©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Bacillus stearothermophilus qcr Operon Encoding Rieske FeS Protein, Cytochrome b, and a Novel-type Cytochrome c of Quinol-cytochrome c Reductase (*)

(Received for publication, November 13, 1995; and in revised form, February 29, 1996)

Nobuhito Sone (§) Naofumi Tsuchiya Masatomo Inoue Shunsuke Noguchi

From the Department of Biochemical Engineering and Science, Kyushu Institute of Technology, 680 Kawazu, Iizuka, Fukuoka-ken 820, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The qcr of Bacillus stearothermophilus K1041 encoding three subunits of the quinol-cytochrome c oxidoreductase (cytochrome reductase, b(6)c(1) complex) was cloned and sequenced. The gene (qcrA) for a Rieske FeS protein of 19,144 Da with 169 amino acid residues, and the gene (qcrC) for cytochrome c(1) of 27,342 Da with 250 amino acid residues were found at adjacent upstream and downstream sides of the previously reported qcrB (petB) for cytochrome b(6) of subunit 25,425 Da with 224 residues (Sone, N., Sawa, G., Sone, T., and Noguchi, S.(1995) J. Biol. Chem. 270, 10612-10617). The three structural genes for thermophilic Bacillus cytochrome reductase form a transcriptional unit.

In the deduced amino acid sequence for the FeS protein, the domain including four cysteines and two histidines binding the 2Fe-2S cluster was conserved. Its N-terminal part more closely resembled the cyanobacteria-plastid type than the proteobacteria-mitochondria type when their sequences were compared. The amino acid sequence of cytochrome c(1) was not similar to either type; the thermophilic Bacillus cytochrome c(1) is composed of an N-terminal part corresponding to subunit IV with three membrane-spanning segments, and a C-terminal part of cytochrome c reminiscent of cytochrome c-551 of thermophilic Bacillus. The subunit IV in the enzyme of cyanobacteria and plastids is the counterpart of C-terminal part of cytochrome b of proteobacteria and mitochondria. These characteristics indicate that Bacillus cytochrome b(6)c(1) complex is unique.


INTRODUCTION

Thermophilic bacilli have menaquinol-cytochrome c oxidoreductase (cytochrome reductase, cytochrome b(6)c(1) complex) and caa(3)-type cytochrome c oxidase as major respiratory complexes(1, 2, 3, 4, 5) , while aa(3)-type menaquinol oxidase is known to oxidize menaquinol in mesophilic bacilli such as Bacillus subtilis(6, 7, 8, 9) and Bacillus cereus(10) . Cytochrome reductase purified from Bacillus PS3, a thermophilic bacterium isolated from a hot spring in Japan closely related to Bacillus stearothermophilus, was found to contain four chromophores in four subunits- 29-kDa cytochrome c(1), 23-kDa Rieske iron-sulfur protein, 21-kDa double-heme cytochrome b(6), and 14-kDa subunit IV (1, 2) .

Cytochrome b/b(6) + subunit IV with two low spin protohemes play important roles in generating proton motive force by the quinone cycle in the reductase(11, 12) . Cytochrome b(6), with two protohemes of the cyanobacterial-plastidal counterparts of cytochrome b of about 420 aa, (^1)is known to be homologous to the N-terminal half of cytochrome b of proteobacteria and mitochondria. Subunit IV of about 160 aa is homologous to the C-terminal half of cytochrome b(13, 14, 15, 16) . The FeS protein and cytochrome c(1)/f are known to be simple electron donors to cytochrome c. Proteobacterial-mitochondrial cytochrome c(1) uses His and Met as axial ligands for heme, while cyanobacterial-plastidal cytochrome f uses His and Tyr(17) ; these two cytochromes exhibit almost no sequence homology(15) . As for the Rieske FeS protein, the C-terminal halves are conserved, while the N-terminal halves are not(15) .

We cloned the cytochrome b locus from transformable B. stearothermophilus K1041 (Bst) and found that the gene for cytochrome b(6) encodes a 224-aa protein with four hydrophobic helices possessing two pairs of His as ligands for the two protoheme, while the gene for subunit IV corresponds to the C-terminal half of cytochrome b(18) . The two genes were adjacent, but separated, indicating that the Bst locus is rather similar to b(6) subunit IV gene organization in cyanobacteria. In addition, several features of deduced amino acid sequences also support evolutionary relatedness to cyanobacterial cytochrome b(6). 1) The two His in the fourth transmembrane segment are separated by 14 aa residues as in cytochrome b(6), not 13 aa as in the proteobacterial and mitochondrial cytochrome b; 2) His of yeast cytochrome b, which may constitute a part of the Qi site, is replaced by Arg as in cytochrome b(6); 3) Lys (as defined in the yeast sequence) is conserved among antimycin A-susceptible forms of the cytochrome but is replaced by Asn as in cytochrome b(6). We found later, however, that the postulated gene for subunit IV downstream from cytochrome b, did not stop after 173 aa residues(18) .

The genes for three subunits of the bc(1) complex form the fbc operon in proteobacteria(20, 21) , while the genes (petBD) for b(6) and subunit IV sit apart from petCD for the FeS protein and cytochrome f in cyanobacteria(14, 22) . A recent paper on Chlorobium limicola showed a third case in which the genes for the FeS protein and cytochrome b are linked, but the gene for cytochrome c(1) is not found downstream of the gene for cytochrome b(23) . Nonetheless, the deduced amino acid sequences of the two C. limicola subunits showed close similarity to the cyanobacterial subunits.

By analyzing upstream and downstream regions of the gene for Bst cytochrome b(6), the genes for FeS protein and cytochrome c(1) are found to form a qcr operon as in proteobacteria. However, the sequence of Bst cytochrome c(1) is quite different from that of purple bacterial cytochrome c(1); the Bst c(1) is composed of a membrane domain homologous to subunit IV and a hydrophilic part homologous to Bacillus small cytochrome c ( (24) and (25) ; see also (26) for review).


EXPERIMENTAL PROCEDURES

Materials

T4-DNA ligase, Klenow fragment, DNA polymerases of Thermus aquaticus (Taq polymerase) and Bacillus caldotenax (Bca best), restriction enzymes, exonuclease III deletion kit, and plasmid vector pUC118, were obtained from Takara Shuzo Co. (Kyoto, Japan). Hybond-N for DNA blotting and [alpha-P]dCTP were purchased from Amersham Corp. DEAE-Toyopearl (DEAE-Fractogel) and a TSK G3000-SW column were products of Tosoh Co. (Tokyo). Cytochrome reductase (cytochrome b(6)c(1) complex) from the thermophilic Bacillus PS3 was prepared as described previously(2) .

Gene Cloning

The Bst cytochrome b gene region containing 1656 bp was described previously(18) . Two adjacent regions of the gene were cloned using Escherichia coli XL-1 blue and pUC118 as a host-vector system as follows. The Bst gene for the FeS protein was cloned by colony hybridization to agarose-sized 2.4-kbp DNA cut with XhoI and ligated into the SalI site of pUC118. The probe was a PCR product of about 400 bp for which partial sequence was obtained. Digoxigenin labeling of the PCR product followed the manufacture's protocol (Boehringer, Mannheim). PCR was carried out with the sense (5`-TCGGA(C/T)AA(C/T)AA(A/G)CA(C/T)(C/A)G) and antisense (5`-GA(C/G)CC(A/G)TG(A/G)CANGG(A/G)CA) oligonucleotides as the primers and PS3 DNA (1 µg) as the template according to the manufacturer's protocol in a thermal cycler (Perkin-Elmer Cetus). A regime of 94 °C/45 °C/72 °C (2 min, 1.5 min, and 1.5 min each) was repeated 25 times. These primers are targeted against an N-terminal peptide of the PS3 FeS protein (-SDNKHR-) and the conserved FeS binding site (-CPCHGS-). The downstream part of the operon was cloned by gene walking using a XhoI-EcoRI fragment of the original 1,656-bp DNA (18) as the probe, and agarose-sized 3.8-kbp SalI fragment of total K1041 genomic DNA as the gene source. Colony hybridizations were carried out at 47-50 °C in 5 times SSC (0.45 M NaCl, 0.045 M sodium citrate buffer, pH 7.0) containing 0.5% blocking reagent (Boehringer Mannheim), 0.1% sodium lauroyl sarcosinate, and 0.02% SDS.

Sequencing of DNA and Peptide

Nucleotide sequencing was carried out by the chain termination method (27) primarily using [alpha-P]dCTP and Bca best DNA polymerase. Deletions were generated with exonuclease III following the manufacture's protocol (Takara). Most parts, especially those that were ambiguous, were also sequenced by the fluorescent dye-primer method using a Shimadzu DNA sequencer (DSQ-1000). Peptide sequences were obtained by Edman degradation with Applied Biosystem's model 473A gas-phase sequencer with proteins transferred to polyvinylidene difluoride membrane after SDS-PAGE. The sequence data were analyzed with software program Genetyx 6.2.0.

RNA Analysis

RNA was prepared from logarithmic-phase cells according to the method of Shiga et al.(28) , electrophoresed, and blotted to Hybond-N. P-Labeled DNA probes were hybridized to the RNA blots in 5 times SSC, 5 times Denhardt's reagent, 50 mM NaPO(4) buffer at pH 6.5, 0.1% SDS, 50% (v/v) formamide at 38 °C for 24 h. DNA fragments for probing were prepared by PCR using qcrB and qcrC, respectively, and labeled by nick-translation method according to the manufacturer's protocol (Takara) with [alpha-P]dCTP.

Other Methods

Membrane fractions from PS3 and Bst, and oligonucleotides for primers were prepared as described previously(18) . Several basic methods, such as SDS-PAGE, protein determination, DNA treatment for molecular cloning, and sequencing were the standard methods as cited in a previous paper (18) .


RESULTS

Preparation of Quinol Reductase and Partial Protein Sequencing

The N-terminal protein sequences of FeS, cytochrome c(1), cytochrome b(6), and subunit IV of cytochrome reductase from the thermophilic Bacillus PS3 are summarized in Table 1. Sequencing of cytochrome b(6) and subunit IV was repeated to confirm our previous results(18) .



Cloning of Bst Genes Encoding the FeS Protein and Cytochrome c(1)

Probes were prepared by PCR using the oligonucleotide mixture targeted to the N-terminal region and the highly conserved FeS-binding motif represented in the PS3 FeS protein. From approximately 300 transformed cells with pUC118 ligated with 2.4-kbp XhoI fragments of Bst DNA, pFBCX3 was isolated. Sequencing of this plasmid revealed that the 3` region of this clone coincides with the upstream sequence of the cytochrome b(6) gene, which we had cloned previously(18) . We also cloned the region of downstream of the cytochrome b(6) gene by the gene-walking method to obtain pFBCSS16.

An Operon Encoding Three Subunits of Bst Cytochrome Reductase

Fig. 1shows a map of the operon encoding three subunits of cytochrome reductase, as well as the sequencing strategy employed. The central part of the gene is the 1.6-kbp SalI-EcoRI fragment reported previously (18) . The 2.4-kbp XhoI fragment in pFBCX3, and the 3.8-kbp SmaI-SalI fragment in pFBCSS16, in addition to BC15, which was previously reported(18) , covers qcrABC encoding the FeS protein, cytochrome b(6), and cytochrome c(1), respectively.


Figure 1: A map of Bst DNA around the qcrABC operon encoding the FeS, cytochrome b(6), and cytochrome c(1), and sequence strategy. Three clones, two new clones (pFBCX3 and pFBCSS16) in addition to bcl5 (SalI-EcoRI fragment) as described previously(18) , cover the whole operon. Putative promoter () and terminator () regions, and primers ( ) used for PCR are shown.



Fig. 2shows typical results of Northern blotting with Bst RNA. Both qrcB and qrcC probes hybridized to main bands at 2.1 kilobases, indicating that the putative promoter and terminator (Fig. 3) are probably assigned correctly, and the transcript is polycistronic for three subunits of the Bst cytochrome reductase. The radioactive bands of a high molecular weight seem to be due to contamination of genomic DNA.


Figure 2: Northern blot analysis of Bst RNA with qcrA and qcrC as the probes. RNA loadings were 5 µg (lanes 1 and 4), 10 µg (lanes 2 and 5), and 20 µg (lanes 3 and 6). Lanes 1-3, probed with qcrB. Lanes 4-6 probed with qcrC. The positions of RNA size standards are as indicated. Exposure time was 12 h.




Figure 3: DNA and deduced amino acid sequences of Bst FeS protein, cytochrome b(6), and cytochrome c(1). Putative Shine-Dalgarno sequences are boxed. The nucleotides that may constitute the putative promoter region are boxed and shaded. The arrows indicate where putative stem-loop structure might form the terminator of the gene. The residues from N-terminal sequence analyses are shown in boldface letters.



DNA and Amino Acid Sequences

Fig. 3shows the DNA and deduced aa sequences of Bst qcr region. From both the N-terminal protein sequences (Table 1) and the deduced amino acid sequences for the three subunits of the Bst cytochrome reductase, 169 residues of 19,144 Da comprise the FeS protein, 224 residues of 25,425 Da are for cytochrome b(6) (as reported previously), and 250 residues of 27,342 Da are for cytochrome c(1). The qcrABC operon is adjacent to the phoR locus of the phosphate regulon, since an ORF translated from nucleotides 1-473 was over 40% identical to the corresponding C-terminal region of phoR of E. coli(29) . ORFs that may be transcribed downstream of qcrC, including those in the complementary strand, have not been assigned. The gene assigned to encode subunit IV in the previous report (18) turns out to be a part of the gene encoding 29-kDa cytochrome c(1), since it is initiated with MHRGKGMKF instead of MKF (Table 1). The residues found in the N-terminal peptides of the subunits of PS3 cytochrome reductase (Table 1) are shown in boldface letters (Fig. 3). The residues found in the peptides of PS3 cytochrome b(6) and cytochrome c(1) coincide with those of the Bst DNA sequences, indicating that no N-terminal processing occurs for these proteins. The only difference between the two strains is found at the 16th residue of the FeS protein, where Pro is found in the PS3 subunit, while Thr is identified in K1041. The initiating f-Met is removed from the FeS protein.

Structure of FeS Protein

Fig. 4shows alignment of the Bst FeS protein with several corresponding proteins from various sources. As pointed out by Hauska et al.(15) , C-terminal halves, including the FeS binding cluster (hatched), are conserved among all 10 proteins displayed here, but N-terminal halves are divided into two groups: proteobacteria-mitochondria and cyanobacteria-plastid types. The Bst protein along with that of Chlorobium limicola(23) have unique sequences, but are still apparently close to those of the cyanobacterial/plastid class. The identities in the alignments were 12.5% (bovine mitochondrion), 11.2% (Paracoccus denitrificans), 12.4% (Rhodobacter sphaeroides), 23.7% (spinach plastid), 23.1% (Synechocystis sp.) 25.4% (Nostoc sp.) and 26.6% (C. limicola). Thus, the Bst FeS protein showed the highest homology with that of the green sulfur bacteria, although it may be still possible for other cyanobacterial FeS proteins to show higher similarity than that of C. limicola. A hydropathy plot of the Bst FeS protein indicated that the protein is mostly hydrophilic and probably has no hydrophobic membrane-spanning segment as is the case for FeS proteins from other organisms, as shown in Fig. 5A.


Figure 4: Multiple alignment of the Bst FeS protein with those from various sources. The proteins from rat mitochondria(37) , yeast mitochondria(38) , R. sphaeroides (Rs, (39) ), spinach plastids (Spi; (40) ), Synechocytosis PCC6803 (Sy; (41) ), Nostoc PCC7906 (No; (22) ), and C. limicola(23) are compared with the Bst protein. The conserved FeS-binding motifs are boxed, and all residues identical with those of Bst are shaded.




Figure 5: Hydropathy profiles of Bst FeS protein (A) and cytochrome c(1) (B). The procedure of Kyte and Doolittle (36) was used with a window of 15 residues. The FeS-binding sites (cf.Fig. 4) in A are hatched. The arrows in B show heme C-binding site.



Structure of Cytochrome c(1)

Cytochrome c(1) is an electron acceptor for the FeS protein and donor to cytochrome c. Cytochrome c(1) and cytochrome f are known to have one hydrophobic segment near the C terminus for membrane anchoring(11, 12) . Fig. 5B shows a hydropathy plot of Bst cytochrome c(1). There are three membrane-spanning hydrophobic segments in the N-terminal portion, but none in the C-terminal portion. The N-terminal hydrophobic part of Bst cytochrome c(1) is the same sequence that was reported previously but misidentified as subunit IV with three hydrophobic segments(18) . There were misreads of the DNA sequence, and its identification as a unique form of subunit IV was subsequently corrected(18) . Fig. 6shows an alignment of Bst cytochrome c(1) with two types of subunit IV from cytochrome b(6)f complexes and three Bacillus small c-type cytochromes. The N-terminal part of Bst cytochrome c(1) shows many identical aa residues (22-25% identity) with subunit IV from Nostoc and maize plastids, while the C-terminal part is analogous to Bacillus small cytochromes c such as PS3 c-551(25) , B. subtilis c-550(24) , and B. licheniformis c-552(30) . The latter portion with about 100 aa residues is hardly homologous to that of cytochrome c(1) or cytochrome f.


Figure 6: Multiple alignment of Bst cytochrome c(1) with subunit IVs (A) and Bacillus small cytochromes c (B). The N-terminal part of Bst cytochrome c(1) is compared with subunit IVs of Nostoc(22) and maize plastids(19) . The C-terminal part is aligned with PS3 cytochrome c-551(25) , B. subtilis c-550(24) , and B. licheniformis c-552(30) . Heme C motifs are boxed, and residues identical with those of Bst cytochrome c(1) are shaded.




DISCUSSION

The presented data demonstrate that the structural genes for three subunits of thermophilic Bacillus cytochrome reductase constitute an operon structure like that of the proteobacterial fbc operon. However, the Bacillus cytochrome b(6) is small, and its sequence is most closely related to cytochrome b(6) of cyanobacteria as reported previously(1, 2, 18) . The petB gene for cytochrome b(6) of cyanobacteria is followed by petD for subunit IV, which has sequence homology with the C-terminal half of cytochrome b of proteobacteria(14, 15) . We reported that Bst cytochrome b(6) was small due to the presence of a stop codon at position 225 leaving the subunit IV part to be encoded on another gene as in cyanobacteria (18) . At first we thought that the Bst subunit IV is equivalent to cyanobacterial subunit IV, since a stop codon for the presumed Bst subunit IV was found at a similar place. The stop codon, however, turned out to be the result of a frameshift due to a misread of the DNA sequence(18) . We previously cloned the PS3 cytochrome b(6) gene by PCR using N-terminal sequences of cytochrome b(6) and subunit IV for designing a set of primers targeting WRDIAD and MKFENT, respectively (18) . The deduced aa residues of qcrC, presumably encoding subunit IV, coincided with the N-terminal sequence only at the first three residues (MKF), but the subsequent residues were totally different, even though the clone (bc15) was obtained by using the probe prepared by PCR(18) . We also determined that the N-terminal sequence of PS3 cytochrome c(1) is MHRGLGMKFV- (Table 1), and found that this corresponds to the ORF previously ascribed for ``subunit IV''(18) , when translational initiation occurs 6 aa residues further upstream. Moreover, there is a heme C-binding motif, CXXCH, at about 580 bp downstream of the initiation codon. Cytochrome c(1), which is SDS-dissociated and fractionated by reverse-phase high pressure liquid chromatography, possesses a heme content of 32 nmol/mg of protein, indicating that the heme-staining protein of about 30 kDa has one heme C. (^2)The qcrC gene encodes a 27,342-Da protein with 250 residues, suggesting that cytochrome c(1) retains subunit IV as an integral N-terminal component. We were also able to prepare and sequence peptides possessing IAQANTXTSXHGENL- by trypsin digestion, and PGGIFKGTDEELQK- by treatment with cyanogen bromide, showing directly that the deduced aa sequence of the c-type cytochrome found in PS3 29-kDa protein is represented in the sequence for Bst qcrC.

What, then, is the identity of the ``band IV protein'' that copurified with cytochrome reductase from the thermophilic Bacillus PS3(2) ? Much of the band IV protein in the PS3 enzyme preparations can be removed without severe loss of quinol-dependent cytochrome c reductase activity by gel filtration in the presence of lauroyl sarcosinate.^2 Thus, this protein may be contaminant or an auxiliary component of the enzyme. On the other hand, it is also noteworthy that some purple bacteria such as Rb. sphaeroides are known to contain subunit IV of 14 kDa(31) , while Rb. capsulatus contains three subunits(32) .

The bacterial cytochrome reductases have been divided into two subclasses; bc(1)-type is found in proteobacteria and mitochondria, while the b(6)f-type occurs in cyanobacteria and plastids having b(6) and the subunit IV instead cytochrome b(13, 14, 15) . The three subunits of bc(1)-type are encoded in qcrABC operon, while petBD for cytochrome b(6)plus subunit IV and petCA for the FeS protein plus cytochrome f are at separate sites in cyanobacterial genomes(14, 22) . It is also pointed out (15) that the N-terminal halves of the FeS proteins and cytochrome c(1) and cytochrome f are not homologous, suggesting their different evolutionary origins for proteobacteria and cyanobacteria. Bacillus cytochrome reductase is apparently a third type, different from both proteobacterial and cyanobacterial counterparts. The structural genes for cytochrome reductase of the thermophilic Bacillus form an operon (qcrABC) similar to the proteobacterial fbcABC operon, but comparison of the corresponding aa sequences of the FeS protein and cytochrome b indicate that Bst sequences are most similar to cyanobacterial counterparts.

The thermophilic Bacillus cytochrome c(1) is quite unique; it consists of a subunit IV-like N terminus with three trans-membrane segments, and a hydrophilic cytochrome c-like C terminus, unlike either proteobacterial cytochrome c(1) or cyanobacterial cytochrome f. A very similar gene, qcrABC, having the same operon structure and close sequence homologies, was very recently found in B. subtilis in the course of studies on F-directed transcription during sporulation(33) . Although the B. subtilis cytochrome reductase, has not been purified, the gene structure suggests the presence of cytochrome reductase very similar to that of the thermophilic bacilli (cytochrome b(6)c(1) complex). A recent report on the green sulfur bacterium C. limicola showed petCB for cyanobacterial type-FeS protein and cytochrome b. The latter is not split but is more similar to the cyanobacterial cytochrome b(6)plus subunit IV gene than proteobacterial fbcB gene(23) . The gene for cytochrome c(1) is missing in this operon of C. limicola. Gene structure of cytochrome reductases of eubacteria described above suggest the following evolutionary scenario. The commonote had a long gene for cytochrome b, while the original genes for the FeS protein and c-type cytochrome occurred at separate places. Then, proteobacteria integrated the genes for their FeS protein and cytochrome c(1) into operon structure, while the green sulfur bacteria recombined with a slightly different FeS gene (note that its N-terminal part is cyano-bacterial type). Gene separation, introducing cytochrome b(6) and subunit IV, due to formation of a stop codon occurred next in a cyanobacterial ancestor. The ancestor of Bacillus recombined the gene for cyanobacterial-type FeS protein upstream of the gene for cytochrome b(6) in addition to obtaining the gene for Bacillus small cytochrome c at the 3`-end of the gene for subunit IV in order to donate electrons to cytochrome c. Although proteobacterial cytochrome c(1), Bacillus cytochrome c(1), and cyanobacterial cytochrome f are similar in size, their origins seem to be quite different. It is interesting to find that cytochrome bc(1)/b(6)f complexes, common for photosynthesis, are composed of different subunits formed through protein domain swapping. In the terminal oxidase superfamily, only subunit I bearing low spin heme and high spin heme-copper binuclear center is completely conserved as the catalytic unit, while the presence and origin of other subunits is variable(34, 35) .


FOOTNOTES

*
This work was supported in part by Grants-in-aid for Scientific Research 04266217 and 07459018 from the Ministry of Education, Science and Culture of Japan (to N. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D83789[GenBank].

§
To whom correspondence should be addressed. Tel.: 81-948-29-7813; Fax: 81-948-29-7801.

(^1)
The abbreviations used are: aa, amino acid(s); Bst, B. stearothermophilus strain K1041; PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; PCR, polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s).

(^2)
N. Sone, N. Tsuchiya, M. Inoue, and S. Noguchi, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Jun Yu, L. Hederstedt, and P. J. Piggot for providing manuscript before publication and Mie Shugyo for skillful technical assistance.


REFERENCES

  1. Sone, N., Sekimachi, M., and Kutoh, E. (1987) J. Biol. Chem. 262, 15386-15391 [Abstract/Free Full Text]
  2. Kutoh, E., and Sone, N. (1988) J. Biol. Chem. 263, 9020-9026 [Abstract/Free Full Text]
  3. Sone, N., and Yanagita, Y. (1982) Biochim. Biophys. Acta 682, 216-226
  4. Ishizuka, M., Machida, K., Shimada, S., Mogi, A., Tsuchiya, T., Ohmori, T., Souma, Y., Gonda, M., and Sone, N. (1990) J. Biochem. (Tokyo) 108, 866-873
  5. Sone, N., and Fujiwara, Y. (1991) J. Biochem. (Tokyo) 110, 1016-1021
  6. Lauraeus, M., Haltia, T., Saraste, M., and Wikström, M. (1991) Eur. J. Biochem. 197, 699-705 [Abstract]
  7. Santana, M., Kunst, F., Hullo, M. F., Rapoport, G., Danchin, A., and Glaser, P. (1992) J. Biol. Chem. 267, 10225-10231 [Abstract/Free Full Text]
  8. Van der Oost, J., von Wachenfeldt, C., and Hederstedt, L. (1991) Mol. Microbiol. 5, 2063-2072 [Medline] [Order article via Infotrieve]
  9. Von Wachenfeldt, C., and Hederstedt, L. (1990) FEMS Microbiol. Lett. 100, 91-100
  10. Garcia-Horsman, J. A., Barquera, B., Gonzalez-Halphen, D., and Escamilla, J. E. (1991) Mol. Microbiol. 5, 197-205 [Medline] [Order article via Infotrieve]
  11. Trumpower, B. L. (1990) J. Biol. Chem. 265, 11409-11412 [Free Full Text]
  12. Trumpower, B. L., and Gennis, R. B. (1994) Annu. Rev. Biochem. 63, 675-716 [CrossRef][Medline] [Order article via Infotrieve]
  13. Widger, W. R., Cramer, W. A., Herrman, R. G., and Trebst, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 674-678 [Abstract]
  14. Kallas, T., Spiller, S., and Malkin, R. (1988) J. Biol. Chem. 263, 14334-14342 [Abstract/Free Full Text]
  15. Hauska, G., Nitschke, W., and Herrmann, R. G. (1988) J. Bioenerg. Biomembr. 20, 211-228 [Medline] [Order article via Infotrieve]
  16. Degli Esposti, M. (1993) Biochim. Biophys. Acta 1143, 243-271 [Medline] [Order article via Infotrieve]
  17. Prince, R. C., and George, G. N. (1995) Trends Biochem. Sci. 20, 217-218 [CrossRef][Medline] [Order article via Infotrieve]
  18. Sone, N., Sawa, G., Sone, T., and Noguchi, S. (1995) J. Biol. Chem. 270,10612-10617; Correction (1995) J. Biol. Chem.270, 22076
  19. Rock, C. D., Barkan, A., and Taylor, W. L. (1987) Curr. Genet. 17, 69-77
  20. Verbis, J., Lang, F., Gabellini, N., and Oesterhelt, D. (1989) Mol. & Gen. Genet. 219, 445-452
  21. Robertson, D. E., Daldal, F., and Dutton, P. L. (1990) Biochemistry 29, 11249-11260 [Medline] [Order article via Infotrieve]
  22. Kallas, T., Spiller, S., and Malkin, R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5794-5798 [Abstract]
  23. Schutz, M., Zirngibl, S., Le Coutre, J., Buttner, M., Xie, D-L., Nelson, N., Deutzman, R., and Hauska, G. (1994) Photosynth. Res. 39, 163-174
  24. Von Wachenfeldt, C., and Hederstedt, L. (1990) J. Biol. Chem. 265, 13939-13948 [Abstract/Free Full Text]
  25. Fujiwara, Y., Oka, M., Hamamoto, T., and Sone, N. (1993) Biochim. Biophys. Acta 1141, 213-219 [Medline] [Order article via Infotrieve]
  26. Sone, N., and Toh, H. (1994) FEMS Microbiol. Lett. 122, 203-210 [Medline] [Order article via Infotrieve]
  27. Sanger, F., Nicklen, S., and Coulson, A. E. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  28. Shiga, Y., Yamagata, H., and Udaka, S. (1993) J. Bacteriol. 175, 7130-7137 [Abstract]
  29. Makino, K., Shinagawa, H., Amemura, M., and Nakata, A. (1986) J. Mol. Biol. 192, 549-556 [Medline] [Order article via Infotrieve]
  30. Hreggvidsson, G. O. (1991) Biochim. Biophys. Acta 1058, 52-55 [Medline] [Order article via Infotrieve]
  31. Usui, S., and Yu, L. (1991) J. Biol. Chem. 266, 15644-15649 [Abstract/Free Full Text]
  32. Gabellini, N. (1988) J. Bioenerg. Biomembr. 20, 59-83 [Medline] [Order article via Infotrieve]
  33. Yu, J., Hederstedt, L., and Piggot, P. J. (1995) J. Bacteriol. 177, 6751-6760 [Abstract]
  34. Castresana, J., Lubben, M., Saraste, M., and Higgins, D. G. (1994) EMBO J. 13, 2516-2525 [Abstract]
  35. Van der Oost, J., de Boer, A. P. N., de Gier, J. L., Zumft, W. G., Stouthamer, A. H., and van Spanning, R. J. M. (1994) FEMS Microbiol. Lett. 121, 1-10 [CrossRef][Medline] [Order article via Infotrieve]
  36. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  37. Nishikimi, M., and Ozawa, T. (1989) Biochem. Biophys. Res. Commun. 159, 19-25 [Medline] [Order article via Infotrieve]
  38. Beckmann, J. D., Ljungdahl, P. O., Lopez, J. L., and Trumpower, B. L. (1987) J. Biol. Chem. 262, 8901-8909 [Abstract/Free Full Text]
  39. Davidson, E., and Daldal, F. (1990) J. Mol. Biol. 195, 23-30
  40. Steppuhn, J., Hermans, J., Jansen, T., Salnikow, J., Hauska, G., and Herrmann, R. G. (1987) Mol. & Gen. Genet. 210, 171-177
  41. Mayes, and Barber, J. (1991) Plant Mol. Biol. 17, 289-293 [Medline] [Order article via Infotrieve]

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