1 Department of Cell and Organism Biology, Lund University, Sölvegatan 35, SE-22362 Lund, Sweden
2 Department of Biological Chemistry, University of Copenhagen, Solvgade 83, DK-1307 Copenhagen K, Denmark
Correspondence
Lars Hederstedt
Lars.Hederstedt{at}cob.lu.se
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ABSTRACT |
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INTRODUCTION |
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B. subtilis cells grown under oxic conditions contain several terminal respiratory oxidases (von Wachenfeldt & Hederstedt, 1992, 2002
). Two of these are a-type cytochromes. Cytochrome caa3 is a cytochrome c oxidase with four polypeptide subunits encoded by the ctaCDEF gene cluster (van der Oost et al., 1991
; Saraste et al., 1991
). The closely related enzyme cytochrome aa3 is a quinol oxidase with four subunits encoded by the qoxABCD operon (Santana et al., 1992
; Lemma et al., 1993
). Subunit I of these oxidases (CtaD and QoxA) contains two haem A groups, haem a and haem a3, and one copper atom designated CuB. Haem a3 and CuB form a binuclear centre where dioxygen is reduced to water (Powers et al., 1994
). The electrons required for the reaction enter the binuclear centre from haem a (Michel et al., 1998
). Subunits II of the two enzymes (CtaC and QoxB) are related proteins but there are major differences. Processed CtaC is a lipoprotein with a membrane-integral N-terminal domain, having two transmembrane
-helical segments, and a C-terminal peripheral domain exposed on the positive (outer) side of the cytoplasmic membrane (Bengtsson et al., 1999
). The peripheral domain contains two subdomains: one with a di-copper centre, CuA, the other being a mono-haem cytochrome c (van der Oost et al., 1991
; von Wachenfeldt et al., 1994
). Electrons enter the enzyme at the cytochrome c and are transferred via the CuA centre to haem a. QoxB of cytochrome aa3 is also a lipoprotein (J. Bengtsson & C. von Wachenfeldt, unpublished data) but contains no haem or copper centers. Subunits III (CtaE and QoxC) and subunits IVB (CtaF and QoxD) contain no known prosthetic groups. Isolated active preparations of the B. subtilis oxidases often lack one or both of subunits III and IVB (cf. Lemma et al., 1993
).
B. subtilis cells offer several experimental advantages in a genetic-based approach to find and identify factors involved in assembly of oxidases. Cytochrome caa3 and cytochrome aa3 are not essential for growth because their basic function can be replaced by that of cytochrome bd (Winstedt & von Wachenfeldt, 2000). Presence of functional cytochrome caa3 can be specifically detected in vivo using the redox dye N,N,N'N'-tetramethyl-p-phenylenediamine (TMPD) (van der Oost et al., 1991
). Colonies containing cytochrome c oxidase activity turn blue in the presence of TMPD. After random or site-specific mutagenesis, colonies with TMPD-oxidation-deficient cells can easily be detected on agar plates. From light absorption spectra of membranes it can then be determined if the mutants are defective in for example cytochrome c synthesis (B. subtilis contains four different cytochromes c) (Schiött et al., 1997a
), or in haem A synthesis (such mutants lack both cytochrome aa3 and cytochrome caa3) (Svensson et al., 1993
). Isolated TMPD-oxidation-deficient mutants that contain cytochrome aa3 might carry mutations in the ctaCDEF gene cluster or in genes encoding assembly factors specific for cytochrome caa3. Mattatall et al. (2000)
have shown that the YpmQ protein plays a role in assembly of cytochrome c oxidase in B. subtilis. YpmQ is a predicted lipoprotein of 177 amino acid residues exposed on the positive side of the cytoplasmic membrane.
We have in this work investigated the role of CtaG in B. subtilis and have identified ctaG and ypmQ mutations by screening mutant libraries for cytochrome caa3-deficient clones. The ctaG gene in the ctaBCDEFG cluster was found and named in the B. subtilis genome sequence project (Kunst et al., 1997) but the functional importance of the gene or the protein it encodes has not previously been addressed. CtaG is a predicted 297 amino acid residue protein with seven
-helical transmembrane segments and the N-terminus exposed on the positive side of the cytoplasmic membrane. It should be noted that B. subtilis CtaG is not similar to the CtaG proteins of Paracoccus denitrificans and Rhodobacter sphaeroides, which are homologous to Cox11p of yeast, and function in the formation of the CuB centre (Hiser et al., 2000
; Carr et al., 2002
). We have analysed CtaG- and YpmQ-deficient strains in parallel and compared their molecular properties.
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METHODS |
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Molecular genetic techniques.
General molecular genetic techniques were as described by Sambrook et al. (1989). Plasmid DNA was isolated using CsCl density-gradient centrifugation (Ish-Horowicz & Burke, 1981
) or by using the Quantum Prep mini preparation kit (Bio-Rad). Chromosomal DNA from B. subtilis strains was isolated as described by Marmur (1961)
. Transformation of B. subtilis using natural competence was done as described by Hoch (1991)
. E. coli cells were transformed by electroporation as before (Le Brun et al., 2000
). PCR was performed using the Expand High Fidelity PCR kit (Roche Molecular Biochemicals).
Fluorescent DNA sequencing was carried out on isolated plasmid DNA or PCR products using the BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems) and an ABI prism 3100 DNA sequencer (PE Biosystems).
Construction of plasmids.
To obtain plasmid pCTAG1 (Fig. 1), a 544 bp DNA fragment was amplified using PCR with B. subtilis 1A1 chromosomal DNA as template and the primers CtaG1 (5'-GGGAATTCAGCTCGGAGCAGTCATGATGA-3') and CtaG11 (5'-GGGTCGACAGTGTTGAAGTTTACTGAAAGCG-3'). The DNA product was cut with EcoRI and SalI (sites underlined in the primer sequences) and ligated into EcoRI/SalI-cut pDG1515. Plasmid CTAG2 (Fig. 1
) was constructed by ligating a XbaI/BamHI-cut 356 bp PCR fragment obtained by using 1A1 chromosomal DNA and primers CtaG13 (5'-GCTCTAGACGTGGCATTTGGGCTTATGTGG-3') and CtaG9 (5'-GGGGATCCATCTCCAACTGATTCATTTCTTCACC-3') into XbaI/BamHI-cut pCTAG1. Plasmid pCTAG5 contains the entire ctaG gene under control of the IPTG-inducible spac promoter. This plasmid was constructed by ligating the PCR fragment obtained using primers CtaG16 (5'-GGCCGTCGACTGATTCAGGACATAGCCAACATG-3') and CtaG17 (5'-CCCAAGCTTGGAGGTGAAGAAATGAATCAGTTGG-3') into HindII/SalI-cut pDG148.
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Construction of strains.
B. subtilis strain LJB107 was obtained by transforming strain 1A1 with pYPMABspc, made linear by digestion with ScaI, and selecting for spectinomycin-resistant clones. Strain LUW202 resulted from the transformation of 1A1 with pCTAG2. Deletion of genes in strains was confirmed by using PCR with isolated chromosomal DNA. Strains LJB109R and LJB110R are isolates that in addition to the introduced deletion-substitutions (Table 1) contain spontaneous, unidentified, mutations that allow these strains to grow better than the original isolates on TBAB plates. LUW143R is also such a better-growing isolate of strain LUW143. The unknown mutations result in the expression of cydABCD also when cells are grown in the absence of glucose in the medium.
Analysis of TMPD-oxidation activity.
Colonies were either stained for TMPD-oxidation activity directly on agar plates as described before (Le Brun et al., 2000), or (in the case of agar plates where the medium had been supplemented with transition metal ions) the colonies were grown on a 0·45 µm pore size filter (Millipore HATF surfactant free) laid on top of the agar. After incubation of the plate overnight the filter was rinsed in water and then stained using the same mixture as for plates except that the agar solution was replaced with a corresponding volume of water.
Quantification of CtaC and CtaD by immunoblot analysis.
SDS-PAGE was performed according to Schägger & von Jagow (1987). The proteins were then transferred to a PVDF membrane (Immobilon-P, Millipore) in a wet blot using the following electrophoresis buffer: 2·4 g Tris l-1, 11·25 g glycine l-1 and 20 % (v/v) methanol. The anti-CtaC rabbit serum used recognizes the C-terminal part of B. subtilis CtaC and has been described before (Bengtsson et al., 1999
). Anti-CtaD serum was obtained by immunizing rabbits with a synthetic peptide (CHIHKEELPNDDKGVKA) conjugated to keyhole limpet haemocyanin. The peptide sequence, except for the cysteinyl residue, corresponds to the 16 most C-terminal residues of B. subtilis CtaD. The secondary antibody was horseradish-peroxidase-conjugated anti-rabbit antibodies. Bound secondary antibodies were detected using the Super Signal reagent (Pierce). Chemiluminescence was recorded using a Kodak Image Station 440CF and relative intensities were analysed using the Kodak 1 D Image Analysis Software, version 3.0.
Miscellaneous methods.
Liquid cultures of B. subtilis were grown in batches of 1 litre in 5 litre Erlenmeyer flasks with indentations. The cultures were incubated at 37 °C on a rotary shaker (200 r.p.m.) and growth was followed by measurement of OD600. Membranes were isolated as described by Hederstedt (1986). Protein concentrations were estimated using the BCA method (Pierce) with bovine serum albumin as the standard. Cytochrome c oxidase activity measurements using reduced S. cerevisiae cytochrome c (Sigma) were performed as described before (van der Oost et al., 1991
). Light absorption spectroscopy at room temperature was performed as described previously (Schiött et al., 1997a
).
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RESULTS |
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In previous work we have, after mutagenesis of spores using ethyl methane sulfonate (EMS), isolated a collection of mutants deficient in TMPD-oxidation activity when grown on TBAB plates (Le Brun et al., 2000; Bengtsson, 2001
). Some of these mutants are blocked in cytochrome c synthesis. As a result of this defect the cytochrome c domain of cytochrome caa3 is not assembled and the mutants therefore lack cytochrome c oxidase activity (Le Brun et al., 2000
). Twenty-four mutants from this collection which were known to contain cytochrome c were transformed with pCTAG5 and pDG148 and then assayed for TMPD-oxidation activity. One mutant strain, CMW24, was complemented by pCTAG5 but not by pDG148. DNA sequence analysis of the ctaG gene in the chromosome of CMW24 showed a nonsense mutation (C to T transition creating a stop codon at position 261 in CtaG). The resulting predicted CtaG protein is truncated, lacking the most C-terminal transmembrane segment (residues 261297). The findings demonstrate that the CtaG protein is important for cytochrome caa3 activity in B. subtilis.
YpmQ-deficient mutants
As part of the systematic B. subtilis Gene Function Analysis Project (Schumann et al., 2001) the plasmid pMUTIN2 was inserted at various known positions in the ypmPQRST gene cluster located at 195° on the chromosomal genetic map (Fig. 2
). Except for YpmQ, the functions of the proteins encoded by the ypmQRST gene cluster are largely unknown. Colonies of strain BFS2217, which has the ypmQ gene disrupted, on TBAB plates were found to be deficient in TMPD-oxidation activity. Strains BSF2218, BSF2219 and BSF2220, with various insertions in the operon downstream of the ypmQ gene, showed wild-type TMPD-oxidation activity. To work with isogenic strains, the pMUTIN2-insertion in the ypmQ gene was moved from BSF2217 to strain 1A1, resulting in strain ORE1. Colonies of ORE1 on TBAB plates were TMPD-oxidation negative as expected.
A promoter region in front of the ypmQ gene has tentatively been identified (Mattatall et al., 2000). Based on expression of lacZ from the integrated pMUTIN2, the ypmQ promoter activity was found to be repressed by the presence of glucose in the medium and showed a peak at the end of the exponential growth phase (data not shown). This pattern is reminiscent of that for ctaC gene expression (Liu & Taber, 1998
).
We deleted the ypmQ gene in strain 1A1 and replaced it with a gene encoding spectinomycin resistance (Fig. 2). Colonies of the resulting mutant strain, LJB107, transformed with plasmid pYPMQ1 (carrying ypmQ and its natural promoter region) were TMPD-oxidation positive on TBAB plates. LJB107 containing only the plasmid vector, pHPSK, was deficient in TMPD-oxidation activity. Plasmids pYPMQ1 and pHPSK were also used to transform the collection of 24 mutants with EMS-induced mutations. Strain CMW19 was complemented by pYPMQ1 but not pHPSK. Sequence analysis of chromosomal DNA from CMW19 showed a G to A substitution within the ypmQ gene, changing residue Cys-68 to Tyr in the YpmQ protein.
YpmQ-deficient mutants show a leaky phenotype
It was observed that strains deficient in YpmQ showed TMPD-oxidation activity when grown on NSMP plates but lacked such activity when grown on TBAB plates. CtaG-deficient mutants, in contrast, showed the same TMPD-negative phenotype on both NSMP and TBAB plates. Expression of the structural genes for cytochrome caa3 (ctaCDEF) in B. subtilis is under catabolite repression and therefore dependent on the composition of the growth medium (Liu & Taber, 1998). The observed difference in TMPD-oxidation activity of the YpmQ-deficient mutants could therefore be due to different cellular levels of functional cytochrome caa3 depending on the growth medium used.
To test this explanation, we grew the parental strain 1A1 and the YpmQ-deficient mutant ORE1 in liquid TB (equivalent to TBAB) and in NSMP. As a control, we also grew strain LUT3, which completely lacks cytochrome caa3 because the ctaCD genes are deleted from the chromosome. The cultures were harvested about 1 h after the end of the exponential growth phase. At this growth stage the cellular level of cytochrome caa3 is at its maximum. Cytochrome c oxidase activity of membranes isolated from the cultured cells is presented in Table 2. The analysis demonstrated that a YpmQ-deficient mutant contains only 1015 % of the normal level of cytochrome c oxidase activity. The parental strain, 1A1, grown in NSMP contained a fivefold higher enzyme activity than cells grown in TB. This difference was also found in the levels of CtaC protein in the membranes (immunoblot not shown). Membranes from strains 1A1 and ORE1 grown in NSMP supplemented with 0·5 % (w/v) glucose (Table 2
) contained only 1030 % of the cytochrome c oxidase activity present in membranes from cells grown in NSMP. This showed that expression of cytochrome caa3 activity in the parental strain and the YpmQ-deficient strain is under catabolite repression to about the same extent.
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Cytochrome c oxidase activity of complemented mutants
The cytochrome c oxidase activities of membranes isolated from LUW202/pCTAG5 and LJB107/pYPMQ1 grown in NSMP are shown in Fig. 3. Strain LJB107 was almost completely complemented by the ypmQ gene with its natural promoter on the plasmid. LUW202, deleted for the ctaG gene, was only complemented to about 50 % by pCTAG5, although the cells were grown in the presence of IPTG to completely induce transcription of ctaG on the plasmid. Transcription of the plasmid-borne ctaG gene from the recombinant spac promoter thus appears to limit the expression of cytochrome c oxidase activity in strain LUW202/pCTAG5.
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Upon SDS-PAGE, the CtaC and CtaD polypeptides of YpmQ- and CtaG-deficient mutants and those of the wild-type migrated identically as determined by the immunoblot experiments. This indicated normal post-translational processing of the CtaC polypeptide in the mutants, e.g. diacylglyceride attachment, cleavage of the signal peptide and covalent attachment of haem (Bengtsson et al., 1999).
The observed near-normal amounts of CtaC polypeptide with normal molecular properties in the mutants were also consistent with our unpublished data available for strains CMW19 (ypmQ19) and CMW24 (ctaG24). These two strains and the parental strain LUW35 grown in NSMP contain normal amounts of CtaC polypeptides as determined by SDS-PAGE and autoradiography of membranes isolated from cells grown in the presence of [14C]aminolaevulinic acid (Le Brun et al., 2000; Bengtsson, 2001
). Growth of B. subtilis in the presence of this radioactive compound specifically labels haem, and only proteins with covalently bound haem appear as radioactive bands after SDS-PAGE (Schiött et al., 1997b
). Using this procedure the relative concentrations of different c-type cytochromes in the sample are determined very accurately since all haem groups have the same specific radioactivity.
Cytochrome a in mutants
The results obtained using immunoblotting and [14C]haem-labelled cytochromes showed that CtaC polypeptide containing cytochrome c and of normal size is present in near-normal amounts in membranes of YpmQ- and CtaG-deficient mutants. This, however, does not provide information on whether subunit I contains haem A.
Fig. 4 shows light absorption difference (reduced minus oxidized) spectra of membranes isolated from strains 1A1, LJB107, LUW202, LJB108 and LUT3 grown in NSMP. Addition of ascorbate to membranes causes preferential reduction of c-type cytochromes and cytochrome a of cytochrome caa3 (Fig. 4a
). Consistently, the parental strain 1A1 showed an absorption peak at 605 nm due to cytochrome a. Strain LUT3, which lacks cytochrome caa3, showed no peak at this wavelength, as expected. The c-type cytochromes, which show an absorption maximum at about 550 nm, were present in similar amounts in membranes from all strains. The YpmQ- and CtaG-deficient mutants LJB107 and LUW202, respectively, and LJB108, deficient in both YpmQ and CtaG, contained close to normal amounts of ascorbate-reducible cytochrome a. Reduction with dithionite, which reduces all B. subtilis membrane-bound cytochromes, showed that the overall cytochrome content is unaffected in YpmQ- and CtaG-deficient mutants (Fig. 4b
).
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Cytochrome aa3 is active in the absence of CtaG and YpmQ
Under oxic conditions, B. subtilis cells require a functional cytochrome aa3 or cytochrome bd for growth (Winstedt & von Wachenfeldt, 2000). We exploited this fact to determine if CtaG and YpmQ are specifically required for the synthesis of active cytochrome caa3 or if they also have a role in synthesis of active cytochrome aa3. Strains LUW207 and LUW208, deleted for cydABCD and the ypmQ or ctaG gene, were constructed (Table 1
). Both these strains grew on TBAB plates like the parental strain only lacking cytochrome bd. This result demonstrated that YpmQ and CtaG are not important for assembly of active cytochrome aa3. Thus, CtaG and YpmQ have no significant role in the assembly of functional quinol oxidases and are not important for general assembly of the haem a3CuB binuclear centre in the haem-copper oxidases.
The oxidase defect in YpmQ-deficient, but not CtaG-deficient, strains is suppressed by copper in the growth medium
Mattatall et al. (2000) showed that copper ions in the growth medium can suppress the phenotype of a YmpQ-deficient mutant. We found that YpmQ-deficient strains showed a TMPD-oxidation positive phenotype on TBAB plates if 0·550 µM CuCl2 was included in the medium. The oxidase defect of CtaG-deficient mutants was in contrast not suppressed by the addition of copper ions to the growth medium.
The YpmQ-deficient strain LJB107 and its parental strain 1A1 were grown in NSMP supplemented with various concentrations of CuCl2. The final total copper concentration in the medium after inoculation with cells was determined by inductively coupled plasma emission mass spectroscopy (ICP-MS). Membranes were isolated from the cells grown to early stationary phase and the cytochrome c oxidase activity was analysed (Fig. 7). The activity of LJB107 grown in the presence of 0·5 µM copper was about 15 % compared to strain 1A1 but increased to 50 % at 1·34·9 µM copper. The activity of 1A1 also increased somewhat with increasing copper concentrations in the medium but this increase was marginal compared to that observed with LJB107.
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Properties of a CtaG YpmQ double mutant
A CtaG and YpmQ double-deficient strain, LJB108, was constructed (Table 1). The phenotype of this strain was found to be indistinguishable from that of strain LUW202 deleted for the ctaG gene only, i.e. isolated membranes of LJB108 lacked cytochrome c oxidase activity (Fig. 3
), contained ascorbate-reducible cytochrome a (Fig. 4
), and contained CtaC polypeptide in amounts that were 60 % or more compared to that in membranes from the parental strain 1A1 grown under the same conditions. Addition of CuCl2 to TBAB plates had no effect on the TMPD-oxidation activity of strain LJB108.
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DISCUSSION |
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Interestingly, in the chromosome of Bacillus halodurans the ctaG gene is located far away from the ctaCDEF gene cluster but adjacent to a gene encoding a YpmQ/Sco1p orthologue. Sco1p in yeast has been shown to interact with subunit II of cytochrome oxidase (Lode et al., 2000) and with Cox17p, a copper-binding protein which functions in delivering copper to mitochondria (for a review see Barrientos et al., 2002
). Cox17p deficiency results in lack of cytochrome c oxidase activity and this can be suppressed by adding copper to the growth medium or by overexpression of SCO1. Sco1p deficiency in eukaryotic cells specifically causes cytochrome c oxidase deficiency. Humans contain two YpmQ orthologues, SCO1 and SCO2, and the cytochrome oxidase deficiency of SCO1-deficient cells in culture can be suppressed by the addition of copper (Salviati et al., 2002
). Sco1p, YpmQ and their homologues, e.g. PrrC in R. sphaeroides, contain two conserved cysteine residues, in a CxxxC motif, and also a conserved histidine residue. Sco1p and PrrC can bind one Cu(I) per monomer and using site-specific mutagenesis the conserved cysteine and histidine residues have been demonstrated to be important for both function and copper binding (Rentzsch et al., 1999
; Mattatall et al., 2000
; Nittis et al., 2001
; McEwan et al., 2002
). The available data from studies in yeast suggest that Cox17p delivers Cu(I) to Sco1p, which functions in the assembly of the CuA centre (Beers et al., 2002
; Punter & Glerum, 2003
).
Based on the complete genome sequence, B. subtilis apparently does not contain a Cox17p orthologue. The functions of CtaG and YpmQ seem related, i.e. both play a role in assembly of active cytochrome caa3, but only in YpmQ-deficient mutants is the deficiency in cytochrome c oxidase activity incomplete and suppressed by increased copper concentration in the medium. A CtaG YpmQ double-deficient mutant showed the same properties as a mutant lacking only CtaG. These results suggest that CtaG protein has a more fundamental role than YpmQ in the formation of active cytochrome caa3. Alternatively, there is some other protein(s) in B. subtilis that in its function partially overlaps with YpmQ and is affected by copper ions in the medium. The low oxidase activity per molecule of cytochrome caa3 in YpmQ-deficient strains was found to be about the same also when the amount of enzyme was varied 10-fold (Table 2 and immunoblots not shown).
Mattatall et al. (2000) originally demonstrated that YpmQ has a role in synthesis of cytochrome caa3 in B. subtilis. Using other types of experiments than we have exploited here, they concluded that cytochrome caa3 is specifically affected by a lack of YpmQ. They showed that the phenotype of a strain deleted for the ypmQ gene is dependent on the copper concentration in the growth medium. The two conserved cysteine residues (Cys-64 and Cys-68 in B. subtilis) and the conserved histidine residue (His-154) in YpmQ were by alanine substitutions shown to be essential for function of the protein. We show here that a replacement of Cys-68 by tyrosine also inactivates YpmQ. From the phenotype of YpmQ-deficient cells, the effect of copper, the sequence similarity of YpmQ to S. cerevisiae ScoIp, and the result of mutating the conserved residues in YpmQ, Mattatall et al. (2000)
suggested that the indicated two cysteine residues and the histidine residue in YpmQ are involved in copper exchange between YpmQ and the CuA site of cytochrome c oxidase. Our results fully agree with this conclusion but there is one notable inconsistency in our experimental data. We found 40100 % of the normal amount of CtaC and CtaD polypeptides in membranes of various YpmQ-deficient strains whereas they found only about 10 % of the wild-type level of CtaC after growth at low copper and about 21 % after growth at high copper concentration. This difference is important for the interpretation of the defect caused by YpmQ deficiency and may be explained by differences between strains, growth conditions and membrane isolation procedures. For example, it is known that the cytochrome c domains in B. subtilis membranes are prone to being shawed off by the action of proteases (von Wachenfeldt & Hederstedt, 1993
; Yu et al., 1995
) and this affects apparent CtaC concentrations in isolated membranes determined using immunoblotting.
In conclusion, YpmQ in B. subtilis and other Gram-positive bacteria might function in delivering copper ions to the CuA site in subunit II on the positive side of the cytoplasmic membrane. This suggested direct or indirect role is of major importance only when the availability of copper is low, i.e. when the concentration of copper ions in the surrounding medium is low (less than 1 µM). High copper concentrations in the medium suppress the phenotype of YpmQ-deficient mutants. Hence, YpmQ might act as a periplasmic copper-binding protein helping to capture ions from the medium. It then remains unexplained why YpmQ is not required also for CuB assembly and why YpmQ orthologues are present in bacteria that apparently lack CuA centre-containing oxidases (Seib et al., 2003). It has been suggested that YpmQ and similar proteins might be thioldisulfide oxidoreductases rather than metal-binding proteins (Chinenov, 2000
). This could explain the CuA specificity because two cysteine residues are metal ligands in that centre but not in the CuB centre. Revealing the exact roles of CtaG and YpmQ in assembly of cytochrome caa3 in Gram-positive bacteria remains a challenge.
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ACKNOWLEDGEMENTS |
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Received 7 August 2003;
revised 7 November 2003;
accepted 10 November 2003.
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