- Email: [email protected]
The Bacillus subtilis ctaB paralogue, yjdK, can complement the heme A synthesis deficiency of a CtaB-deficient mutant
FEMS Microbiology Letters 183 (2000) 247^251
www.fems-microbiology.org
The Bacillus subtilis ctaB paralogue, yjdK, can complement the heme A synthesis de¢ciency of a CtaB-de¢cient mutant Mimmi Throne-Holst *, Lars Hederstedt Department of Microbiology, Lund University, So«lvegatan 12, S-22362 Lund, Sweden Received 19 November 1999; received in revised form 22 December 1999; accepted 23 December 1999
Abstract Heme A is a prosthetic group in many respiratory oxidases. It is synthesised from heme B (protoheme IX) with heme O as an intermediate. In Bacillus subtilis two genes required for heme A synthesis, ctaA and ctaB, have been identified. CtaB is the heme O synthase and CtaA is involved in the conversion of heme O to heme A. A ctaB paralogue, yjdK, has been identified through the B. subtilis genome sequencing project. In this study we show that when carried on a low copy number plasmid, the yjdK gene can complement a ctaB deletion mutant with respect to heme A synthesis. Our results indicate that YjdK has heme O synthase activity. We therefore suggest that yjdK be renamed as ctaO. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cytochrome a; Heme O; Respiratory oxidase
1. Introduction Heme A is found exclusively in terminal oxidases of aerobic respiratory chains. In eukaryotes, heme A is an essential component of the mitochondrial terminal oxidase, cytochrome aa3 . The Gram-positive bacterium Bacillus subtilis can synthesise at least four di¡erent terminal oxidases of which two contain heme A; cytochrome aa3 and cytochrome caa3 . The polypeptides of the cytochrome aa3 and cytochrome caa3 are encoded by the qoxABCD operon and the ctaCDEF genes, respectively. Heme A is synthesised from heme B (protoheme IX) with heme O as a stable intermediate. Heme O di¡ers from heme B by having a hydroxyethylfarnesyl chain instead of a vinyl group on carbon 2 of the porphyrin ring (Fisher nomenclature). Heme A di¡ers from heme O by having a formyl group instead of a methyl group on carbon 8 of the porphyrin ring. The ctaA and ctaB genes are required for heme A synthesis in B. subtilis [1,2]. These genes are located at 133³ on the chromosome, immediately upstream of the ctaCDEFG gene cluster and ctaB is cotranscribed with ctaCDEF [3]. CtaA and CtaB are both polytopic membrane proteins [4]. Mutants lacking CtaA and/or CtaB contain no detect-
able heme A [2]. CtaB has been shown to be a heme O synthase (protoheme IX hydroxyethylfarnesyltransferase) ([5], our unpublished data), very similar to its well studied Escherichia coli homologue, CyoE [6,7]. CtaA is involved in the conversion of heme O to heme A and contains heme B as a prosthetic group [4,8]. Genes encoding proteins similar to CtaA and CtaB can be found in e.g. bacteria, yeast and man. The yjdK gene, found through the B. subtilis genome sequencing project [9], encodes a putative paralogue to CtaB. To analyse whether YjdK has a function in heme A synthesis we have constructed a YjdK-de¢cient mutant and used the yjdK gene on a low copy number plasmid in complementation experiments with a ctaB deletion mutant. 2. Materials and methods 2.1. Bacterial strains and plasmids The B. subtilis strains and plasmids used in this work are listed in Table 1. Recombinant DNA work with ctaB and yjdK was carried out in B. subtilis, because the wildtype B. subtilis ctaB gene is toxic to E. coli [8]. 2.2. Media and general growth of bacteria
* Corresponding author. Tel. : +46 (46) 222 41 41; Fax: +46 (46) 15 78 39; E-mail : [email protected]
B. subtilis strains were kept on tryptose blood agar base
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 6 6 6 - 7
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(TBAB) plates (Difco). For the preparation of membranes, B. subtilis strains were grown in nutrient sporulation medium supplemented with phosphate (NSMP), pH 7.0, containing 0.5% (w/v) glucose as described before [2]. Antibiotics at the following concentrations were used when appropriate : chloramphenicol, 3 to 5 mg l31 ; spectinomycin 150 mg l31 . 2.3. Construction of pYJDK4, pYJDK4Es, pYJDK5 and pCTAB1 For the construction of pYJDK4, a 1.4 kb EcoRI-BamHI fragment containing the yjdK gene was ampli¢ed using chromosomal DNA from B. subtilis 1A1 as template and the proof-reading Pwo DNA polymerase (Roche Molecular Biochemicals) in a PCR using a Perkin Elmer GeneAmp PCR system 2400 reactor. EcoRI and BamHI restriction sites were added via the primers 5P-GGGGAATTCATTGCCTTTAAGTCATCCTATCG-3P and 5P-GGGGGATCCAGGTTACGGTTACTAGTCGCCAC-3P (restriction sites are underlined). The ampli¢ed fragment was cut with EcoRI and BamHI and ligated into pHP13 resulting in plasmid pYJDK4. The EcoRI-BamHI fragment from pYJDK4, containing the yjdK gene, was moved into pHP13Es resulting in pYJDK4Es. pYJDK5 was constructed by ligating a 1.2 kb SmaI-EcoRV fragment containing the spc gene derived from pDG1726 [10] into pYJDK4Es digested with HpaI. For the construction of pCTAB1, a 1.5 kb fragment containing the ctaB gene was ampli¢ed using pCTA1305 as template in a PCR. A BamHI restriction site was added via one of the primers 5P-GAGAGAGGATCCGGGTCTGATAGAAATAAG-3P and 5P-AGCAGGCTTGAGCGTGGA-3P (the restriction site is underlined). The ampli¢ed fragment was cut with SpeI and BamHI and a 1173 bp fragment was puri¢ed by agarose gel electrophoresis and ligated into pHPKS resulting in plasmid pCTAB1.
2.4. Construction of strains LMT60, LOA10 and LOA30 For the construction of the YjdK-de¢cient mutant, LMT60, B. subtilis 1A1 was transformed with a 3.1 kb SpeI-NcoI fragment from pYJDK5, containing yjdK with the spc gene inserted at position 85 484 in the genome sequence [9] (accession no. Z99110). Southern blot analysis of chromosomal DNA from transformants, using the AlkPhos Direct (Amersham) detection system and pYJDK5 as probe, con¢rmed the insertion (data not shown). For the construction of LOA10, a yjdK ctaB double mutant, LMT60 was transformed to chloramphenicol resistance with chromosomal DNA from B. subtilis LUB29R. A ctaB deletion mutant, LOA30, was obtained by transformation of B. subtilis 1A1 to chloramphenicol resistance with chromosomal DNA from LOA10. 2.5. Other methods B. subtilis was grown to competence essentially as described by Arwert and Venema [12]. Membranes from B. subtilis strains were isolated essentially as described by Hederstedt [13] and stored at 380³C. Light absorption spectroscopy was carried out as described before [14]. The protein concentration of membrane fractions was determined using the bicinchoninic acid protein assay (Pierce Chemical Co.), with bovine serum albumin as the standard. Heme was extracted from membranes and analysed by reversed-phase HPLC as described before [2]. 3. Results and discussion 3.1. Sequence analyses There are two possible translation initiation codons for yjdK, an AUG at position 85 583 and a UUG at position
Table 1 B. subtilis strains and plasmids Strain or plasmid Propertiesa B. subtilis strain 1A1 LUB29R LMT60 LOA10 LOA30 Plasmids pHP13 pHP13Es pHPKS pYJDK4 pYJDK4Es pYJDK5 pCTA1305 pCTAB1 a
Source/reference
trpC2 met ade trpC2 vctaB; Camr trpC2 yjdK: :spc; Spcr trpC2 yjdK: :spc vctaB; Camr , Spcr trpC2 vctaB; Camr
Bacillus Genetic Stock Center, OH, USA [2,8] This work This work This work
Shuttle vector; Camr , Eryr pHP13 derivative ; Camr pHP13 derivative ; Camr , Eryr pHP13 with yjdK on a 1.4 kb EcoRI-BamHI fragment ; Camr , Eryr pHP13Es with yjdK on a 1.4 kb EcoRI-BamHI fragment; Camr pYJDK4Es with a 1.2 kb fragment from pDG1726 inserted into yjdK; Camr Spcr pHP13 with ctaABC on a 4.9 kb fragment ; Camr , Eryr pHPKS with ctaB on a 1.2 kb SpeI-BamHI fragment; Camr , Eryr
[11] J. Bengtsson, Lund University P. Johansson, Lund University This work This work This work [8] This work
Spcr , Camr and Eryr indicate resistance to spectinomycin, chloramphenicol and erythromycin, respectively.
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medium. LMT60 contained normal amounts of cytochrome a, as determined by light absorption spectroscopy of membranes (Table 2). 3.3. Complementation of a CtaB-de¢cient mutant with yjdK on plasmid The B. subtilis ctaB deletion mutant LOA30, the yjdK ctaB double mutant LOA10, and the parental strain 1A1, were transformed with plasmids pYJDK4 (containing yjdK), pCTAB1 (containing ctaB) and pHP13. The various plasmid-containing strains were grown in NSMP supplemented with glucose, to maximise the cytochrome a content, and membranes were isolated. Visible light absorption spectroscopy of membranes from 1A1/
Fig. 1. Sequence comparison of CtaB and YjdK from B. subtilis and CyoE from E. coli. Residues invariant in all three proteins are indicated under the sequences and those underlined are known to be functionally important in CyoE. Horizontal lines indicate putative transmembrane segments.
85 557 (accession no. Z99110). Only the latter codon is preceded, at a distance of 10 bp, by a potential ShineDalgarno sequence. The calculated [15] vG³ value of interaction between this sequence and the 3P-end of B. subtilis 16S rRNA is 317.4 kcal/mol. Based on these observations we conclude that translation of yjdK mRNA is initiated at the UUG codon, resulting in a protein predicted to have 320 residues. YjdK shows strong sequence similarity to B. subtilis CtaB and E. coli CyoE (Fig. 1). The size and the hydrophobicity pro¢les (data not shown) of the three proteins are also similar. Many of the amino acid residues that have been shown to be essential for catalytic function of CyoE, e.g. the residues in the proposed farnesylpyrophosphate binding motif (residues 57^ 71 in CyoE) [7], are invariant in CtaB and YjdK. 3.2. Properties of a YjdK-de¢cient mutant Strain LMT60, containing a disrupted yjdK gene, grew like the parental strain 1A1 on TBAB plates and in NSMP
Fig. 2. Di¡erence (dithionite reduced minus potassium ferricyanide oxidised) light absorption spectra of B. subtilis membranes isolated from cells grown in NSMP with 0.5% (w/v) glucose and harvested at the end of the exponential growth phase. A, 1A1/pHP13 ; B, 1A1/pYJDK4 ; C, 1A1/pCTAB1; D, LOA30/pHP13; E, LOA30/pYJDK4; F, LOA30/ pCTAB1 ; G, LOA10/pHP13; H, LOA10/pYJDK4; I, LOA10/pCTAB1. The membranes, 4 mg protein ml31 , were in 20 mM sodium-3-(N-morpholino)propanesulfonic acid bu¡er, pH 7.4. The spectra were recorded at room temperature. The absorbance scale is indicated by the bar.
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Table 2 Occurrence of cytochrome a and heme A in membranes from di¡erent B. subtilis strains Strain
Presence of cytochrome aa
Presence of heme Ab
1A1 LMT60 1A1/pHP13 1A1/pYJDK4 1A1/pCTAB1 LOA30/pHP13 LOA30/pYJDK4 LOA30/pCTAB1 LOA10/pHP13 LOA10/pYJDK4 LOA10/pCTAB1
+ + + + + 3 + + 3 + +
n.d. n.d. + n.d. n.d. 3 + + 3 + +
a b
As determined by light absorption spectroscopy of membranes : +, cytochrome a present; 3, cytochrome a absent. As determined by HPLC analysis of heme extracted from membranes: +, heme A present; 3, heme A absent; n.d., not determined.
pHP13, 1A1/pYJDK4, 1A1/pCTAB1, LOA30/pYJDK4, LOA30/pCTAB1, LOA10/pYJDK4 and LOA10/pCTAB1 showed that they all contained cytochrome a as judged from the absorption peak at about 600 nm (Fig. 2; traces A, B, C, E, F, H and I). Essentially no cytochrome absorbing at 600 nm was present in membranes of LOA30/ pHP13 and LOA10/pHP13 (Fig. 2; traces D and G), as expected because of the CtaB de¢ciency. The membranes contain the cytochrome bd terminal oxidase, which shows an absorption maximum at about 625 nm. The small absorption peak seen at about 595 nm, in the spectra of membranes from LOA30/pHP13 and LOA10/pHP13, is due to the cytochrome b595 component of the cytochrome bd. The heme content of membranes was determined by reversed-phase HPLC (Table 2). Membranes from 1A1/ pHP13, LOA30/pYJDK4, LOA30/pCTAB1, LOA10/ pYJDK4 and LOA10/pCTAB1 all contained heme A and heme B. No heme A was found in membranes from LOA30/pHP13 and LOA10/pHP13, con¢rming the results from the spectroscopy. Heme O was not found in membranes from any of the strains. 4. Conclusions Our experimental results demonstrate that the yjdK gene is not needed for growth or heme A synthesis in B. subtilis. However, the defect in heme A synthesis of a B. subtilis CtaB-de¢cient mutant can be complemented by yjdK on a low copy number plasmid (about ¢ve copies per genome [11]), showing that YjdK can function as a heme O synthase. From these results and the sequence similarity between CtaB and YjdK, we suggest that yjdK is renamed to ctaO. Heme A is not found in B. subtilis CtaB-de¢cient mutants ([2], and this work), although they carry one copy of the yjdK (ctaO) gene in the chromosome. This may be due to a low expression of yjdK (ctaO) under the growth conditions used in the experiments. Another explanation can
be that the YjdK (CtaO) protein has a low heme O synthase activity compared to that of CtaB. Such de¢ciencies are likely to be overcome by having multiple copies of yjdK (ctaO) in the cell, as in our experiments. Acknowledgements î gerstam for We are grateful to Ingrid Sta®hl and Oskar A technical assistance, and to Jenny Bengtsson and Dr Per Johansson for providing plasmids. This work was supported by grants from Emil och Wera Cornells Stiftelse and the Swedish Natural Science Research Council.
References [1] Mueller, J.P. and Taber, H.W. (1989) Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis. J. Bacteriol. 171, 4967^4978. [2] Svensson, B., Lu«bben, M. and Hederstedt, L. (1993) Bacillus subtilis CtaA and CtaB function in heme A biosynthesis. Mol. Microbiol. 10, 193^201. [3] Liu, X. and Taber, H.W. (1998) Catabolite regulation of the Bacillus subtilis ctaBCDEF gene cluster. J. Bacteriol. 180, 6154^6163. [4] Svensson, B. and Hederstedt, L. (1994) Bacillus subtilis CtaA is a heme-containing membrane protein involved in heme A biosynthesis. J. Bacteriol. 176, 6663^6671. [5] Saiki, K., Mogi, T., Ishizuka, M. and Anraku, Y. (1994) An Escherichia coli cyoE homologue in thermophilic Bacillus PS3 encodes a thermotolerant heme O synthase. FEBS Lett. 351, 385^388. [6] Chepuri, V. and Gennis, R.B. (1990) The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli. J. Biol. Chem. 265, 12978^12986. [7] Saiki, H., Mogi, T., Hori, H., Tsubaki, M. and Anraku, Y. (1993) Identi¢cation of the functional domains in heme O synthase. J. Biol. Chem. 268, 26927^26934. [8] Svensson, B., Andersson, K.K. and Hederstedt, L. (1996) Low-spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis. Eur. J. Biochem. 238, 287^295. [9] Kunst, 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,
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M. Throne-Holst, L. Hederstedt / FEMS Microbiology Letters 183 (2000) 247^251 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., Go¡eau, 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., Sco¡one, 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., Weitzeneg-
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[15]
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ger, T., Winters, P., Wipat, A., Yamamoto, H., Yamane, K., Yasumoto, K., Yata, K., Yoshida, K., Yoshikawa, H.F., Zumstein, E., Yoshikawa, H. and Danchin, A. (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249^256. Gue¨rout-Fleury, A-M., Shazand, K., Frandzen, N. and Stragier, P. (1995) Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167, 335^336. Haima, P., Bron, S. and Venema, G. (1987) The e¡ects of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg. Mol. Gen. Genet. 209, 335^342. Arwert, F. and Venema, G. (1973) Transformation in B. subtilis. Fate of newly introduced transforming DNA. Mol. Gen. Genet. 123, 185^ 198. Hederstedt, L. (1986) Molecular properties, genetics and biosynthesis of Bacillus subtilis succinate dehydrogenase complex. Methods Enzymol. 126, 339^414. Schio«tt, T., von Wachenfeldt, C. and Hederstedt, L. (1997) Identi¢cation and characterization of the ccdA gene, required for cytochrome c synthesis in Bacillus subtilis. J. Bacteriol. 179, 1962^1973. Tinoco, I., Borer, P.L., Dengler, B. and Levine, M.D. (1973) Improved estimation of secondary structure in ribonucleic acids. Nature 246, 40^41.
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The Bacillus subtilis ctaB paralogue, yjdK, can complement the heme A synthesis de¢ciency of a CtaB-de¢cient mutant Mimmi Throne-Holst *, Lars Hederstedt Department of Microbiology, Lund University, So«lvegatan 12, S-22362 Lund, Sweden Received 19 November 1999; received in revised form 22 December 1999; accepted 23 December 1999
Abstract Heme A is a prosthetic group in many respiratory oxidases. It is synthesised from heme B (protoheme IX) with heme O as an intermediate. In Bacillus subtilis two genes required for heme A synthesis, ctaA and ctaB, have been identified. CtaB is the heme O synthase and CtaA is involved in the conversion of heme O to heme A. A ctaB paralogue, yjdK, has been identified through the B. subtilis genome sequencing project. In this study we show that when carried on a low copy number plasmid, the yjdK gene can complement a ctaB deletion mutant with respect to heme A synthesis. Our results indicate that YjdK has heme O synthase activity. We therefore suggest that yjdK be renamed as ctaO. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cytochrome a; Heme O; Respiratory oxidase
1. Introduction Heme A is found exclusively in terminal oxidases of aerobic respiratory chains. In eukaryotes, heme A is an essential component of the mitochondrial terminal oxidase, cytochrome aa3 . The Gram-positive bacterium Bacillus subtilis can synthesise at least four di¡erent terminal oxidases of which two contain heme A; cytochrome aa3 and cytochrome caa3 . The polypeptides of the cytochrome aa3 and cytochrome caa3 are encoded by the qoxABCD operon and the ctaCDEF genes, respectively. Heme A is synthesised from heme B (protoheme IX) with heme O as a stable intermediate. Heme O di¡ers from heme B by having a hydroxyethylfarnesyl chain instead of a vinyl group on carbon 2 of the porphyrin ring (Fisher nomenclature). Heme A di¡ers from heme O by having a formyl group instead of a methyl group on carbon 8 of the porphyrin ring. The ctaA and ctaB genes are required for heme A synthesis in B. subtilis [1,2]. These genes are located at 133³ on the chromosome, immediately upstream of the ctaCDEFG gene cluster and ctaB is cotranscribed with ctaCDEF [3]. CtaA and CtaB are both polytopic membrane proteins [4]. Mutants lacking CtaA and/or CtaB contain no detect-
able heme A [2]. CtaB has been shown to be a heme O synthase (protoheme IX hydroxyethylfarnesyltransferase) ([5], our unpublished data), very similar to its well studied Escherichia coli homologue, CyoE [6,7]. CtaA is involved in the conversion of heme O to heme A and contains heme B as a prosthetic group [4,8]. Genes encoding proteins similar to CtaA and CtaB can be found in e.g. bacteria, yeast and man. The yjdK gene, found through the B. subtilis genome sequencing project [9], encodes a putative paralogue to CtaB. To analyse whether YjdK has a function in heme A synthesis we have constructed a YjdK-de¢cient mutant and used the yjdK gene on a low copy number plasmid in complementation experiments with a ctaB deletion mutant. 2. Materials and methods 2.1. Bacterial strains and plasmids The B. subtilis strains and plasmids used in this work are listed in Table 1. Recombinant DNA work with ctaB and yjdK was carried out in B. subtilis, because the wildtype B. subtilis ctaB gene is toxic to E. coli [8]. 2.2. Media and general growth of bacteria
* Corresponding author. Tel. : +46 (46) 222 41 41; Fax: +46 (46) 15 78 39; E-mail : [email protected]
B. subtilis strains were kept on tryptose blood agar base
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 6 6 6 - 7
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(TBAB) plates (Difco). For the preparation of membranes, B. subtilis strains were grown in nutrient sporulation medium supplemented with phosphate (NSMP), pH 7.0, containing 0.5% (w/v) glucose as described before [2]. Antibiotics at the following concentrations were used when appropriate : chloramphenicol, 3 to 5 mg l31 ; spectinomycin 150 mg l31 . 2.3. Construction of pYJDK4, pYJDK4Es, pYJDK5 and pCTAB1 For the construction of pYJDK4, a 1.4 kb EcoRI-BamHI fragment containing the yjdK gene was ampli¢ed using chromosomal DNA from B. subtilis 1A1 as template and the proof-reading Pwo DNA polymerase (Roche Molecular Biochemicals) in a PCR using a Perkin Elmer GeneAmp PCR system 2400 reactor. EcoRI and BamHI restriction sites were added via the primers 5P-GGGGAATTCATTGCCTTTAAGTCATCCTATCG-3P and 5P-GGGGGATCCAGGTTACGGTTACTAGTCGCCAC-3P (restriction sites are underlined). The ampli¢ed fragment was cut with EcoRI and BamHI and ligated into pHP13 resulting in plasmid pYJDK4. The EcoRI-BamHI fragment from pYJDK4, containing the yjdK gene, was moved into pHP13Es resulting in pYJDK4Es. pYJDK5 was constructed by ligating a 1.2 kb SmaI-EcoRV fragment containing the spc gene derived from pDG1726 [10] into pYJDK4Es digested with HpaI. For the construction of pCTAB1, a 1.5 kb fragment containing the ctaB gene was ampli¢ed using pCTA1305 as template in a PCR. A BamHI restriction site was added via one of the primers 5P-GAGAGAGGATCCGGGTCTGATAGAAATAAG-3P and 5P-AGCAGGCTTGAGCGTGGA-3P (the restriction site is underlined). The ampli¢ed fragment was cut with SpeI and BamHI and a 1173 bp fragment was puri¢ed by agarose gel electrophoresis and ligated into pHPKS resulting in plasmid pCTAB1.
2.4. Construction of strains LMT60, LOA10 and LOA30 For the construction of the YjdK-de¢cient mutant, LMT60, B. subtilis 1A1 was transformed with a 3.1 kb SpeI-NcoI fragment from pYJDK5, containing yjdK with the spc gene inserted at position 85 484 in the genome sequence [9] (accession no. Z99110). Southern blot analysis of chromosomal DNA from transformants, using the AlkPhos Direct (Amersham) detection system and pYJDK5 as probe, con¢rmed the insertion (data not shown). For the construction of LOA10, a yjdK ctaB double mutant, LMT60 was transformed to chloramphenicol resistance with chromosomal DNA from B. subtilis LUB29R. A ctaB deletion mutant, LOA30, was obtained by transformation of B. subtilis 1A1 to chloramphenicol resistance with chromosomal DNA from LOA10. 2.5. Other methods B. subtilis was grown to competence essentially as described by Arwert and Venema [12]. Membranes from B. subtilis strains were isolated essentially as described by Hederstedt [13] and stored at 380³C. Light absorption spectroscopy was carried out as described before [14]. The protein concentration of membrane fractions was determined using the bicinchoninic acid protein assay (Pierce Chemical Co.), with bovine serum albumin as the standard. Heme was extracted from membranes and analysed by reversed-phase HPLC as described before [2]. 3. Results and discussion 3.1. Sequence analyses There are two possible translation initiation codons for yjdK, an AUG at position 85 583 and a UUG at position
Table 1 B. subtilis strains and plasmids Strain or plasmid Propertiesa B. subtilis strain 1A1 LUB29R LMT60 LOA10 LOA30 Plasmids pHP13 pHP13Es pHPKS pYJDK4 pYJDK4Es pYJDK5 pCTA1305 pCTAB1 a
Source/reference
trpC2 met ade trpC2 vctaB; Camr trpC2 yjdK: :spc; Spcr trpC2 yjdK: :spc vctaB; Camr , Spcr trpC2 vctaB; Camr
Bacillus Genetic Stock Center, OH, USA [2,8] This work This work This work
Shuttle vector; Camr , Eryr pHP13 derivative ; Camr pHP13 derivative ; Camr , Eryr pHP13 with yjdK on a 1.4 kb EcoRI-BamHI fragment ; Camr , Eryr pHP13Es with yjdK on a 1.4 kb EcoRI-BamHI fragment; Camr pYJDK4Es with a 1.2 kb fragment from pDG1726 inserted into yjdK; Camr Spcr pHP13 with ctaABC on a 4.9 kb fragment ; Camr , Eryr pHPKS with ctaB on a 1.2 kb SpeI-BamHI fragment; Camr , Eryr
[11] J. Bengtsson, Lund University P. Johansson, Lund University This work This work This work [8] This work
Spcr , Camr and Eryr indicate resistance to spectinomycin, chloramphenicol and erythromycin, respectively.
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medium. LMT60 contained normal amounts of cytochrome a, as determined by light absorption spectroscopy of membranes (Table 2). 3.3. Complementation of a CtaB-de¢cient mutant with yjdK on plasmid The B. subtilis ctaB deletion mutant LOA30, the yjdK ctaB double mutant LOA10, and the parental strain 1A1, were transformed with plasmids pYJDK4 (containing yjdK), pCTAB1 (containing ctaB) and pHP13. The various plasmid-containing strains were grown in NSMP supplemented with glucose, to maximise the cytochrome a content, and membranes were isolated. Visible light absorption spectroscopy of membranes from 1A1/
Fig. 1. Sequence comparison of CtaB and YjdK from B. subtilis and CyoE from E. coli. Residues invariant in all three proteins are indicated under the sequences and those underlined are known to be functionally important in CyoE. Horizontal lines indicate putative transmembrane segments.
85 557 (accession no. Z99110). Only the latter codon is preceded, at a distance of 10 bp, by a potential ShineDalgarno sequence. The calculated [15] vG³ value of interaction between this sequence and the 3P-end of B. subtilis 16S rRNA is 317.4 kcal/mol. Based on these observations we conclude that translation of yjdK mRNA is initiated at the UUG codon, resulting in a protein predicted to have 320 residues. YjdK shows strong sequence similarity to B. subtilis CtaB and E. coli CyoE (Fig. 1). The size and the hydrophobicity pro¢les (data not shown) of the three proteins are also similar. Many of the amino acid residues that have been shown to be essential for catalytic function of CyoE, e.g. the residues in the proposed farnesylpyrophosphate binding motif (residues 57^ 71 in CyoE) [7], are invariant in CtaB and YjdK. 3.2. Properties of a YjdK-de¢cient mutant Strain LMT60, containing a disrupted yjdK gene, grew like the parental strain 1A1 on TBAB plates and in NSMP
Fig. 2. Di¡erence (dithionite reduced minus potassium ferricyanide oxidised) light absorption spectra of B. subtilis membranes isolated from cells grown in NSMP with 0.5% (w/v) glucose and harvested at the end of the exponential growth phase. A, 1A1/pHP13 ; B, 1A1/pYJDK4 ; C, 1A1/pCTAB1; D, LOA30/pHP13; E, LOA30/pYJDK4; F, LOA30/ pCTAB1 ; G, LOA10/pHP13; H, LOA10/pYJDK4; I, LOA10/pCTAB1. The membranes, 4 mg protein ml31 , were in 20 mM sodium-3-(N-morpholino)propanesulfonic acid bu¡er, pH 7.4. The spectra were recorded at room temperature. The absorbance scale is indicated by the bar.
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Table 2 Occurrence of cytochrome a and heme A in membranes from di¡erent B. subtilis strains Strain
Presence of cytochrome aa
Presence of heme Ab
1A1 LMT60 1A1/pHP13 1A1/pYJDK4 1A1/pCTAB1 LOA30/pHP13 LOA30/pYJDK4 LOA30/pCTAB1 LOA10/pHP13 LOA10/pYJDK4 LOA10/pCTAB1
+ + + + + 3 + + 3 + +
n.d. n.d. + n.d. n.d. 3 + + 3 + +
a b
As determined by light absorption spectroscopy of membranes : +, cytochrome a present; 3, cytochrome a absent. As determined by HPLC analysis of heme extracted from membranes: +, heme A present; 3, heme A absent; n.d., not determined.
pHP13, 1A1/pYJDK4, 1A1/pCTAB1, LOA30/pYJDK4, LOA30/pCTAB1, LOA10/pYJDK4 and LOA10/pCTAB1 showed that they all contained cytochrome a as judged from the absorption peak at about 600 nm (Fig. 2; traces A, B, C, E, F, H and I). Essentially no cytochrome absorbing at 600 nm was present in membranes of LOA30/ pHP13 and LOA10/pHP13 (Fig. 2; traces D and G), as expected because of the CtaB de¢ciency. The membranes contain the cytochrome bd terminal oxidase, which shows an absorption maximum at about 625 nm. The small absorption peak seen at about 595 nm, in the spectra of membranes from LOA30/pHP13 and LOA10/pHP13, is due to the cytochrome b595 component of the cytochrome bd. The heme content of membranes was determined by reversed-phase HPLC (Table 2). Membranes from 1A1/ pHP13, LOA30/pYJDK4, LOA30/pCTAB1, LOA10/ pYJDK4 and LOA10/pCTAB1 all contained heme A and heme B. No heme A was found in membranes from LOA30/pHP13 and LOA10/pHP13, con¢rming the results from the spectroscopy. Heme O was not found in membranes from any of the strains. 4. Conclusions Our experimental results demonstrate that the yjdK gene is not needed for growth or heme A synthesis in B. subtilis. However, the defect in heme A synthesis of a B. subtilis CtaB-de¢cient mutant can be complemented by yjdK on a low copy number plasmid (about ¢ve copies per genome [11]), showing that YjdK can function as a heme O synthase. From these results and the sequence similarity between CtaB and YjdK, we suggest that yjdK is renamed to ctaO. Heme A is not found in B. subtilis CtaB-de¢cient mutants ([2], and this work), although they carry one copy of the yjdK (ctaO) gene in the chromosome. This may be due to a low expression of yjdK (ctaO) under the growth conditions used in the experiments. Another explanation can
be that the YjdK (CtaO) protein has a low heme O synthase activity compared to that of CtaB. Such de¢ciencies are likely to be overcome by having multiple copies of yjdK (ctaO) in the cell, as in our experiments. Acknowledgements î gerstam for We are grateful to Ingrid Sta®hl and Oskar A technical assistance, and to Jenny Bengtsson and Dr Per Johansson for providing plasmids. This work was supported by grants from Emil och Wera Cornells Stiftelse and the Swedish Natural Science Research Council.
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