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• W2057963987 abstract "We demonstrate that the cccB gene, identified in the Bacillus subtilis genome sequence project, is the structural gene for a 10-kDa membrane-bound cytochrome c551 lipoprotein described for the first time in B. subtilis. Apparently, CccB corresponds to cytochrome c551 of the thermophilic bacterium Bacillus PS3. The heme domain of B. subtilis cytochrome c551 is very similar to that of cytochrome c550 , a protein encoded by the cccA gene and anchored to the membrane by a single transmembrane polypeptide segment. Thus, B. subtilis contains two small, very similar, c-type cytochromes with different types of membrane anchors. The cccB gene is cotranscribed with the yvjA gene, and transcription is repressed by glucose. Mutants deleted for cccB or yvjA-cccB show no apparent growth, sporulation, or germination defect. YvjA is not required for the synthesis of cytochrome c551, and its function remains unknown. We demonstrate that the cccB gene, identified in the Bacillus subtilis genome sequence project, is the structural gene for a 10-kDa membrane-bound cytochrome c551 lipoprotein described for the first time in B. subtilis. Apparently, CccB corresponds to cytochrome c551 of the thermophilic bacterium Bacillus PS3. The heme domain of B. subtilis cytochrome c551 is very similar to that of cytochrome c550 , a protein encoded by the cccA gene and anchored to the membrane by a single transmembrane polypeptide segment. Thus, B. subtilis contains two small, very similar, c-type cytochromes with different types of membrane anchors. The cccB gene is cotranscribed with the yvjA gene, and transcription is repressed by glucose. Mutants deleted for cccB or yvjA-cccB show no apparent growth, sporulation, or germination defect. YvjA is not required for the synthesis of cytochrome c551, and its function remains unknown. nutrient sporulation medium with phosphate polymerase chain reaction 5-aminolevulinic acid base pair(s) The cytoplasmic membrane of the Gram-positive bacterium Bacillus subtilis contains cytochromes of a-, b-, c-, and d-type (1von Wachenfeldt C. Hederstedt L. FEMS Microbiol. Lett. 1992; 100: 91-100Crossref PubMed Google Scholar). The c-type cytochromes differ from other cytochromes by having heme covalently bound to the polypeptide via cysteine residues in a consensus motif, Cys-Xaa-Xaa-Cys-His, in which the His residue functions as the fifth axial ligand to the heme iron. Three different membrane-bound c-type cytochromes have been described in B. subtilis. They are all dispensable for growth, repressed by glucose, and expressed in the early stationary phase (1von Wachenfeldt C. Hederstedt L. FEMS Microbiol. Lett. 1992; 100: 91-100Crossref PubMed Google Scholar). These cytochromes c are subunit II of the cytochrome caa3 complex (encoded by the ctaC gene) (2Saraste M. Metso T. Nakari T. Jalli T. Lauraeus M. van der Oost J. Eur. J. Biochem. 1991; 195: 517-525Crossref PubMed Scopus (99) Google Scholar), cytochrome c of the cytochrome bc complex (encoded by the qcrC gene) (3Yu J. Hederstedt L. Piggot P.J. J. Bacteriol. 1995; 177: 6751-6760Crossref PubMed Scopus (76) Google Scholar), and the monomeric cytochrome c550 (encoded by the cccA gene) (4von Wachenfeldt C. Hederstedt L. J. Biol. Chem. 1990; 265: 13939-13948Abstract Full Text PDF PubMed Google Scholar). Cytochrome caa3 is a cytochrome c oxidase. The cytochrome bc complex oxidizes menaquinol and transfers electrons to cytochrome c. Cytochrome c550 is a 13-kDa protein with a membrane anchor domain consisting of a single α-helical transmembrane segment of about 30 residues and a heme domain of about 74 residues (4von Wachenfeldt C. Hederstedt L. J. Biol. Chem. 1990; 265: 13939-13948Abstract Full Text PDF PubMed Google Scholar). The latter domain, like that of all bacterial c-type cytochromes, is located on the outer surface of the cytoplasmic membrane (5von Wachenfeldt C. Hederstedt L. FEBS Lett. 1990; 270: 147-151Crossref PubMed Scopus (44) Google Scholar). At pH 7.0, cytochrome c550 has a midpoint redox potential of +178 mV (6von Wachenfeldt C. Hederstedt L. Eur. J. Biochem. 1993; 212: 499-509Crossref PubMed Scopus (30) Google Scholar). The function of this cytochrome is not known, and deletion or overexpression of the cccA gene does not affect the respiration activity of the cell (4von Wachenfeldt C. Hederstedt L. J. Biol. Chem. 1990; 265: 13939-13948Abstract Full Text PDF PubMed Google Scholar). Understanding the respiratory system and energy metabolism of B. subtilis requires detailed knowledge of the cytochromes and their specific biological roles. Sequence analysis of the entire B. subtilis genome revealed the cccB gene encoding a possible novel cytochrome c in B. subtilis. The deduced CccB sequence shows about 35% identity to CccA and has the cytochrome c consensus motif in the C-terminal part of the polypeptide. This was the only new c-type cytochrome found in the B. subtilis genome sequencing project. The cccB gene is located at 310° on the chromosome far away from the cccA gene at 222° (7Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Cummings N.J. Daniel R.A. Denizot F. Devine K.M. Dusterhoft A. Ehrlich S.D. Emmerson P.T. Entian K.D. Errington J. Fabret C. Ferrari E. Foulger D. Fritz C. Fujita M. Fujita Y. Fuma S. Galizzi A. Galleron N. Ghim S.Y. Glaser P. Goffeau A. Golightly E.J. Grandi G. Guiseppi G. Guy B.J. Haga K. Haiech J. Harwood C.R. Henaut A. Hilbert H. Holsappel S. Hosono S. Hullo M.F. Itaya M. Jones L. Joris B. Karamata D. Kasahara Y. Klaerr-Blanchard M. Klein C. Kobayashi Y. Koetter P. Koningstein G. Krogh S. Kumano M. Kurita K. Lapidus A. Lardinois S. Lauber J. Lazarevic V. Lee S.M. Levine A. Liu H. Masuda S. Mauel C. Medigue C. Medina N. Mellado R.P. Mizuno M. Moestl D. Nakai S. Noback M. Noone D. O'Reilly M. Ogawa K. Ogiwara A. Oudega B. Park S.H. Parro V. Pohl T.M. Portetelle D. Porwollik S. Prescott A.M. Presecan E. Pujic P. Purnelle B. Rapoport G. Rey M. Reynolds S. Rieger M. Rivolta C. Rocha E. Roche B. Rose M. Sadaie Y. Sato T. Scanlan E. Schleich S. Schroeter R. Scoffone F. Sekiguchi J. Sekowska A. Seror S.J. Serror P. Shin B.S. Soldo B. Sorokin A. Tacconi E. Takagi T. Takahashi H. Takemaru K. Takeuchi M. Tamakoshi A. Tanaka T. Terpstra P. Tognoni A. Tosato V. Uchiyama S. Vandenbol M. Vannier F. Vassarotti A. Viari A. Wambutt R. Wedler E. Wedler H. Weitzenegger T. Winters P. Wipat A. Yamamoto H. Yamane K. Yasumoto K. Yata K. Yoshida K. Yoshikawa H.F. Zumstein E. Yoshikawa H. Danchin A. Nature. 1997; 390: 249-256Crossref PubMed Scopus (3126) Google Scholar). In this paper we demonstrate that cccB is the structural gene for a membrane-anchored cytochrome c551. As compared with the other c-type cytochromes in wild type cells, CccB is present in very low amounts, i.e. less than 103 molecules/cell. We have also analyzed the transcription of cccB and the properties of cccB null mutants. This new B. subtilis cytochrome has been purified and some of its characteristics are presented. Bacterial strains and plasmids used in this work are presented in Table I.Table IBacterial strains and plasmids used in this workStrains and plasmidsGenotype or relevant properties1-aApr, ampicillin resistance; Emr, erythromycin resistance; Cmr, chloramphenicol resistance; and Tetr, tetracycline resistance.Origin or Ref.E. coli SUREe14−(mcrA) Δ(mcrCD− hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC:Tn5 (kanr) uvrC [F′ proAB lacI qZΔM15 Tn:10 (tetr)]StratageneB. subtilis 168trpC2Laboratory stockB. subtilis L16205trpC2 ΔcccB::tetThis workB. subtilis L16224trpC2 Δ(yvjA-cccB)::tetThis workB. subtilis L16225trpC2 Δ(yvjA-cccB)::tet ΔcccA::catThis workB. subtilis L16238trpC2, amyE::pCR977 (yvjA-lacZ)This workB. subtilis LUH20trpC2 ΔctaCD::ble ΔcccA::catThis workB. subtilis LUH36trpC2 ΔctaCD::ble ΔcccA::cat ΔcccB::tetThis workpBluescript SK(−)AprStratagenepUC18, pUC19Apr8Yanisch-Perron C. Viera J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11460) Google ScholarpHP13Emr Cmr9Haima P. Bron S. Venema G. Mol. Gen. Genet. 1987; 209: 335-342Crossref PubMed Scopus (161) Google ScholarpMY2B. subtilis sdh promoter on a 792-bp fragment in pHP1310Hansson M. Gustafsson M.C.V. Kannangara G. Hederstedt L. Biochim. Biophys. Acta. 1997; 1340: 97-104Crossref PubMed Scopus (18) Google ScholarpLUT191part of cccA on a 600-bp Eco RI-Kpn I fragment in pUC1911Schiött T. Throne-Holst M. Hederstedt L. J. Bacteriol. 1997; 179: 4523-4529Crossref PubMed Google Scholarp4303pMTL20EC with cccB on a 1671-bp Pst I fragment12Rivolta C. Molecular genetics of Bacillus subtilis: From Genome Sequence to Protein Function, Ph.D. thesis. University of Lausanne, Lausanne, Switzerland1999Google ScholarpCR977transcriptional fusion of B. subtilis yvjA-cccB promoter and E. coli lacZ in pDH32This work (Fig. 1)pCRΔcccBΔcccB::Tetrin pUC18This work (Fig. 1)pCRΔ972Δ(yvjA-cccB)::Tetr in pUC18This work (Fig. 1)pLUJ104cccB on a 1010-bp BamHI-HindIII fragment in pMY2This workpLUJ105cccA-cccB hybrid gene on a 915-bp EcoRI-BamHI fragment in pHP13This work1-a Apr, ampicillin resistance; Emr, erythromycin resistance; Cmr, chloramphenicol resistance; and Tetr, tetracycline resistance. Open table in a new tab Escherichia coli cells were grown on Luria agar plates or in LB (13Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Unless otherwise stated, B. subtilis cells were grown on tryptose blood agar base (Difco) plates or in nutrient sporulation medium with phosphate (NSMP)1, pH 7.0 (14Fortnagel P. Freese E. J. Bacteriol. 1968; 95: 1431-1438Crossref PubMed Google Scholar). The concentration of antibiotics used for B. subtilis was 4 μg/ml chloramphenicol and erythromycin, 15 μg/ml tetracycline, and the concentration used for E. coli was 100 μg/ml ampicillin, 12.5 μg/ml chloramphenicol, 15 μg/ml tetracycline. Plasmids were isolated using CsCl density gradient centrifugation (15Ish-Horowicz D. Burke J.F. Nucleic Acids Res. 1981; 9: 2989-2998Crossref PubMed Scopus (1079) Google Scholar) or by using the Quantum Prep® plasmid mini preparation kit (Bio-Rad). General DNA techniques were as described by Sambrook et al. (13Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The procedure for transformation of B. subtilis was based on a method described by Arwert and Venema (16Arwert F. Venema G. Mol. Gen. Genet. 1973; 123: 185-198Crossref PubMed Scopus (50) Google Scholar) or according to Karamata and Gross (17Karamata D. Gross J.D. Mol. Gen. Genet. 1970; 108: 277-287Crossref PubMed Scopus (113) Google Scholar). E. coli competent cells were prepared and transformed according to the calcium chloride method (13Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) or by electroporation as described in Ref. 18Hanahan D. Jessee J. Bloom F.R. Methods Enzymol. 1991; 204: 63-113Crossref PubMed Scopus (452) Google Scholar. PCR was done using the AmpliTaq polymerase (Perkin-Elmer) or Pwo DNA polymerase (Roche Molecular Biochemicals) according to the suppliers' instructions. PCR was used to investigate the presence of mRNA molecules carrying the sequence corresponding to the yvjA-cccB intergenic region. For this purpose, the following oligonucleotides were prepared: CR108, 5′-GTC CGA TTT TAA TGT GCG TGG TTG-3′, whose sequence is identical to the distal part of the yvjA- coding DNA strand; and CR109, 5′-GCT TCC GTC TTG CTG CCA GTG TCT-3′, complementary to the mRNA encoding a proximal part of cccB. 32 μg of total RNA were extracted from 22 ml of a late exponential phase LB culture of B. subtilis 168 by using the RNeasy Mini Kit (Qiagen). The extract was incubated for 60 min with 5 units of DNase I at 37 °C. After heat inactivation of the DNase (65 °C for 20 min), 5 ng of the RNA preparation were incubated for 20 min at 60 °C in reverse transcription buffer containing primer CR109 (2 μm), 0.9 mm MnCl2, 3.2 mm dNTP mixture, and 4 units of Tth DNA polymerase (Roche Molecular Biochemicals). Under these conditions and in the presence of Mn2+, the Tth DNA polymerase can perform reverse transcription and thus catalyze the synthesis of the cDNA strand complementary to the cccB mRNA. Subsequently, the mixture was supplemented with primer CR108 (2 μm), 0.75 mm EGTA, and PCR buffer according to the manufacturer's instructions. The PCR was performed in the same tube, because the Tth enzyme can act as a thermostable DNA polymerase in the presence of the Mg2+ present in the PCR buffer. To confirm that the resulting product originated from template mRNA and not from eventual chromosomal DNA contamination, a negative control was performed by running in parallel the same RNA preparation previously incubated for 120 min at 37 °C with 5 mg/ml DNase-free RNase A. Plasmid pCRΔcccB was constructed in several steps. Basically it is a derivative of pUC18 into which the two DNA fragments from the B. subtilis chromosome (Fig. 1), obtained by using PCR, and the tetracycline resistance gene from the plasmid pBEST307 (19Itaya M. Tanaka T. J. Mol. Biol. 1991; 220: 631-648Crossref PubMed Scopus (122) Google Scholar) were introduced. pCRΔ972 was obtained from pCRΔcccB by substituting the distal part of yvjA with a PCR-obtained fragment homologous to the chromosomal region located upstream of yvjA (Fig. 1). Plasmid pCR977 carries a transcriptional fusion of the yvjA-cccB promoter region with the lacZ gene from E. coli (Fig. 1). It was obtained by cloning the PCR-derived DNA fragment used for the pCRΔ972 construction into pDH32. The latter plasmid allows the ectopical integration of the gene fusion into the B. subtilis amyE locus (20Perego M. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology and Molecular Genetics. American Society for Microbiology, Washington, D. C.1993: 615-624Google Scholar). Plasmid pLUJ104, used for overproduction of CccB, was constructed as follows. Plasmid p4303 was cleaved by Eco RI and Hin dIII, and the obtained 990-bp fragment containing the cccB gene was ligated into pBluescript SK(-). From this plasmid, multiplied in E. coli SURE, a 1010-bp Bam HI-Hin dIII fragment containing the cccB gene was ligated into pMY2 downstream of the B. subtilis sdh promoter. Plasmid pLUJ105 was constructed as follows. Plasmid pLUT191, which is a pUC19 derivative and contains 600 bp of the B. subtilis cccA gene region corresponding to the promoter and the part of the gene encoding amino acid residues 1–33, was cleaved by Kpn I and Bam HI and treated with alkaline phosphatase. The part of the cccB region that encodes residues 28–112 of CccB and contains the proposed transcription termination loop (Fig. 1.) was amplified by PCR using two primers, 03III, 5′-CG GGT ACCAAG ACA GAC ACT GGC AGC AAG (the Kpn I site is underlined), and 03IV, 5′-CG GGA TCC ATA TTG TCA AGG CAT AAA AAC ATC (the Bam HI site is underlined). Plasmid p4303 was used as the template. The PCR was performed using Pwo DNA polymerase and buffer from Roche Molecular Biochemicals containing 4 mm MgSO4. The PCR product was cleaved with Kpn I and Bam HI, and the 315-bp fragment was ligated into pLUT191. The resulting pLUJ105 has the cccA-cccB hybrid gene under the native B. subtilis cccA promoter. The cccB gene was deleted by gene replacement consisting of the integration of linearized pCRΔcccB into the B. subtilis 168 chromosome via a double crossover event resulting in strain L16205. The deletion of the yvjA-cccB segment was performed in a similar way by using linearized pCRΔ972. Strain LUH20 was obtained by the transformation of strain 168 to phleomycin resistance with chromosomal DNA containing a ΔctaCD::ble gene replacement (21van der Oost J. von Wachenfeldt C. Hederstedt L. Saraste M. Mol. Microbiol. 1991; 5: 2063-2072Crossref PubMed Scopus (59) Google Scholar) and then to chloramphenicol resistance with DNA containing a ΔcccA::cat gene replacement (5von Wachenfeldt C. Hederstedt L. FEBS Lett. 1990; 270: 147-151Crossref PubMed Scopus (44) Google Scholar). LUH36 was obtained by the transformation of LUH20 to tetracycline resistance with L16205 (ΔcccB::tet) chromosomal DNA. Membranes isolated from LUH36/pLUJ104 and LUH36/pLUJ105 were diluted to 1.5 mg protein/ml in solubilization buffer (30 mm Tris/SO4, pH 8, 0.5 m Na2SO4, and 1 mmNa-EDTA, pH 8) containing 2% (w/v) cholate, Triton X-100, or no detergent. Phenylmethylsulfonyl fluoride was added to 0.5 mm, and the samples were sonicated and then centrifuged for 40 min at 140,000 × g at 4 °C. The supernatants and the pellets, homogenized in 2 ml of buffer without detergent, were analyzed by light absorption spectroscopy. Membranes isolated from B. subtilis LUH20/pLUJ104 were diluted to 5 mg protein/ml in solubilization buffer containing 2% (w/v) cholate. Phenylmethylsulfonyl fluoride was added and the samples were incubated and centrifuged as for the differential solubilization described above. The supernatant was supplemented with polyethylene glycol (Mr 20,000) to a final concentration of 8% (w/v) and centrifuged at 32,000 × g for 20 min at room temperature. To the supernatant, polyethylene glycol was added to a final concentration of 30% (w/v), and MgSO4 was added to 5 mm. After mixing, the sample was centrifuged at 43,700 × g for 20 min at room temperature. The pellet was suspended in 10 mm Tris/HCl, pH 8, containing 1% (w/v) Thesit and then dialyzed at 4 °C against the same buffer using Spectrapor® tubing with a 3.5-kDa cut-off. The sample was applied on a QMA MemSep® 1010 Ion Exchange Membrane Chromatography Cartridge (Millipore) connected to an FPLC® system (flow rate 5 ml/min). After two washing steps with 10 mm Tris/HCl, pH 8, 0.1% Thesit, containing 5 and 20 mm NaCl, respectively, the CccB cytochrome was eluted with 10 mm Tris/HCl, pH 8, containing 0.1% Thesit and 100 mm NaCl. The 5-ml eluate was dialyzed as above against 10 mm Tris/HCl, pH 8, 0.1% Thesit. The purification procedure up to this point was based on a method described by Sone et al. (22Sone N. Kutoh E. Yanagita Y. Biochim. Biophys. Acta. 1989; 977: 329-334Crossref PubMed Scopus (31) Google Scholar) to purify cytochrome c551 from Bacillus PS3. The cytochrome c was further purified using isoelectric focusing with the Rotorfor® System (Bio-Rad) in the presence of 0.1% Thesit. Twenty fractions were collected, and the absorption at 414 nm was determined. The fractions with high absorption at 414 nm (pH 3.7–4.0) were diluted in 5 volumes of 0.1 m Tris/HCl, pH 8, containing 0.1% Thesit, pooled, and concentrated using Microcon 10-kDa cut-off concentrators. Light absorption spectroscopy at room temperature, in vivo labeling of heme using 2 μm and 0.1 μCi/ml of 5-[4-14C]aminolevulinic acid ([14C]ALA) and SDS-polyacrylamide gel electrophoresis were performed as described in Ref. 23Schiött T. von Wachenfeldt C. Hederstedt L. J. Bacteriol. 1997; 179: 1962-1973Crossref PubMed Google Scholar except that the Schägger/von Jagow gel system (24Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10475) Google Scholar) was used. B. subtilis membranes were isolated according to Ref.25Hederstedt L. Methods Enzymol. 1986; 126: 399-414Crossref PubMed Scopus (61) Google Scholar. Low temperature (77 K) light absorption spectroscopy was done as described in Ref. 21van der Oost J. von Wachenfeldt C. Hederstedt L. Saraste M. Mol. Microbiol. 1991; 5: 2063-2072Crossref PubMed Scopus (59) Google Scholar. Protein concentrations were determined using the BCA protein assay reagent (Pierce) with bovine serum albumin as standard. β-galactosidase assays were performed according to Ref. 26Ferrari E. Howard S.M.H. Hoch J.A. J. Bacteriol. 1986; 166: 173-179Crossref PubMed Google Scholar. Heme C was determined from the pyridine hemochromogen difference (reduced minus oxidized) spectrum in alkaline solution using the absorption coefficient 23.97 mm−1cm−1 (550 nm minus 535 nm) (27Berry E.A. Trumpower B.L. Anal. Biochem. 1987; 161: 1-15Crossref PubMed Scopus (746) Google Scholar). Inspection of the B. subtilis genome sequence reveals that the cccB gene is flanked by the genes yvjA and ftsE (Fig.1). Like cccB, these flanking genes are transcribed in the direction of DNA replication. The fstE gene encodes a 25.5-kDa protein with sequence similarities to FstE from E. coli, which is an ATP-binding protein involved in cell division. The putative 29.8-kDa polypeptide encoded by the yvjA gene shows about 30% sequence identity to several proteins of unknown function in B. subtilis, e.g. YgfU, YxkD, and YpjC. Judging from the sequence, there is no obvious promoter located immediately upstream of the cccB gene and no transcription terminator between yvjA and cccB. Downstream of cccB there is an inverted repeat followed by a run of Ts that probably functions as a rho-independent transcription terminator. The DNA sequence upstream of yvjA shows the features of a transcription terminator followed by a promoter region. Together, these observations suggest that yvjA and cccB are co-transcribed as an approximately 1.55-kilobase mRNA. Northern blot analysis of total B. subtilis RNA, using cccB as the probe, has also shown a 1.6-kilobase transcript. 2T. Schiött, personal communication. The presence of such a di-cistronic mRNA was confirmed by reverse transcription PCR on total RNA extracted from strain 168 (Fig.2). The obtained cDNA product showed that yvjA and cccB mRNA is contiguous (Fig.1). To study the expression pattern of yvjA-cccB during growth, a transcriptional yvjA-lacZ fusion was constructed (Fig. 1) and inserted into the chromosome at the amyE locus in strain 168 resulting in strain L16238. β-galactosidase activity was analyzed in cells growing at 37 °C in NSMP with or without 0.5% glucose. Activities were low and decreased in the presence of 0.5% glucose (Fig. 3). In L16238 cells grown in unsupplemented NSMP, the β-galactosidase activity reached a maximum at the end of the exponential growth phase. The results indicate that the cccB gene is expressed under exponential growth but at a low level. To analyze the role of YvjA and CccB, deletion mutants L16224 (ΔyvjA-cccB) and L16205 (ΔcccB) were constructed. No apparent growth defect was detected, i.e. the mutants grew as wild type on solid and liquid media including minimal medium. It can be noted that mutants deficient in cytochrome c synthesis also do not show any growth defect (23Schiött T. von Wachenfeldt C. Hederstedt L. J. Bacteriol. 1997; 179: 1962-1973Crossref PubMed Google Scholar). The cccB deletion mutant showed normal sporulation, spore outgrowth, and sensitivity to lysozyme (data not shown). The amino acid sequence of the C-terminal part of CccB is very similar to that of CccA, the B. subtilis cytochrome c550 polypeptide (Fig.4.). This part constitutes the heme domain of cytochrome c550 (6von Wachenfeldt C. Hederstedt L. Eur. J. Biochem. 1993; 212: 499-509Crossref PubMed Scopus (30) Google Scholar). The N-terminal parts of the two proteins are clearly different. In CccA, the first 32 residues are known to function as a noncleaved signal sequence for membrane insertion and peptide membrane anchor for the cytochrome domain (5von Wachenfeldt C. Hederstedt L. FEBS Lett. 1990; 270: 147-151Crossref PubMed Scopus (44) Google Scholar). The N-terminal part of CccB also has the features of a signal peptide but contains the bacterial lipoprotein consensus sequence, Leu-Ala-Ala-Cys. This suggests that it is modified at the Cys residue by the addition of a diacylglycerol moiety and subsequently is cleaved by type II signal peptidase resulting in the modified Cys at the N-terminal end of the protein (28Hayashi S. Wu H.C. J. Bioenerg. Biomembr. 1990; 22: 451-471Crossref PubMed Scopus (427) Google Scholar). CccB is therefore most likely a lipoprotein anchored to the membrane by fatty acid residues. The thermophilic bacterium Bacillus PS3 contains a 10-kDa cytochrome c, which has been shown to be a lipoprotein containing two palmitic acid (C16:0) residues/molecule of cytochrome (29Noguchi S. Yamazaki T. Yaginuma A. Sakamoto J. Sone N. Biochim. Biophys. Acta. 1994; 1188: 302-310Crossref PubMed Scopus (19) Google Scholar). This cytochrome shows an absorbance maximum at 551 nm and has therefore been named cytochrome c551. The structural gene for this cytochrome in Bacillus PS3 is called cccA (30Fujiwara Y. Oka M. Hamamoto T. Sone N. Biochim. Biophys. Acta. 1993; 1144: 213-218Crossref PubMed Scopus (24) Google Scholar). Sequence similarities strongly suggest that B. subtilis CccB corresponds to CccA of Bacillus PS3 (Fig. 4). This conclusion is supported by the fact that B. subtilis YvjA and the protein encoded by the open reading frame located immediately upstream of cccA in the chromosome of Bacillus PS3 (30Fujiwara Y. Oka M. Hamamoto T. Sone N. Biochim. Biophys. Acta. 1993; 1144: 213-218Crossref PubMed Scopus (24) Google Scholar) show 70% sequence identity. It has been demonstrated that Bacillus PS3 cytochrome c551 can be synthesized from the cloned gene in both Bacillus stearothermophilus K1041 (29Noguchi S. Yamazaki T. Yaginuma A. Sakamoto J. Sone N. Biochim. Biophys. Acta. 1994; 1188: 302-310Crossref PubMed Scopus (19) Google Scholar) and B. subtilis (31Kai K. Noguchi S. Sone N. J. Ferment. Bioeng. 1997; 84: 190-194Crossref Scopus (6) Google Scholar). Membrane-bound cytochromes with covalently bound heme can be identified by a combination of in vivo radioactive labeling of heme using ALA, a precursor to heme, and SDS-polyacrylamide gel electrophoresis of isolated membranes followed by autoradiography, cf. Ref. 23Schiött T. von Wachenfeldt C. Hederstedt L. J. Bacteriol. 1997; 179: 1962-1973Crossref PubMed Google Scholar. In wild type B. subtilis strains, four cytochromes are visualized by this method (Fig.5, lane 1). These are the 39-kDa subunit II of cytochrome caa3 (CtaC), the 28-kDa cytochrome c of the bc complex (QcrC), the 25-kDa cytochrome b subunit of the cytochrome bc complex (QcrB) (32Yu J. Le Brun N.E. J. Biol. Chem. 1998; 273: 8860-8866Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and the 13-kDa cytochrome c550 (CccA). The predicted mass of the mature CccB lipoprotein with covalently bound heme is about 10 kDa. CccA protein in membranes of the parental strain 168 labeled with [14C]ALA gives rise to a diffuse but rather strong, radioactive cytochrome c band in the 15-kDa region of the gel. This band can hide other small cytochrome polypeptides. Therefore, to assess the presence of CccB, we constructed and analyzed strain LUH20, in which both cccA and ctaC are deleted. As expected, the QcrC and QcrB polypeptides were present in this strain, whereas a very faint, diffuse, radioactive polypeptide was found in the 14-kDa region of the gel. This polypeptide is most likely CccB because it was not present in labeled membranes from strain LUH36, which in addition to cccA and ctaCD has been deleted for the cccB gene (Fig. 5). To facilitate the detection of14C-heme labeled CccB as well as the isolation of the protein for biochemical characterization we have constructed pLUJ104. This plasmid is a derivative of pHP13, an E. coli/B. subtilis shuttle vector with a copy number of about 5 in B. subtilis (9Haima P. Bron S. Venema G. Mol. Gen. Genet. 1987; 209: 335-342Crossref PubMed Scopus (161) Google Scholar), containing the cccB gene cloned downstream of the sdh promoter. [14C]ALA-labeled membranes obtained from B. subtilis strain LUH36 containing pLUJ104 presented a strong, diffuse band migrating faster than CccA but at the same position as the weak band observed with LUH20 (Fig. 5, lane 5). The results show that CccB contains covalently bound heme, i.e. is a cytochrome c. The diffuse polypeptide bands observed with CccA and CccB are because of inherent properties of these cytochromes (not to the electrophoresis system as previously shown for CccA (6von Wachenfeldt C. Hederstedt L. Eur. J. Biochem. 1993; 212: 499-509Crossref PubMed Scopus (30) Google Scholar)). Genes that are organized in one operon often encode functionally related proteins. To determine if the YvjA protein plays a role in the maturation of the CccB cytochrome a yvjA-cccB, cccA deletion strain L16225 was constructed. Membranes of L16225 containing pLUJ104 or the plasmid vector, pHP13, were analyzed for cytochrome c (Fig. 5, lanes 6 and 7). The results showed that YvjA is not required for the synthesis of the membrane-bound CccB cytochrome or any other cytochrome with covalently bound heme. To investigate the domain structure of CccB, a cccA-cccB in frame gene fusion was constructed and cloned into pHP13 resulting in pLUJ105. The hybrid gene is transcribed from the native cccA promoter and is expected to encode a protein with the CccA membrane anchor domain (residues 1–33) fused to the predicted heme domain of CccB (residues 28–112). Membranes from strain LUH36 containing pLUJ105 and grown in the presence of [14C]ALA contained a radioactive polypeptide corresponding to the CccA-CccB hybrid protein, which in the polyacrylamide gel migrated slightly slower than CccA (Fig. 5, lane 4). The results define the heme domain of CccB and demonstrate that the membrane anchor domain of CccA and CccB is functionally interchangeable. Membranes from strain LUH36/pLUJ104 grown in NSMP were analyzed by light absorption spectroscopy. LUH36 lacks cytochrome c550 and cytochrome caa3, which are the dominant high potential B. subtilis cytochromes absorbing in the 550-nm region of the spectrum (11Schiött T. Throne-Holst M. Hederstedt L. J. Bacteriol. 1997; 179: 4523-4529Crossref PubMed Google Scholar) and is deleted for the cccB gene. Ascorbate-reduced minus ferricyanide-oxidized difference spectra at 77 K of membranes from LUH36/pLUJ104 showed an α-band absorption peak at 547 nm and a β-band peak at 519 nm (Fig.6, asc. spectrum). These peaks are because of CccB, because they were not seen with membranes from LUH36/pHP13 (Fig. 6). Only cytochromes of high (>100 mV) midpoint redox potential are reduced by the ascorbate. Difference spectra of dithionite-reduced membranes, where all cytochromes are reduced, indicated that the CccB cytochrome is present in relatively large amounts in LUH36/pLUJ104 (Fig. 6, dit. spectra). At room temperature, reduced CccB cytochrome showed absorption maxima at 551 ± 0.7 nm and 522 ± 0.7 nm (not shown). Because of its spectral properties and other similarities to the small cytochrome of Bacillus PS3, we name B. subtilis CccB cytochrome c551. Membranes from strain LUH36 containing pLUJ105, which encodes the CccA-CccB hybrid protein, showed an absorbance maximum at 551 nm at room temperature after reduction with ascorbate. This confirmed that residues 28–112 of CccB (amino acid numbering according to the unprocessed CccB) constitute the entire heme domain of cytochrome c551. Cytochrome c551 was overproduced to about 0.36 nmol/mg membrane protein in strain LUH20/pLUJ104. The cytochrome was extracted from these membranes using cholate and purified according to steps 1 and 2 of a method described by Noguchi et al. (29Noguchi S. Yamazaki T. Yaginuma A. Sakamoto J. Sone N. Biochim. Biophys. Acta. 1994; 1188: 302-310Crossref PubMed Scopus (19) Google Scholar), except that we used 1% (w/v) Thesit instead of Triton X-100. A final isoelectric focusing step in the presence of 0.1% Thesit was performed to obtain pure B. subtilis cytochrome c as determined by SDS-polyacrylamide gel electrophoresis and staining for protein and covalently bound heme. The cytochrome polypeptide gave rise to a diffuse band in the gels (not shown) like that observed with14C-heme labeled cytochrome (Fig. 5). The properties of isolated B. subtilis cytochrome c551 are very similar to those of cytochrome c551 from Bacillus PS3 (TableII). The latter cytochrome has been demonstrated to be a lipoprotein (29Noguchi S. Yamazaki T. Yaginuma A. Sakamoto J. Sone N. Biochim. Biophys. Acta. 1994; 1188: 302-310Crossref PubMed Scopus (19) Google Scholar). That B. subtilis cytochrome c551 is a lipoprotein also, as suggested from the amino acid sequence, was confirmed by the finding that it could be efficiently extracted from LUH36/pLUJ104 membranes using cholate. In contrast, the CccA-CccB fusion protein, which contains a peptide membrane anchor, was poorly extractable from membranes of LUH36/pLUJ105 by cholate but, as expected, was solubilized by Triton X-100.Table IIBiochemical properties of cytochrome c551 of B. subtilis compared to that of Bacillus PS3PropertiesB. subtilisBacillus PS3Number of amino acid residues2-aIn the processed polypeptide.9293Isoelectric point (pI)3.84.0Extinction coefficient32 mm−1 cm−120.9 mm−1 cm−1(Reduced minus oxidized)(A551 −A535)(A551 −A535)Midpoint redox potential>100 mV225 mVAbsorption maxima at room temperature Oxidized409 nm409 nm Reduced416, 522, 551 nm2-bThe numbers given are within an experimental error of ±0.7 nm.416, 522, 551 nm2-a In the processed polypeptide.2-b The numbers given are within an experimental error of ±0.7 nm. Open table in a new tab A small cytochrome c has recently been isolated and characterized from the Gram-positive photosynthetic bacterium Heliobacterium gestii. This 18-kDa cytochrome c553 is a lipoprotein similar to CccB, the function of which, like that of CccB, remains unknown. It contains palmitate and stearate in the lipid moiety at the N terminus (33Albert I. Rutherford A.W. Grav H. Kellermann J. Michel H. Biochemistry. 1998; 37: 9001-9008Crossref PubMed Scopus (28) Google Scholar). Cytochrome c551 of Bacillus PS3 has been shown to contain two palmitate residues. We have not been able to detect radioactivity in CccB polypeptide after growth of LUH20/pLUJ104 in the presence of [3H]palmitate followed by SDS-polyacrylamide gel electrophoresis of isolated membranes and autoradiography. This negative result can be explained by the low amount of CccB in the membrane and/or by the fact that the cytochrome contains fatty acid residues with an acyl chain shorter than that of palmitate. The heme domains of CccA and CccB seem from the amino acid sequence to belong to a family of small c-type cytochromes found in Bacillus species (34 and this work).B. subtilis cytochrome c550 and c551 differ essentially only in the way they are anchored to the membrane (Fig. 7). The very similar amino acid sequence and redox properties of the heme domain of these two cytochromes indicate that they might serve the same, yet unknown, function in electron transfer in the membrane. If so, B. subtilis would be endowed with two different membrane-anchoring systems for a conserved cytochrome c domain, each of which may offer a distinct advantage under specific growth conditions. Mutants, e.g. LUH36, lacking both these cytochromes grow as well as the parental strain, suggesting that the growth conditions used in the laboratory do not require any of the two cytochromes. It is possible that, under certain natural environmental conditions, B. subtilis may preferentially resort to lipid mediated anchoring to the cytoplasmic membrane, i.e. use CccB rather than CccA. To the best of our knowledge, this is the first example of two homologous membrane proteins with different types of membrane anchors that coexist in one organism. The close similarity between cytochrome c551 of B. subtilis and Bacillus PS3 suggests that they fulfill the same function in their respective bacterium. In Bacillus PS3, c551 is a major cytochrome, whereas a cytochrome c corresponding to B. subtilis cytochrome c550 has not been found. The function of cytochrome c551 in Bacillus PS3 has been investigated by Sone et al. (35Sone N. Kutoh E. Sato K. J. Biochem. (Tokyo). 1990; 107: 597-602Crossref PubMed Scopus (12) Google Scholar). This cytochrome is mainly synthesized under air-limited conditions and is efficiently oxidized by a novel cytochrome c oxidase, cytochrome ba3/bo3 (36Sakamoto J. Handa Y. Sone N. J. Biochem. (Tokyo). 1997; 122: 764-771Crossref PubMed Scopus (26) Google Scholar). The structural genes, cbaAB, of this oxidase have recently been sequenced (37Nikaido K. Noguchi S. Sakamoto J. Sone N. Biochim. Biophys. Acta. 1998; 1397: 262-267Crossref PubMed Scopus (21) Google Scholar). If cytochrome c551 specifically interacts with cytochrome ba3/bo3 to donate electrons, B. subtilis would contain such an oxidase also. However, genes corresponding to cbaA or cbaB were not found in the genome of B. subtilis 168. This leaves open the question whether cytochrome c551 is required for the reduction of cytochrome ba3/bo3 only or whether it may have other functions." @default.
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• W2057963987 title "Bacillus subtilis Contains Two Small c-Type Cytochromes with Homologous Heme Domains but Different Types of Membrane Anchors" @default.
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