c oxidase. Based on results of the peptide sequence analyses, we are currently trying to clone the genes for the B. stearothermophilus bd-type oxidase.
Acknowledgments 4. Discussion A cytochrome bd-type quinol oxidase was purified from a mutant of Bacillus stearothermophilus K1041. The oxidase is composed of two subunits with molecular masses of 52 and 40 kDa at a 1: 1 ratio, and contains protoheme IX and heme D as chromophores. Cytochrome bd-type quinol oxidases have been purified from E. coli [6], A. vinelundii [18], Photobacterium phosphoreum [19] and Klebsiella pneumoniae [20], all of which belong to the gamma subclass of proteobacteria. Structural genes, cydAB, for the oxidase have been cloned and sequenced from E. coli and A. vinelundii [&lo]. In addition to cydAB, E. coli is known to possess genes, appCB, which encode proteins very similar in sequence to those of the bd-type oxidase [9]. It was recently reported that a second bd-type quinol oxidase is expressed in E. coli when the cell was transformed with some DNA from Bacillus firmus OF4 [21]. The Bacillus bd complex is similar to the oxidases of these Gram-negative bacteria in its subunit com-
We thank Dr. S. Noguchi of this institute and Prof. M. Kawamura of the University of Occupational and Environmental Health for helping us in peptide sequence analysis. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan.
References 111Sone, N. and Yanagita,
Y. (1982) A cytochrome aas-type terminal oxidase of a thermophilic bacterium purification. properties and proton pumping. Biochim. Biophys. Acta 682, 216226. 121Kusano, T., Kuge, S., Sakamoto, J., Noguchi, S. and Sone, N. (1996) Nucleotide and amino acid sequences for cytochrome caas-type oxidase of Bacillus stearothermophilus K1041 and non-Michaelis-type kinetics with cytochrome c. Biochim. Biophys. Acta 1273, 129-138. J.A., Barquera, B., Rumbley, J., Ma, J. and [31Garcia-Horsman, Gennis, R.B. (1994) The superfamily of heme-copper respiratory oxidase. J. Bacterial. 176, 5587-5600.
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[4] Van der Oost, J., de Boer, A.P.N., de Gier, .I.-W.L., Zumft, W.G., Stouthamer, A.H. and van Spanning, R.J.M. (1994) The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase. FEMS Microbial. Lett. 121, I-10. [5] Miller, M.J. and Gennis, R.B. (1983) The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain. J. Biol. Chem. 258, 915999165. [6] Kita, K., Konishi, K. and Anraku, Y. (1984) Terminal oxidases of Escherichia coli aerobic respiratory chain. II. Purification and properties of cytochrome bs,?a-dcomplex from cells grown with limited oxygen and evidence of branched electroncarrying systems. J. Biol. Chem. 259, 337553381. [7] Sane, N. and Fujiwara, Y. (1991) Haem 0 can replace haem A in the active site of cytochrome c oxidase from thermophilic bacterium PS3. FEBS Lett. 288, 154158. [S] Green, G.N., Fang, H., Lin, R.-J., Newton, G., Mather, M. Georgiou, CD. and Gennis, R.B. (1988) The nucleotide sequence of the cyd locus encoding the two subunits of the cytochrome d terminal oxidase complex of Escherichia coli. J. Biol. Chem. 263, 13138-13143. [9] Dassa, J., Fsihi, H., Marck. C., Dion, M., Kieffer-Bontemps. M., and Boquet, P.L. (1991) A new oxygen-regulated operon in Escherichia co/i comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (uppA). Mol. Gen. Genet. 229, 341-352. [IO] Moshiri, F., Chawla, A. and Maier, R.J. (1991) Cloning, characterization, and expression in Escherichia co/i of the genes encoding the cytochrome d oxidase complex from Azotohacter vinelandii. J. Bacterial. 173, 623s-6241. [ll] Narumi, I., Sawakami. K., Nakamoto, S.. Nakayama, N.. Yanagisawa, T., Takahashi, N. and Kihara, H. (1992) A newly isolated Bacillus stearothermophilus K1041 and its transformation by electroporation. Biotechnol. Techniques 6, 83-86. [12] Mueller, J.P. and Taber H.W. (1989) Isolation and sequence of ctaA, a gene required for cytochrome (14s biosynthesis and sporulation in Bacillus subtilis. J. Bacterial. 171, 49674978. [13] Sane, N. (1986) Cytochrome oxidase from thermophilic bacterium PS3. Methods Enzymol. 126, 145-152.
[14] Tsubaki, M., Hori, H., Mogi, T. and Anraku, Y. (1995) Cyanide-binding site of bd-type ubiquinol oxidase from Escherichia coli. J. Biol. Chem. 270, 28565-28569. [15] Hedrick, J.L. and Smith, A.J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126, 155164. [16] Ikeuchi, M. and Inoue, Y. (1988) A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome. FEBS Lett. 241, 99-104. [17] Hagerhall, C, Aasa, R, von Wachenfeldt, C., and Hederstedt, L. (1992) Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase. Biochemistry 31, 7411-7421. [18] Kolonay, Jr., J.F., Moshiri, F., Gennis, R.B., Kaysser, T.M. and Maier, R.J. (1994) Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: comparison to the complex from Escherichia coli. J. Bacterial. 176, 41774181. [19] Konishi, K., Ouchi, M., Kita, K. and Horikoshi, I. (1986) Purification and properties of a cytochrome b56O-dcomplex, a terminal oxidase of the aerobic respiratory chain of Photobacterium phosphorem. J. Biochem. 99, 1227-1236. [20] Smith, A., Hill, S. and Anthony, C. (1990) The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation. J. Gen. Microbiol. 136, 171-180. [21] Sturr, M.G., Krulwich, T.A. and Hicks, D.B. (1996) Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a Acyo Acyd strain complemented by genes from Eacillusjirmus 0F4. J. Bacterial. 176, 17422 1749. [22] Escamilla, J.E., Ramirez, R., Del Arenal, I.P., Zarzoza, G. and Linares, V. (1987) Expression of cytochrome oxidases in Bacillus cereus: effects of oxygen tension and carbon source. J. Gen. Microbial. 133, 3549-3555. [23] Lauraeus, M. and Wikstrom, M. (1993) The terminal quinol oxidases of Bacilius subtifis have different energy conservation properties. J. Biol. Chem. 268, 11470-11473.
ELSEVIER
FEMS Microbiology Letters 143 (1996) 151-158
Cytochrome M-type quinol oxidase in a mutant of Bacillus stearothermophilus deficient in caas-type cytochrome c oxidase Junshi Sakamoto *, Akira Matsumoto,
Kenji Oobuchi, Nobuhito
Sone
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Kawazu 680-4. Iizuka. Fukuoka-ken 820. Japan
Received 1 July 1996; revised 28 July 1996; accepted 28 July 1996
Abstract
Gram-positive thermophilic Bacillus species contain cytochrome caas-type cytochrome c oxidase as a terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated B. stearothermophilus mutants defective in the cuus-type oxidase activity. One mutant contained little cytochrome a and had low cytochrome c oxidase activity. However, growth and the respiratory activity of membranes in the presence of NADH were close to normal, suggesting that the mutant contains an alternative electron transfer pathway. A novel oxidase was isolated from the membrane fraction of the mutant. The enzyme is a cytochrome bd-type qulnol oxidase composed of two subunits of 52 and 40 kDa, whose N-terminal regions show sequence similarity to polypeptides of the bd-type oxidase from Escherichiu colt’ and Azotobucter vinelundii. This is the first report of a bd-type terminal oxidase purified from a Gram-positive bacterium. Keywords: Thermophilic
Bacillus stearothermophilus; bd-type quinol oxidase;
bacteria;
1. Introduction Gram-positive spore-forming thermophilic bacilli such as Bacillus PS3 and B. stearothermophilus contain cytochrome caas-type cytochrome c oxidases as the terminal oxidases in the respiratory chain [ 1,2]. Based on the prosthetic groups present, peptide se-
* Corresponding author. Tel.: +81 (948) 297823; Fax: +81 (948) 297801; E-mail: [email protected] Abbreviations: MEGA 9+10, 1:l mixture of n-nonanoyl Nmethylglucamide and n-decanoyl N-methylglueamide; NTG, lmethyl-3-nitro-1-nitrosoguanidine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel N, N, N’, N’-tetramethyl-p-phenylene 03781097/96/$12.00 Copyright PIISO378-1097(96)00312-6
electrophoresis; diamine
TMPD,
0 1996 Federation
of European
Cytochrome
d
quence data and function, the enzyme is regarded as a member of the heme-copper respiratory oxidase family [3,4]. This family includes many members such as cbba-type cytochrome c oxidase of Bradyrhizobium japonicum, aas-type cytochrome c oxidase of Paracoccus denitrljicans and bo-type quinol oxidase of Escherichia coli. In addition to the main terminal oxidases, which function under regular growth conditions, alternative oxidases have been found in many species. For example, E. coli expresses a bdtype oxidase in stationary phase [5,6] in addition to the bo-type oxidase expressed in exponential growth phase. The former enzyme does not have copper in its active site and shows no appreciable sequence similarity to the oxidases of the heme-copper terminal oxidase family. In the case of thermophilic bacilli, a caos-type cyMicrobiological
Societies. Published
by Elsevier Science B.V.
152
J. Sukumoto
CI ul. IFEMS
Microbiology
tochrome c oxidase has been identified in cells grown under air-limited conditions. Its four polypeptide subunits are the same as those of the caas-type enzyme, but high spin heme 0 is replacing the heme A at the 02-reducing binuclear site [7]. The presence of additional oxidases in thermophilic bacilli has been suggested but not identified yet. In order to identify alternative oxidases in thermophilic bacilli, we isolated B. stearothermophilus mutants defective in oxidase activity. In one of the mutants, named K17, the content of heme A is low while the respiratory activity of its membrane fraction is comparable to that of the wild strain when NADH was used as the substrate, suggesting that an alternative electron transfer pathway operates in the mutant. Here, we report that a cytochrome bd-type quinol oxidase exists in the mutant and that the purified enzyme shows clear sequence similarity to the hd-type oxidases of E. coli [lO,l l] and Azotobucter vinelandii [12]. To our best knowledge, this is the first bd-type terminal oxidase purified from a bacterium outside the gamma subclass of proteobacteria.
2. Materials and methods 2.1. Mutagenesis und screening jtir mutants B. steurothermophilus K1041 [l l] was aerobically cultured at 60°C in 5 ml of medium containing 0.8% polypeptone, 0.4% yeast extract, 0.3% NaCl, and 0.1% dipotassium phosphate with pH adjusted to 7.0 (medium A). At an optical density of 1.5 at 650 nm, the cells were sedimented, washed with medium A, and gently suspended in medium A containing 50 or 100 ug ml-’ 1-methyl-3-nitro- 1-nitrosoguanidine (NTG) with or without streptomycin (0.1 or 0.3 pg ml-‘), followed by incubation with shaking at 30°C for 20 min. After being washed three times, the cells were again suspended in medium A and cultured at 50°C for 3 h. The cells were then diluted, spread on 2.0% agar plates containing medium A and incubated overnight at 60°C. The TMPD plate assay described by Mueller and Taber [12] was employed with some modifications as a screening method for mutants with low TMPD oxidase activity. Soft agar at 0.6% containing 0.5% NaCl, 20% glycerol, and 10 mM N-2-hydroxyethylpiperazine-N’-2-ethane sulfo-
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nit acid-HCl, pH 7.0 was autoclaved, and cooled to about 60°C then supplemented with N,N,N’,N’tetramethyl-p-phenylene diamine (TMPD) to a final concentration at 1.9 mM, and immediately poured over the culture plate as evenly as possible. Most of the colonies quickly turned purple while some of them stayed white for a while. The latter colonies were picked (1st screening). Each colony was streaked on another plate and screened for TMPD oxidation as above (2nd screening). Mutant clones were cultured in 200 ml of medium A, and cells were harvested to prepare the membrane fraction as described previously [13]. The isolates whose membrane preparations showed low TMPD oxidase activity and modified cytochrome content were selected for further study (3rd screening). 2.2. Enzyme preparation Growth and membrane preparation of B. steurothermophilus were performed as previously described [13]. The membranes (1 g of protein) were washed twice with 2% (w/v) Na-cholate at 10 mg protein ml-‘, and then solubilized with 1% (w/v) of a 1 : 1 mixture of n-nonanoyl N-metylglucamide (MEGA 9) and n-decanoyl N-methylglucamide (MEGA 10) in 100 mM NaCl, 1 mM EDTA, and 20 mM sodium phosphate buffer, pH 6.0. The mixture was centrifuged at 140 000 Xg for 40 min, and the supernatant was dialyzed against 20 mM sodium phosphate buffer, pH 6.0, supplemented with 1% (w/v) MEGA 9+10, and then applied to a DEAEToyopearl column (1 .OX 6.4 cm) equilibrated with 0.5% (w/v) MEGA 9+10, 1 mM EDTA, 20 mM sodium phosphate, pH 6.0. The column was washed with 300 ml of the same solution and proteins were then eluted using a lOO-ml linear gradient from 0 to 100 mM NaCl in the buffer followed by 30 ml of the buffer containing 500 mM NaCl. Peak fractions were combined and applied to a hydroxylapatite column (0.5 X 1.5 cm) and proteins were stepwise eluted with buffer containing 0.5% (w/v) MEGA 9+10 and increasing concentration of sodium phosphate. The peak fraction was concentrated using a Centriconconcentrator (Amicon Inc., Beverly) and applied to a G3000SW glass gel filtration column (Tosoh Co., Tokyo).
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550
Wavelength
600
153
650
(nm)
Fig. 1. Redox difference spectra of B. stearothermophilus membranes and the quinol oxidase. Samples were membrane fractions prepared from wild-type cells (A) and mutant K17 (B) at 5 mg protein/ml and the purified bd-type quinol oxidase (C) at 2.1 PM. Spectroscopy was done as described in Section 2.
2.3. Enzymatic assay and optical spectroscopy Oxygen consumption was measured using a Clarktype oxygen electrode at 40°C [13]. TMPD oxidase activity was spectrophotometrically measured at 22°C by monitoring the increase in Asss. The buffer for these two measurements was 100 mM NaCl, 1 mM EDTA (sodium salt), and 50 mM sodium phosphate buffer, pH 6.0. Duroquinol oxidase activity was measured at 40°C in the 20 mM sodium phosphate buffer, pH 6.7 by monitoring the increase in A264.5minus A2ss.s. Enzyme activities were calculated millimolar extinction coefficients using the 10.5 mM_’ cm-’ and A&zs4.5__2ss.5 = 19.5 A&562 = mM_’ cm-‘, respectively. Redox difference spectra were recorded using a Beckman DU-70 spectrophotometer at room tem-
perature. Air-oxidized sample was taken as the baseline and a few grains of solid sodium dithionite were added to the oxidized sample, followed by spectral measurement. Contents of hemes were calculated from redox difference spectra of cytochromes using the millimolar extinction coefficients Aassi--58s = 21 .O M-’ cm-’ for protoheme IX and A~+~ss-s~~= 27.9 M-’ cm-’ for heme D, determined for E. coli bdtype quinol oxidase [14]. 2.4. Electrophoresis
and peptide sequencing
SDS-PAGE was performed according to the method described by Laemmli except that boiling of protein samples was omitted. Molecular masses of polypeptides were estimated by Ferguson plot analysis [ 151 using 10, 12, 14, and 16% (w/v) acrylamide
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gels. For sequence analysis, proteins were separated by SDS-PAGE and electro-transferred to a polyvinylidene difluoride membrane for 3 h in the presence of 25 mM tris(hydroxymethyl)aminomethane, 192 mM glycine, 0.05% (w/v) SDS and 20% (v/v) methanol. The membrane was washed extensively with water to remove glycine, treated with 0.6 N HCl at room temperature for 24 h to cleave a possible Nterminal formyl group [16], and applied on a pulseliquid peptide sequencer (Applied Biosystems, model 477A). 2.5. Materials N-2-Hydroxyethylpiperazine-N’-2-ethane sulfonic acid, MEGA 9 and MEGA 10 were purchased from Dojin Co. (Kumamoto, Japan). DEAE-Toyopearl anion exchange gel and hydroxylapatite were obtained from Tosoh (Tokyo) and Bio-Rad (Hercules). Proteins used as molecular mass standards. polyvinylidene difluoride membranes and NTG were obtained from Sigma, Millipore (Bedford, MA) and Aldrich Chemical (Milwaukee), respectively. TMPD was purchased from Wako (Kyoto). Other reagents were analytical grade.
3. Results 3.1. Isolation of mutants with low TMPD oxidase activity Since the presence of high oxidase activity due to the cytochrome caas-type complex might affect the synthesis and/or detection of alternative oxidases with much lower activity, it was decided to isolate Table I Properties Strain
wild type K7 K17 K21 K22 K25 K41
of B. stearothermophilus Growth
+++ ++ ++ + ++ + +
rate
mutant
mutants with low or no caa3-type oxidase activity and a low content of cytochrome c or a. Cells treated with NTG were spread on culture plates, which sometimes contained streptomycin since mutants deficient in caas-type oxidase would be more resistant to the antibiotic because its uptake into cells is suggested to be dependent on proton motive force across the cell membrane. The TMPD plate assay as described by Mueller and Taber [12] was employed as a screening method for mutants. Sixteen mutant colonies were isolated at the second screening and grown in liquid culture medium to obtain membrane fractions of the cells. All mutant strains showed less than 50% TMPD oxidase activity compared to that of the wild strain. To focus on mutants substantially different from the wild type, we further screened for those which showed clearly modified redox difference spectra and less than 25% of TMPD oxidase activity. Table 1 summarizes the properties of six mutants selected on those criteria. The overall frequency of mutants obtained in the presence of streptomycin (K7 through K25) was 0.07% (5/6900) and higher than that (O.OOS%, l/19400) of mutants obtained in its absence (K41). Mutant K17 showed no apparent absorption peak of cytochrome a at about 600 nm (Fig. lB), suggesting that it lacks the cytochrome caas-type oxidase. However, it grew aerobically at a near normal rate and isolated membranes consumed oxygen in the presence of NADH at wild-type rates, suggesting that it possesses an alternative pathway of respiration. 3.2. IdentiJication of a cytochrome bd-type quinol oxidase In order to identify the alternative
oxidase operat-
strains Cytochrome
Oxygen uptake rate in membranes
a
h
‘
NADH
++ ++ _
++ + ++ ++ + + _
++ ++ + _
480 7 536 27 46 II 73
+ ++ + +
++ + ++
(200 PM)
TMPD 553 45 115 19 67 9 4
(ng atom mg-’
(100FM)
min-‘)
cyt. c (20 FM) 133 4 44 0 9 3 6
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J. Sakamoto et al. I FEMS Microbiology Letters 143 (1996) 151-158
0.08 c V
o^ 0.04 V
1.0
I’
100
3 0.06
B 5 0.03 >r .z
h
5
^n V 0.5 T
ti t 2
0.02
g
0.01
50 s & C w f.
a
0
z ,O
0
1
20
30
40
50
Fraction Number
-4 0,
1 1
0.08
I.5 100
E 0.06
B s E :S *t; $ d g
3-
2-
,_
1.0
g 2 $ 2 s 0 i;
a^
”
V
0.04
N
2
50:
w z
a 0.5 0.0; 0
t
0
Fraction Number Fig. 2. Elution profiles of B. stearothermophilus oxidases in DEAE-anion-exchange chromatography. Membranes from mutant K17 (A) and the wild type (B) were solubilized with MEGA 9+10, applied to a DEAE-Toyopearl anion-exchange column and eluted with a linear gradient of NaCl. TMPD (w) and duroquinol oxidase activity (0) were measured as described in Section 2. Absorbance at 412 nm (A ).
ing in mutant K17, isolated membranes were solubilized with non-ionic detergents and subjected to anion-exchange chromatography. Fig. 2 compares the elution patterns of such chromatography of membrane proteins from the wild type and mutant K17. In the case of wild type (Fig. 2B), TMPD oxidase activity was found in the fraction eluted with buffer
containing about 70 mM NaCl, and corresponded to caas-type cytochrome c oxidase. In the case of mutant K17 (Fig. 2A), TMPD oxidase activity in the 70 mM NaCl eluate was less than 1% of that of the wild type. Instead, quinol oxidase activity was found in the 50 mM NaCl eluate. Practically no quinol oxidase activity was detected in the corresponding frac-
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tions of the wild type (Fig. 2B). The apparent duroquinol oxidation activity found in the fractions eluted with 90 mM NaCl might be due to succinate dehydrogenase, since the polypeptide profile after SDS-PAGE was very similar to that of Bacillus subtilis succinate dehydrogenase [17] and the fraction mainly contained cytochrome b,5:,8which was reducible with Na-succinate. Mutant K17 showed duroquino1 oxidation activity due to succinate dehydrogenase at the corresponding fractions but with a lower activity. The quinol oxidase in mutant K17 was further purified using a hydroxylapatite column and a gel filtration column as described in Section 2. Table 2 shows a summary of the purification. The oxidase activity was not very stable after solubilization even though MEGA 9+10 was more suitable than other detergents tested. The purified oxidase in the reduced state showed absorption peaks at 560 and 618 nm (Fig. 1C). The redox difference spectrum of its pyridine hemochrome showed peaks at 557 and 613 nm, indicating that the chromophores are protoheme IX and heme D. The contents of protoheme IX and heme D were 14.5 and 8.5 nmol (mg protein))‘, calculated from redox difference spectra of cytochromes. The turnover number, measured as duroquinol oxidase activity, was 0.39 ss’ based on the assumption that the enzyme contained two protoheme IX and one heme D as purple bacterial bdtype oxidases. Upon SDS-PAGE the oxidase preparation showed three polypeptide bands migrating at 65, 39, and 27 kDa (Fig. 3). The stoichiometry of the 39 and 27 kDa bands was close to 1: 1 as judged by densitometric scanning, while the relative amount of the 65 kDa band to those of the two lower bands was much less than unity and varied from preparation to preparation. Molecular masses of the two main polypeptides were determined to be 52 and 40 Table 2 Purification Experimental
of cytochrome step
Letters 143 (19961 151-158
66 45 36 29
20
Ml
23
4
Fig. 3. SDS-PAGE of the hd-type quinol oxidase isolated from R .rre~lrothrrmophilus. The concentration of acrylamide was 13.5%. The samples are membranes from mutant K17 (lane I). the MEGA 9+10 extract (lane 2), the peak fraction from DEAEToyopedrl chromatography (lane 3) and the peak fraction from hydroxylapatite chromatography (lane 4). The protein amounts in these lanes are 20, 3, 1.5 and 3 pg. respectively. The proteins used as molecular mass standards (lane M) are bovine serum abumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa) and trypsin inhibitor (20 kDa).
kDa by Ferguson plot analysis. This is consistent with the fact that hydrophobic subunits of oxidases generally migrated faster than hydrophilic proteins of similar size used as molecular mass standards. The three polypeptides were subjected to N-terminal protein sequence analyses. The largest polypep-
h&type quinol oxidase from B. .stc,uuothPrmo)/)hllu.s
Total protein (mg)
Total activity (unit)
Recovery (‘%I)
Specific activity
Purification
(X IO-” unit/mg)
Membranes Cholate wash MEGA extract DEAE fraction Purified enzyme
882 280 138 2.51 2.13
0.676 0.563 1.014 0.200 0.074
100 83 150 29.6 IO.9
0.77 2.01 7.34 79.1 34.5
I 2.62 9.58 104 45.0
(-fold)
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20
B. s. 52 kDa subunit E. co/i
cydA
E.coli appC A. vinelandii
cydA
Fig. 4. Comparison of the N-terminal amino acid sequences of the subunits of B. stearothermophilus bd-type quinol oxidase with the corresponding polypeptides of E. coli and A. vinelandii bdtype quinol oxidases.
tide gave comparable amounts of two main amino acids per every Edman reaction cycle and they were identical to the combined N-terminal sequences of the two smaller polypeptides, indicating that the band found at 65 kDa is composed of the two true subunits of the oxidase which have not been dissociated in the presence of SDS. In Fig. 4 the N-terminal sequences of the 52 and 40 kDa subunits are compared with those of subunits of the bd-type quino1 oxidases from E. coli [8,9] and A. vinelundii [lo].
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position, molecular mass and tendency to resist dissociation by SDS. The N-terminal sequences of the two subunits show similarity to those of E. coli and A. vinelandii, although the similarity is lower than that between the proteobacterial enzymes (Fig. 4). These oxidases might compose an oxidase family distinct from the heme-copper terminal oxidase family [41. The bd-type oxidase was not detected in the wild strain of B. stearothermophilus K1041 (Fig. 2B), or in other thermophilic bacilli grown under highly aerated or air-limited conditions. Cytochrome d has been found in the mesophilic bacteria Bacillus cereus [22] and B. subtilis [23] in stationary growth phase or at low oxygen tensions. It is thus likely that K17 is a mutant that can grow because repression on the operon for the bd-type oxidase is released in some way and that the consequently synthesized bd-type oxidase complements the deficiency in caas-type cytochrome c oxidase. Based on results of the peptide sequence analyses, we are currently trying to clone the genes for the B. stearothermophilus bd-type oxidase.
Acknowledgments 4. Discussion A cytochrome bd-type quinol oxidase was purified from a mutant of Bacillus stearothermophilus K1041. The oxidase is composed of two subunits with molecular masses of 52 and 40 kDa at a 1: 1 ratio, and contains protoheme IX and heme D as chromophores. Cytochrome bd-type quinol oxidases have been purified from E. coli [6], A. vinelundii [18], Photobacterium phosphoreum [19] and Klebsiella pneumoniae [20], all of which belong to the gamma subclass of proteobacteria. Structural genes, cydAB, for the oxidase have been cloned and sequenced from E. coli and A. vinelundii [&lo]. In addition to cydAB, E. coli is known to possess genes, appCB, which encode proteins very similar in sequence to those of the bd-type oxidase [9]. It was recently reported that a second bd-type quinol oxidase is expressed in E. coli when the cell was transformed with some DNA from Bacillus firmus OF4 [21]. The Bacillus bd complex is similar to the oxidases of these Gram-negative bacteria in its subunit com-
We thank Dr. S. Noguchi of this institute and Prof. M. Kawamura of the University of Occupational and Environmental Health for helping us in peptide sequence analysis. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan.
References 111Sone, N. and Yanagita,
Y. (1982) A cytochrome aas-type terminal oxidase of a thermophilic bacterium purification. properties and proton pumping. Biochim. Biophys. Acta 682, 216226. 121Kusano, T., Kuge, S., Sakamoto, J., Noguchi, S. and Sone, N. (1996) Nucleotide and amino acid sequences for cytochrome caas-type oxidase of Bacillus stearothermophilus K1041 and non-Michaelis-type kinetics with cytochrome c. Biochim. Biophys. Acta 1273, 129-138. J.A., Barquera, B., Rumbley, J., Ma, J. and [31Garcia-Horsman, Gennis, R.B. (1994) The superfamily of heme-copper respiratory oxidase. J. Bacterial. 176, 5587-5600.
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[4] Van der Oost, J., de Boer, A.P.N., de Gier, .I.-W.L., Zumft, W.G., Stouthamer, A.H. and van Spanning, R.J.M. (1994) The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase. FEMS Microbial. Lett. 121, I-10. [5] Miller, M.J. and Gennis, R.B. (1983) The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain. J. Biol. Chem. 258, 915999165. [6] Kita, K., Konishi, K. and Anraku, Y. (1984) Terminal oxidases of Escherichia coli aerobic respiratory chain. II. Purification and properties of cytochrome bs,?a-dcomplex from cells grown with limited oxygen and evidence of branched electroncarrying systems. J. Biol. Chem. 259, 337553381. [7] Sane, N. and Fujiwara, Y. (1991) Haem 0 can replace haem A in the active site of cytochrome c oxidase from thermophilic bacterium PS3. FEBS Lett. 288, 154158. [S] Green, G.N., Fang, H., Lin, R.-J., Newton, G., Mather, M. Georgiou, CD. and Gennis, R.B. (1988) The nucleotide sequence of the cyd locus encoding the two subunits of the cytochrome d terminal oxidase complex of Escherichia coli. J. Biol. Chem. 263, 13138-13143. [9] Dassa, J., Fsihi, H., Marck. C., Dion, M., Kieffer-Bontemps. M., and Boquet, P.L. (1991) A new oxygen-regulated operon in Escherichia co/i comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (uppA). Mol. Gen. Genet. 229, 341-352. [IO] Moshiri, F., Chawla, A. and Maier, R.J. (1991) Cloning, characterization, and expression in Escherichia co/i of the genes encoding the cytochrome d oxidase complex from Azotohacter vinelandii. J. Bacterial. 173, 623s-6241. [ll] Narumi, I., Sawakami. K., Nakamoto, S.. Nakayama, N.. Yanagisawa, T., Takahashi, N. and Kihara, H. (1992) A newly isolated Bacillus stearothermophilus K1041 and its transformation by electroporation. Biotechnol. Techniques 6, 83-86. [12] Mueller, J.P. and Taber H.W. (1989) Isolation and sequence of ctaA, a gene required for cytochrome (14s biosynthesis and sporulation in Bacillus subtilis. J. Bacterial. 171, 49674978. [13] Sane, N. (1986) Cytochrome oxidase from thermophilic bacterium PS3. Methods Enzymol. 126, 145-152.
[14] Tsubaki, M., Hori, H., Mogi, T. and Anraku, Y. (1995) Cyanide-binding site of bd-type ubiquinol oxidase from Escherichia coli. J. Biol. Chem. 270, 28565-28569. [15] Hedrick, J.L. and Smith, A.J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126, 155164. [16] Ikeuchi, M. and Inoue, Y. (1988) A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome. FEBS Lett. 241, 99-104. [17] Hagerhall, C, Aasa, R, von Wachenfeldt, C., and Hederstedt, L. (1992) Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase. Biochemistry 31, 7411-7421. [18] Kolonay, Jr., J.F., Moshiri, F., Gennis, R.B., Kaysser, T.M. and Maier, R.J. (1994) Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: comparison to the complex from Escherichia coli. J. Bacterial. 176, 41774181. [19] Konishi, K., Ouchi, M., Kita, K. and Horikoshi, I. (1986) Purification and properties of a cytochrome b56O-dcomplex, a terminal oxidase of the aerobic respiratory chain of Photobacterium phosphorem. J. Biochem. 99, 1227-1236. [20] Smith, A., Hill, S. and Anthony, C. (1990) The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation. J. Gen. Microbiol. 136, 171-180. [21] Sturr, M.G., Krulwich, T.A. and Hicks, D.B. (1996) Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a Acyo Acyd strain complemented by genes from Eacillusjirmus 0F4. J. Bacterial. 176, 17422 1749. [22] Escamilla, J.E., Ramirez, R., Del Arenal, I.P., Zarzoza, G. and Linares, V. (1987) Expression of cytochrome oxidases in Bacillus cereus: effects of oxygen tension and carbon source. J. Gen. Microbial. 133, 3549-3555. [23] Lauraeus, M. and Wikstrom, M. (1993) The terminal quinol oxidases of Bacilius subtifis have different energy conservation properties. J. Biol. Chem. 268, 11470-11473.