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Journal of Bacteriology, February 2007, p. 789-800, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01441-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Membrane-Spanning and Periplasmic Segments of CcmI Have Distinct Functions during Cytochrome c Biogenesis in Rhodobacter capsulatus

Carsten Sanders, Clémence Boulay, and Fevzi Daldal^*

Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received 11 September 2006/ Accepted 13 November 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
In gram-negative bacteria, like Rhodobacter capsulatus, about 10 membrane-bound components (CcmABCDEFGHI and CcdA) are required for periplasmic maturation of c-type cytochromes. These components perform the chaperoning and thio-oxidoreduction of the apoproteins as well as the delivery and ligation of the heme cofactors. In the absence of any of these components, including CcmI, proposed to act as an apocytochrome c chaperone, R. capsulatus does not have the ability to produce holocytochromes c or consequently to exhibit photosynthetic growth and cytochrome cbb₃ oxidase activity. Previously, we have demonstrated that null mutants of CcmI partially overcome cytochrome c deficiency phenotypes upon overproduction of the CcmF-R. capsulatus CcmH (CcmF-CcmH_Rc) couple in a growth medium-dependent manner and fully bypass these defects by additional overproduction of CcmG. Here, we show that overproduction of the CcmF-CcmH_Rc couple and overproduction of the N-terminal membrane-spanning segment of CcmI (CcmI-1) have similar suppression effects of cytochrome c maturation defects in CcmI-null mutants. Likewise, additional overproduction of CcmG, the C-terminal periplasmic segment of CcmI (CcmI-2), or even of apocytochrome c₂ also provides complementation abilities similar to those of these mutants. These results indicate that the two segments of CcmI have different functions and support our earlier findings that two independent steps are required for full recovery of the loss of CcmI function. We therefore propose that CcmI-1 is part of the CcmF-CcmH_Rc-dependent heme ligation, while CcmI-2 is involved in the CcdA- and CcmG-dependent apoprotein thioreduction steps, which intersect at the level of CcmI during cytochrome c biogenesis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The c-type cytochromes (cyts) have been found in virtually all organisms. In most microorganisms, they function primarily as electron carriers in energy transduction pathways like respiration (Res) and photosynthesis (Ps) (41). In some eukaryotes, they are also involved in other fundamental cellular processes, such as programmed cell death (apoptosis) (27). They carry at least one covalently and stereospecifically attached heme (iron protoporphyrin IX) moiety via thioether bonds between the heme vinyl groups and the cysteine thiols of a highly conserved Cys-Xxx-Yyy-Cys-His signature motif within the apoproteins (41). In recent years, this complex posttranslation/posttranslocation protein modification process has been under intense study. So far, three evolutionarily distinct modes of holocytochrome (holocyt) c formation have been found in various organisms and are called cyt c maturation systems I, II, and III (33). The most sophisticated of these modes (Ccm system I) is used by - and -proteobacteria, deinococci, and mitochondria of plants and protozoa. In gram-negative bacteria, the Ccm system I consists of at least 10 genes, designated ccmABCDEFGHI and dsbD or ccdA. All of these genes encode membrane-bound components that act on c-type apocytochromes (apocyts) or heme moieties following their translocation across the cytoplasmic membrane to produce holocyts c (2, 33, 51).

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus produces a variety of membrane-bound and soluble c-type cyts, including cyts c₁, c₂, c', c_y, c_o, and c_p, to sustain its versatile growth modes (15, 25, 31, 55). Of these proteins, cyt c₁ and either cyt c₂ or c_y are required for Ps growth (55), while cyts c_o and c_p are involved in Res growth as subunits of the cbb₃-type cyt c oxidase (31), whose activity can be detected by monitoring the staining of colonies following the Nadi (-naphthol + dimethylphenylenediamine indophenol blue + H₂O) reaction (30). Studies of R. capsulatus mutants which exhibit Ps^–/Nadi^– dual phenotypes (32) have identified various components required for cyt c maturation in this species (16, 22, 36, 38). Of these components, CcmABCD form an ATP-binding cassette (ABC)-containing transporter complex, which is thought to be required for the translocation of heme groups across the cytoplasmic membrane and for their attachment to the heme chaperone CcmE in the periplasm (24, 42, 52). Recent works indicate that CcmABCD are also involved in subsequent ATP-dependent release of holo-CcmE providing heme groups for the putative apocyt c heme lyase component CcmF (21, 24, 42, 52).

As disulfide bonds are formed between the cysteines of exported proteins via the DsbA-DsbB pathway for oxidative protein folding (11, 28), similar bonds are also assumed to be formed between the cysteine thiols of the apocyt c heme binding motif (37, 43). Thus, a specific thioreduction pathway (38) is thought to shuttle electrons across the cytoplasmic membrane via CcdA (homologous to the central part of Escherichia coli DsbD) to CcmG and then to CcmH (R. capsulatus CcmH [CcmH_Rc] or E. coli CcmH [CcmH_Ec]) in order to reduce the disulfide bond at the apocyt c heme binding site prior to heme ligation (20, 29, 38). Finally, CcmI is proposed to chaperone the apocyts c to their heme ligation sites following their translocation into the periplasm (36).

In R. capsulatus and in some other species, CcmI is a bipartite membrane protein with two amino-terminal transmembrane helices encompassing a leucine zipper-like motif in its cytoplasmic loop (CcmI-1 segment of 112 amino acid residues in length) and a large periplasmic carboxyl-terminal extension with four tetratricopeptide repeat (TPR)-like motifs (CcmI-2 segment of 312 amino acid residues in length) (36, 45) (Fig. 1). In R. capsulatus, CcmI mutants lacking the CcmI-2 segment cyt c maturation become growth medium dependent. These mutants produce several c-type holocyts (e.g., cyts c₁, c_p, c_y, and c_o) during growth on minimal medium, but they contain only holocyt c₁ during their growth on enriched medium (36). This indicates that CcmI-1 is required for the production of all cyts c, whereas CcmI-2 is to some extent dispensable for cyt c₁ (36, 45). No homologue of the CcmI-1 segment is found in E. coli whereas that of CcmI-2 is naturally fused C terminally to the homologue of CcmH_Rc, yielding a bifunctional component referred to here as CcmH_Ec. Remarkably, in E. coli, the lack of a CcmI-1 segment coincides with the absence of any carboxyl terminally membrane-attached c-type cyt, such as cyt c₁. Recent genetic analyses suggest that R. capsulatus CcmI might form together with CcmF and CcmH_Rc a heme ligation core complex catalyzing the final steps of heme attachment to apocyts c (45, 46).

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FIG. 1. Membrane-embedded CcmI-1 and periplasmic CcmI-2 segments of CcmI. The CcmI-1 segment is 112 amino acids long and comprises the first and second transmembrane helices (black and gray cylinders) of CcmI encompassing a cytoplasmic loop with a leucine zipper-like motif (light-gray oval). The CcmI-2 segment refers to the 312-amino-acid-long periplasmic portion of CcmI, which contains four TPR-like motifs (dark-gray rectangles). Q38, S64, L79, A96, and P121 are landmark amino acid residues of CcmI that define modified variants of CcmI, such as CcmIQ38-L79::FLAG, CcmI-1S64::FLAG, CcmI-1A96::FLAG, and CcmI-1::FLAG, produced by the plasmids pCS1570, pCB2, pCS1588, pCB1, pCB6, and pCS1590, respectively. T84M designates the engineered start codon in CcmI to yield a CcmI-2::FLAG derivative produced by pCS1579 or pCS1583, and F175 marks the location of the spectinomycin cassette inserted into ccmI carried by pSEL12 and yielding a CcmI-F175::spe derivative, as described in the text.

Interestingly, complete suppression of holocyt c production defects due to the absence of CcmI requires two distinct and additive events: overproduction of the CcmF-CcmH_Rc couple that overcomes the function of CcmI on minimal but not enriched medium (17) and additional overproduction of CcmG that further renders cyt c biogenesis fully proficient in all media (45). The requirement of two genetically independent steps together with the bipartite structure of CcmI (Fig. 1) led us to further investigate possible functional relationships between the CcmI segments CcmF, CcmH_Rc, and CcmG. In this work, we demonstrate that overproduction of either the CcmF-CcmH_Rc couple or CcmI-1, supplemented with the overproduction of CcmG, CcmI-2, or even apocyt c₂, fully complements CcmI-null mutants for all defects in holocyt c formation. Moreover, we show that the hyperproduction of the CcmF-CcmH_Rc couple and hyperproduction of CcmI-1 also suppress cyt c deficiency to comparable extents. These findings indicate that on one hand CcmF, CcmH_Rc, and CcmI-1 and on the other CcmG and CcmI-2 act together during cyt c maturation. Thus, the membrane-spanning and periplasmic segments of CcmI interconnect the CcdA- and CcmG-dependent periplasmic apoprotein thioreduction pathway to the heme ligation pathway involving the CcmF-CcmH_Rc couple.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this work are described in Table 1. R. capsulatus strains were grown at 35°C on enriched (MPYE) (13) or minimal (Med A) (49) medium, supplemented with appropriate antibiotics (tetracycline, kanamycin, and spectinomycin at final concentrations of 2.5, 10, and 10 µg/ml, respectively), either chemoheterotrophically (Res growth) or photoheterotrophically (Ps growth) in anaerobic jars with H₂-CO₂-generating gas packs from BBL Microbiology Systems (Cockeysville, MD). The ability of the strains to form visible colonies after 3 days of incubation under appropriate growth conditions is denoted by "+" (plus), after 5 days by "slow," and in other cases by "–" (minus). E. coli strains were grown on Luria-Bertani (LB) broth supplemented as needed with appropriate antibiotics (tetracycline, kanamycin, spectinomycin, and ampicillin at final concentrations of 12.5, 50, 10, and 100 µg/ml, respectively), as published earlier (13, 26).

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TABLE 1. Bacterial strains and plasmids used in this study

Molecular genetic techniques. Standard molecular genetic techniques (44) and conjugal transfer of plasmids from E. coli to R. capsulatus (13) were performed as described elsewhere. The primers (designations and nucleotide sequences) used in this study are listed in Table 2. Plasmid pSVEN was restricted with BamHI and HindIII, and the 1.66-kb fragment containing ccmI and its native promoter sequence was cloned into pBluescriptKS+ to yield pSVEN-KS(+). The nucleotide sequence of a FLAG epitope (DYKDDDDK) was added in frame to the 3' end of ccmI by PCR with the primers CcmI-FLAGfwd and CcmI-FLAGrev using a QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions, yielding pNJ1. The ccmI::FLAG-harboring allele was excised from pNJ1 with BamHI and HindIII (1.69 kb) and ligated into the respective sites of pRK415 and pBluescriptIISK+ to create pNJ2 and pCS1564, respectively. Plasmid pCS1570 was constructed by digesting pNJ2 with PstI and then religating the remaining vector fragment to delete the in-frame 126 bp of ccmI (ccmIQ38-L79::FLAG) corresponding to a CcmI derivative lacking 42 amino acid residues of its cytoplasmic loop. Plasmid pCS1580 harboring ccmI1::FLAG, which produces a CcmI derivative corresponding to its N-terminal 121 amino acid residues, fused C terminally with a FLAG tag, was constructed by deleting 912 bp of its 3' end in pCS1564 via PCR amplification using the primers CS1580fwd and CS1580rev and again a QuikChange XL site-directed mutagenesis kit. Plasmid pCS1580 was restricted with HindIII and ligated into the same site of pRK415 to obtain pCB1. Similarly, pCS1580 was cloned into pCS1540 carrying an overexpressing ccmG allele (45) to generate pCS1589. Furthermore, a 1,396-bp PCR product containing a 3' fragment of ccmI (ccmI2::FLAG) that corresponds to a gene product lacking the N-terminal 84 amino acid residues of CcmI (i.e., its first transmembrane helix and almost its entire cytoplasmic segment) was amplified from pCS1564 by using the primers CS1579fwd, introducing a ribosomal binding site and a start codon, and M13-forward. This PCR product was phosphorylated, restricted with HindIII, and cloned into pBluescriptSK+ digested with EcoRV and HindIII, yielding pCS1576. Plasmid pCS1579 was then constructed by restricting pCS1576 with XbaI and KpnI and by cloning of the 1,332-bp ccmI2::FLAG-containing fragment into the respective sites of pCHB500. For pCS1566, a 604-bp PCR product harboring ccmG was amplified by using the primers helXforward-XbaI and 1566rev, carrying the recognition sites for XbaI and KpnI at their 5' ends, and cloned into the respective sites of pCHB500 containing ccmG::His₆ with its native ribosomal binding site and expressed from the R. capsulatus cycA promoter.

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TABLE 2. Designations and nucleotide sequences of primers used in this study

Two-step PCR amplifications were carried out to construct pCS1583, pCB2, pCS1588, pCS1590, and pCB5. As does pCS1579, pCS1583 contains ccmI2::FLAG but expresses this CcmI allele from its native ccmI promoter. The first-round PCR used pCS1564 as a template and the primers M13-reverse and CS1583rev to yield a 466-bp fragment and the primers CS1583fwd and M13fwd to generate a 1,304-bp fragment. These fragments were purified with a QIAquick PCR purification kit (QIAGEN, Valencia, CA) and used as DNA templates with complementary sequences of at least 15 bp for a second-round PCR using the primers M13-reverse and M13-forward. The latter PCR fragment of 1,755 bp was restricted with HindIII and XbaI and ligated into the respective site of pRK415 to finally create pCS1583. For pCB2, the first round of PCR used pCS1580 as a template and the primers M13-reverse and CB2rev or the primers CB2fwd and M13-forward to produce a 658-bp and a 284-bp fragment, respectively. The first-round PCR amplification for pCS1588 was also done using pCS1580 as a template and the primers M13-reverse and 1588rev or the primers 1588fwd and M13-forward to generate a 754-bp and a 284-bp fragment, respectively. In both cases, these fragments were purified, as described for pCS1583 above, and used as DNA templates for a second round of PCR using the primers M13-reverse and M13-forward. The latter PCR fragments for the construction of pCB2 (927 bp) and pCS1588 (1,023 bp) were restricted with HindIII and XbaI and then ligated into the same sites of pRK415 containing ccmI derivatives (ccmIS64::FLAG or ccmIA96::FLAG) that correspond to gene products of the N-terminal 64 and 96 amino acid residues of CcmI with C-terminal FLAG tags. The plasmids pCS1590 and pCB5 were constructed similarly. In the first round of PCR, a 414-bp fragment was amplified using pCS1580 as a template and the primers RccycH-XbaIf and CB5rev, and a 594-bp fragment was amplified using pCS1566 as a template and the primers CB5fwd and M13-forward. This yielded a PCR product corresponding to a CcmG derivative without its native N-terminal signal sequence and membrane anchor. These PCR fragments were purified and used as DNA templates for a second round of PCR using the primers RccycH-XbaIf and M13-forward, generating a 1,003-bp fragment, which was then restricted with XbaI and KpnI, ligated into the same sites of pRK415 and pCHB500 harboring a ccmI1::FLAG-ccmG::His₆ gene fusion with the ccmI ribosomal binding site, and expressed from different promoters.

Finally, pCS1591 and pCB6 were created by PCR amplification from pCS1580 using the primers RccycH-XbaIf and M13-forward, restricting the produced 660-bp fragment with XbaI and KpnI and ligating it into the corresponding sites of the pRK415 and pCHB500. These constructs contained ccmI1::FLAG with its native ribosomal binding site but expressed from two different promoters, as described above. All plasmids were verified by DNA sequencing using appropriate primers, and DNA sequence analyses, homology searches, and genome sequence comparisons were done using MacVector from Accelrys (San Diego, CA) and BLAST software packages (3). Note that the ccmI alleles in pNJ2, pCS1570, pCB2, pCS1588, pCB1, pSEL12, pCS1583, and pCS1589 are expressed from the native ccmI promoter, those in pCB6, pCS1579, and pCB5 from the R. capsulatus cycA promoter of pCB500 derivatives, and those in pCS1590 and pCS1591 from the E. coli lacZ promoter of pRK415 derivatives.

Biochemical techniques. Small-scale crude cell extracts were prepared by using CelLytic B2X cell lysis reagent (Sigma-Aldrich, St Louis, MO) according to the manufacturer's suggestions, modified as follows. Ten-ml cultures were centrifuged for 10 min at 5,000 x g and 4°C, and cell pellets were washed with 2 ml of TNE buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM EDTA, 0.1 mM amino-caproic acid, and 0.1 mM Pefabloc SC [Sigma-Aldrich, St. Louis, MO]). CelLytic B2X cell lysis reagent (200 µl) supplemented with a protease inhibitor mixture (final concentration, 50 mM EDTA, 0.1 mM amino-caproic acid, and 0.1 mM Pefabloc SC) was added to the pellets, which were resuspended by vortexing for 2 min before lysozyme (final concentration, 0.2 mg/ml) was added. After incubation for 10 min at 25°C, the solution was sonicated (microtip, 60% power, 50% pulse) for 30 s using a Sonic Dismembrator 550 (Fisher Scientific, Pittsburgh, PA) and centrifuged for 10 min at 20,000 x g and 4°C. The supernatants were used for further protein analyses.

Intracytoplasmic membrane vesicles (chromatophores) were prepared in TNE buffer by two passages through a French pressure cell at 18,000 lb/in². After elimination of the disrupted cells by centrifugation for 1 h at 20,000 x g and 4°C, supernatants were recentrifuged for 2 h at 150,000 x g and 4°C and membrane pellets used for further analyses. Protein concentrations were determined according to the method of Wiechelman et al. (53) or Bradford (7) by use of a bicinchoninic acid kit (Sigma-Aldrich, St. Louis, MO) or bio-safe Coomassie solution (Bio-Rad, Hercules, CA), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (34) or Schaegger and von Jagow (47) as appropriate, and the c-type cyts were revealed by determining their endogenous peroxidase activity using 3,3',5,5'-tetramethylbenzidine (TMBZ) and H₂O₂ (50). Cyt c oxidase activities of colonies were detected on agar plates using the Nadi reaction as described above (30). Rabbit antisera against CcmG and CcmH_Rc used for immunoblot analyses were described elsewhere (23, 38). Rabbit anti-FLAG immunoglobulin G polyclonal antibody (Sigma, St. Louis, MO) was used to detect the FLAG epitope fused to various CcmI derivatives. Monoclonal anti-rabbit immunoglobulin clone RG-16 alkaline phosphatase conjugate with BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium liquid substrate (Sigma-Aldrich, St. Louis, MO) and stabilized goat anti-rabbit immunoglobulin G horseradish peroxidase conjugate with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) were used as secondary antibodies.

Protein purification. Intracytoplasmic membrane vesicles were resuspended and homogenized in TNE buffer and solubilized for 1 h at 4°C by addition of dodecylmaltoside (DDM) to the continuously stirring membrane protein solution (1 mg protein/mg DDM; final protein concentration, 1 mg/ml). After centrifugation for 30 min at 20,000 x g and 4°C, TNE buffer was exchanged with TNED buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 0.1% [wt/vol] DDM) by using a PD-10 column (Amersham-Pharmacia, Piscataway, NJ). Then, the solution was applied to an anti-FLAG agarose column (Sigma-Aldrich, St. Louis, MO), preequilibrated with 20 ml TNED buffer per ml of column matrix. Solubilized membrane protein suspensions were passed through the column three times, after which the column was washed twice with 5 ml of TNED buffer. FLAG-tagged CcmI derivatives were subsequently eluted with 5 column volumes of TNED buffer supplemented with 0.1 mg per ml of FLAG peptide (Sigma-Aldrich, St. Louis, MO).

Chemicals. All chemicals were of reagent grade and obtained from commercial sources.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cytoplasmic loop of the membrane-spanning segment of CcmI is required for efficient cytochrome c biogenesis. In order to investigate the specific roles of the different CcmI segments and motifs (Fig. 1) during cyt c biogenesis, we have constructed various ccmI alleles fused to FLAG tag sequences at their 3' ends (Fig. 2) and introduced them into ccmI mutants MT-SRP1 (CcmI^–) and MT-SRP1.r1 (CcmI^– CcmF^up CcmH_Rc^up) (where CcmF^up and CcmH_Rc^up denote up-regulated alleles of CcmF and CcmH_Rc, respectively) (Table 3), as appropriate. The cytoplasmic loop of CcmI contains a leucine zipper-like motif, which is implicated in protein-protein interactions (35, 40), but its importance for CcmI function is unknown. Two plasmids, pNJ2 carrying an intact ccmI (ccmI::FLAG) and pCS1570 harboring an allele (ccmIQ38-L79::FLAG) encoding a gene product lacking 42 amino acid residues (Fig. 1, Q38 to L79) of the cytoplasmic loop within the CcmI-1 segment (Fig. 2, rows 1 and 2), were constructed, and both plasmids mediated Ps⁺ and Nadi⁺ phenotypes to a CcmI-null mutant (MT-SRP1) on enriched and minimal media. We noted that the latter plasmid exhibited a weaker Ps growth phenotype (Ps^slow) on enriched medium than the former and that only pNJ2, not pCS1570, conferred a Ps⁺ phenotype to a double mutant (SEL22) lacking both the CcmI-2 segment and cyt c₂ (36) on all media (Table 3). Thus, the cytoplasmic loop of CcmI-1, although not absolutely essential for cyt c maturation, becomes important under Ps growth in the absence of cyt c₂, which then relies on adequate production of the membrane-bound cyts c₁ and c_y.

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FIG. 2. Restriction maps of various plasmids carrying different alleles of ccmI, ccmF-ccmHRc, ccmG, or cycA, and their related phenotypes in selected CcmI-null mutants. Restriction maps of various plasmids and Nadi+, Ps+, or Psslow phenotypes that they confer to the CcmI-null mutants MT-SRP1 and MT-SRP1.r1 (Table 1) on minimal (Med A) or enriched (MPYE) media are shown on the left and right, respectively. Plasmid pNJ2 carries the entire 1,272-bp ccmI gene (ccmI::FLAG) and pCS1570 a 126-bp in-frame deletion (ccmIQ38-L79::FLAG). Plasmids pCB2 and pCS1588 contain 192-bp and 288-bp 5' fragments of ccmI (ccmI1S64::FLAG and ccmI1A96::FLAG), respectively, and pCB1, pCB6, and pCS1590 harbor a 363-bp 5' fragment of ccmI (ccmI1::FLAG) corresponding to the entire CcmI-1 segment. Plasmid pSEL12 carries a spectinomycin cassette inserted into the XhoI site of ccmI and expresses a truncated allele (ccmIF175::spe). Plasmids pCS1583 and pCS1579 both contain a ccmI fragment lacking its first 249 bp, with an exogenous ribosomal binding site and a start codon created at its position T84 to ensure the expression of the remaining part of ccmI to yield a derivative corresponding to its CcmI-2 segment. Plasmid pCS1537 carries an up-regulated ccmFHRc (ccmFHRcup) allele, and pCS1540 and pHM14 harbor ccmG and cycA, respectively. Finally, pCS1589 carries both a ccmG allele as in pCS1540 and a ccmI1 allele as in pCB1. All ccmI derivatives, except that produced by pSEL12, are fused to a FLAG tag sequence at their 3' ends (indicated by a white rectangle with a black X). The promoters of ccmI, E. coli lacZ, cycA, and up-regulated ccmFHRc that drive the expression of downstream genes are indicated as white arrows, black arrows, gray arrows, and striped arrows, respectively. Note that the ccmI derivatives in pNJ2, pCS1570, pCB2, pCS1588, pCB1, pSEL12, pCS1583, and pCS1589 are expressed from their native ccmI promoter, while those in pCB6, pCS1579, and pCB5 are from the R. capsulatus cycA promoter and those in pCS1590 and pCS1591 are from the E. coli lacZ promoter. A, B, E, H, K, P, and X refer to cleavage sites of the restriction enzymes XhoI, BamHI, EcoRI, HindIII, KpnI, PstI, and XbaI, respectively. Numbers 1 and 2 indicate the patterns of restriction sites E, K, and B and X, B, P, E, K, and B, respectively, and double dashes refer to truncations used to fit the figure, as appropriate.

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TABLE 3. Phenotypes of various CcmI-null mutants and their derivatives

Immunoblot analyses using polyclonal antibodies against the FLAG epitope, CcmH_Rc, and CcmG were carried out, and the data indicate that both pNJ2 and pCS1570 produced similar levels of CcmI in MT-SRP1 (Fig. 3A, lanes 3 and 4). We note that, although the theoretical molecular masses for CcmI::FLAG and CcmI-Q38-L79::FLAG are 45.5 and 40.7 kDa, FLAG-specific signals were detected at higher molecular masses of 55 and 50 kDa, respectively. Interestingly, much higher levels of CcmI::FLAG were present in MT-SRP1.r1, which overproduces CcmF and CcmH_Rc and substitutes partially for the function of CcmI during cyt c maturation (Fig. 4A, lanes 3 and 4). Additional experiments suggest that the levels of CcmH_Rc and CcmG, known to have different suppression effects on holocyt c production when CcmI is absent (17, 45), were comparable to wild type-levels (Fig. 3B and C and 4C, lanes 1, 3, and 4), except for the levels of CcmH_Rc in the transconjugant of MT-SRP1.r1, which are much higher, as this strain already overexpresses ccmH_Rc genomically (Fig. 4B, lanes 1, 3, and 4).

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FIG. 3. Cytochrome c profiles and immunoblot analyses of various CcmI mutants grown on minimal medium. Crude cell extracts of R. capsulatus strains grown in minimal medium under Res conditions were subjected to SDS-PAGE using approximately 100 µg of proteins per lane. Immunoblot analyses using polyclonal antibodies against the FLAG epitope of CcmI derivatives (A), CcmHRc (B), and CcmG (C) were performed, and holocyts c were detected via their endogenous peroxidase activity using TMBZ and H2O2 (D and E) as described in Materials and Methods. Lanes 1 to 9 correspond to crude cell extracts of the wild type (MT1131/pRK415), ccmI (MT-SRP1/pRK415), ccmI::FLAG (MT-SRP1/pNJ2), ccmIQ38-L79::FLAG (MT-SRP1/pCS1570), ccmI1S64::FLAG (MT-SRP1/pCB2), ccmI1A96::FLAG (MT-SRP1/pCS1588), ccmI1::FLAGccmI expressed by its native ccmI promoter from a composite plasmid (MT-SRP1/pCB1), ccmI1::FLAGcycA expressed by the cycA promoter from a pRK415 derivative (MT-SRP1/pCB6), and ccmFHRcup (MT-SRP1/pCS1537), respectively. Molecular mass markers (in kDa) are indicated on the left. Note that the two bands around the 30-kDa marker, indicated by an asterisk on the left, were detected due to their peroxidase activities and are not specific to the antibodies used.

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FIG. 4. Cytochrome c profiles and immunoblot analyses of various CcmI mutants overproducing the CcmF-CcmHRc couple grown on enriched medium. Crude cell extracts of R. capsulatus strains grown in enriched medium under Res conditions were subjected to SDS-PAGE using approximately 100 µg of proteins per lane. Immunoblot analyses using polyclonal antibodies against the FLAG epitope of CcmI derivatives (A), CcmHRc (B), and CcmG (C) were performed, and holocyts c were detected via their endogenous peroxidase activity using TMBZ and H2O2 (D and E), as described in Materials and Methods. Lanes 1 to 9 correspond to crude cell extracts of the wild type (MT1131/pRK415), ccmI ccmFHRcup (MT-SRP1.r1/pRK415), ccmI::FLAG (MT-SRP1.r1/pNJ2), ccmIQ38-L79::FLAG (MT-SRP1.r1/pCS1570), ccmI::FLAGccmI expressed by its native ccmI promoter from a composite plasmid (MT-SRP1.r1/pCB1), ccmI1::FLAGcycA expressed by the cycA promoter from a pRK415 derivative (MT-SRP1.r1/pCB6), ccmI2::FLAG (MT-SRP1.r1/pCS1579), ccmFHRcup (MT-SRP1.r1/pCS1537), and ccmG (MT-SRP1.r1/pCS1540), respectively. Molecular mass markers (in kDa) are shown on the left. Note that the two bands around the 30-kDa marker, indicated by an asterisk on the left, were detected due to their peroxidase activities and are not specific to the antibodies used.

Next, the cyt c profiles of strains harboring either pNJ2 or pCS1570 were analyzed using TMBZ and SDS-PAGE. The amounts of membrane-bound cyts c_p, c₁, c_y, and c_o were lower in the CcmI-null mutants expressing ccmIQ38-L79::FLAG from pCS1570 than in those expressing ccmI::FLAG from pNJ2 and the wild type (Fig. 3D and 4D, lanes 1, 3, and 4), while the amounts of soluble cyt c₂ were similar in the MT-SRP1 transconjugants but still lower than amounts in the wild type (Fig. 3E and 4E, lanes 3 and 4). Therefore, overall data indicate that the cytoplasmic segment of CcmI with its leucine zipper-like motif is important for efficient holocyt c production and is more critical for sufficient production of membrane-bound holocyt c_y than soluble holocyt c₂.

An intact CcmI-1 segment is necessary to suppress porphyrin excretion and to partially bypass cyt c deficiency phenotypes of a CcmI-null mutant. Various R. capsulatus mutants deficient in cyt c maturation excrete heme precursors (uroporphyrins and protoporphyrins) when grown under Res conditions (16, 23). A similar phenotype is also observed with CcmI-null mutants, like MT-SRP1 and SEL11, but not with CcmI mutants lacking only its CcmI-2 segment, such as SEL12, SEL22, or FJM13, which produce at least holocyt c₁ (36; also this work). For Sinorhizobium meliloti, CcmI-null mutants expressing the N-terminal transmembrane helix and the adjacent cytoplasmic loop of the CcmI-1 segment, although still defective for cyt c maturation, alleviate the associated protoporphyrin IX accumulation phenotype (9). We therefore tested whether in R. capsulatus the N-terminal part of CcmI is also involved in porphyrin excretion. Of the three plasmids constructed, pCB2 and pCS1588 (Fig. 2, rows 3 and 4) produce the N-terminal 64 and 96 amino acid residues of CcmI (CcmI-S64::FLAG and CcmI-A96::FLAG), respectively. These CcmI derivatives contain only the first transmembrane helix of CcmI-1 and portions of the cytoplasmic loop (Fig. 1, S64 and A96). The third plasmid, pCB6 (Fig. 2, row 7), expresses via the exogenous cycA promoter the first 121 residues of CcmI, which include both of the transmembrane helices and the entire cytoplasmic loop of CcmI-1 (CcmI-1::FLAG) (Fig. 1, P121). While pCB2 and pCS1588 could not complement the CcmI-null mutant MT-SRP1 for Ps⁺ or Nadi⁺ phenotypes on either minimal or enriched medium, pCB6 did so for Nadi⁺ on both media and for Ps⁺ on minimal medium only. When the porphyrin excretion phenotype was monitored under UV light at 312 nm by the formation of red fluorescence surrounding colonies, the transconjugants carrying pCB2 and pCS1588 still excreted heme precursors, while those harboring pCB6 did not exhibit this phenotype, like the CcmI-2-null mutants.

Next, immunoblot analyses were carried out using crude extracts of cells grown by respiration in minimal medium to reassure that various FLAG-tagged CcmI derivatives were produced adequately in appropriate strains tested. Proteins of apparent molecular masses of 12, 14, and 15 kDa (Fig. 3A and 4A, lanes 5, 6, and 8), in all cases higher than the respective theoretical molecular masses of 7.9 kDa for CcmI-S64::FLAG, 11.2 kDa for CcmI-A96::FLAG, and 13.5 kDa for CcmI-P121::FLAG, respectively, were detected. In addition, similar analyses indicate that, in the strains tested, the levels of CcmH_Rc (Fig. 3B, lanes 5, 6, and 8) were similar to those seen in a wild-type control (Fig. 3B, lanes 1 and 2). In contrast, the levels of CcmG were lower in the wild-type strain and in the MT-SRP1 transconjugant containing pCB6 (Fig. 3C, lanes 1 and 8) than in those harboring pCB2 and pCS1588 (Fig. 3C, lanes 5 and 6). This observation indicates that the CcmG levels are increased in the absence of CcmI and hence of cyt c production. Finally, determination of the cyt c profiles of appropriate strains confirmed that the membrane-bound cyts c_p, c₁, c_y, and c_o as well as the soluble cyt c₂ could be detected in cell extracts of MT-SRP1 transconjugants carrying only pCB6 and not pCB2 and pCS1588 (Fig. 3D and E, lanes 8, 5, and 6, respectively), in agreement with their Nadi, Ps, and heme precursor excretion phenotypes. Therefore, overall data provide evidence that the presence of an intact CcmI-1 segment comprised of the first and second transmembrane helices and the leucine zipper-like motif-containing cytoplasmic loop is the minimal topological unit necessary to alleviate porphyrin excretion and to confer cyt c production in R. capsulatus.

Overproduction of CcmI-1, like that of CcmF and CcmH_Rc, partially overcomes the cyt c maturation defects of CcmI-null mutants. The fact that the Nadi⁺ and Ps⁺ phenotypes of a CcmI-null mutant overproducing CcmI-1 (MT-SRP1/pCB6) are identical to those of a CcmI-null mutant overproducing CcmF and CcmH_Rc, like MT-SRP1.r1 (17), suggests that the membrane-spanning segment of CcmI and the CcmF-CcmH_Rc couple might play similar roles during cyt c maturation. Thus, three differently replicating plasmids, pCB1, pSEL12, and pCS1591 (Fig. 2, rows 5, 6, and 8), expressing different amounts of CcmI-1 (due to different promoters and plasmid copy numbers) were constructed to further probe to what extent these components might substitute for each other, depending on their production levels. Plasmid pSEL12 (36) contains a spectinomycin cassette insertion in the periplasmic segment of CcmI and produces a CcmI derivative with its entire CcmI-1 segment and a short stretch of CcmI-2 but lacking the four TPR-like motifs (Fig. 1, F175). Plasmids pCB1 and pCS1591 express a CcmI derivative identical to that produced by pCB6 (Fig. 1, P121), but the latter plasmid lacks the exogenous cycA promoter, while the former is a composite plasmid assumed to be of higher copy number, due to the cointegration of ColE1 and RP4 replicons (Fig. 2). All three plasmids conferred to a CcmI-null mutant like MT-SRP1 Nadi and Ps phenotypes similar to that conferred by pCB6, indicating that they all produced CcmI-1. However, pCB1 was also able to mediate to MT-SRP1 a Ps^slow phenotype on enriched medium. Moreover, all plasmids rendered MT-SRP1.r1 (CcmI^– CcmF^up CcmH_Rc^up) Nadi⁺/Ps⁺ on both media, but only pCB1 complemented SEL22, a double mutant lacking the CcmI-2 segment and cyt c₂ (21), for Ps⁺ growth on minimal medium and Ps^slow growth on enriched medium. This suggests that pCB1, which also acted differently than pCS1537 carrying an up-regulated allele of ccmFH_Rc (55), might overproduce CcmI-1. In addition, comparison of the cyt c profiles of strains harboring pCB1 or pCB6 grown by respiration reveals that membrane-bound cyts c_p, c₁, c_y, and c_o and soluble cyt c₂ (Fig. 3D and E, lanes 7 and 8, and Fig. 4D and E, lanes 5 and 6) were all produced, although their levels were lower than those detected in a wild-type strain (Fig. 3D and E and 4D and E, lanes 1), as previously seen with strains overproducing CcmF and CcmH_Rc (17).

Immunoblot analyses using polyclonal antibodies against the FLAG epitope indeed showed that the strains harboring pCB1 produced a CcmI derivative with an apparent molecular mass of 15 kDa at an amount higher than that seen with pCB6 (Fig. 3A, lanes 7 and 8, and 4A, lanes 5 and 6), confirming that this overproduction was responsible for the additional Ps^slow phenotype mediated by pCB1. Additional immunoblot analyses indicated that the levels of CcmH_Rc in strains carrying pCB1 or pCB6 differed neither among each other (Fig. 3B, lanes 7 and 8, and Fig. 4B, lanes 5 and 6) nor compared to their respective controls (Fig. 3B and 4B, lane 2). These levels were significantly higher in strains like MT-SRP1.r1 (Fig. 3B, lane 9, and Fig. 4B, lane 8) that contained higher levels of CcmF and CcmH_Rc than did a wild-type strain (Fig. 4B, lanes 1 and 2), due to the overexpression of the respective chromosomal gene copies (21). On the other hand, the levels of CcmG in the CcmI-null mutants harboring pCB1, pCB6, or pCS1537 remained comparable to those seen in a wild-type strain (Fig. 3C, lanes 1, 7, 8, and 9, and 4C, lanes 1, 5, 6, and 8). Clearly, overall data indicate that overproduction of either CcmI-1 or the CcmF-CcmH_Rc couple suppresses, either partially or fully (depending on the produced amounts), cyt c maturation defects encountered in CcmI-null mutants, suggesting that the CcmI-1 segment and the CcmF-CcmH_Rc couple function together during cyt c biogenesis.

The CcmI-2 segment and CcmG play similar roles to bypass completely the need for CcmI during cyt c biogenesis. The findings that the CcmF-CcmH_Rc couple and CcmI-1 act similarly and that additional overproduction of CcmG overcomes CcmI defects in all growth media led us to inquire whether the CcmI-2 segment and CcmG might also have similar functions during cyt c maturation. Plasmids pCS1583 and pCS1579 (Fig. 2, rows 9 and 10), producing different amounts of the same CcmI-2 derivatives via different promoters, were constructed. These derivatives start at position 84 (Fig. 1, T84M) and contain the second transmembrane helix and the periplasmic extension of CcmI with its four TPR-like motifs, thought to facilitate protein-protein interactions (6, 14). Neither pCS1583 nor pCS1579 complemented a CcmI-null mutant like MT-SRP1, but they both conferred Nadi⁺/Ps⁺ phenotypes on enriched media to MT-SRP1.r1 (CcmI^– CcmF^up CcmH_Rc^up), like the overproduction of CcmG (45).

The cyt c profiles of appropriate strains carrying pCS1579 grown on enriched medium under Res conditions indicate that the amounts of membrane-bound cyts c_p, c₁, c_y, and c_o as well as soluble cyt c₂ were similar to those carrying pCS1540 (overproducing CcmG) (Fig. 4D and E, lanes 7 and 9). Immunoblot analyses show that the levels of CcmH_Rc were not distinguishable between the strains carrying pCS1579 and pCS1540 (Fig. 4B, lanes 7 and 9), while, as expected, those of CcmG were drastically increased in strains containing pCS1540 (Fig. 4C, lanes 7 and 9) (45). However, the FLAG epitope-tagged CcmI-2 segment of CcmI could not be detected in crude cell extracts of appropriate strains harboring pCS1579 despite their Ps⁺ phenotypes (Fig. 4A, lane 7), possibly due to its rapid proteolytic degradation. This difficulty was circumvented by using intracytoplasmic membrane vesicles from an appropriate strain carrying pCS1579 grown under Ps conditions on enriched medium and affinity-purified FLAG-tagged CcmI-2, as described in Materials and Methods. CcmI-2::FLAG migrated at an apparent molecular mass of 45 kDa (larger than its theoretical molecular mass of 36.5 kDa) (Fig. 5, lanes 6 to 8). The data establish that the CcmI-2 segment and CcmG play very similar roles in enhancing cyt c maturation in appropriate CcmI suppressors.

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FIG. 5. Production of CcmI-2 in MT-SRP1.r1 grown under photosynthetic conditions on enriched medium. Intracytoplasmic membrane vesicles prepared from R. capsulatus strain MT-SRP1.r1/pCS1579 [(ccmI::kan) ccmFHRcup ccmI2::FLAG expressed by the cycA promoter] grown on enriched medium under Ps conditions were solubilized using DDM, and FLAG-tagged CcmI-2 was purified as described in Materials and Methods. Samples from different purification steps (CM, intracytoplasmic membrane vesicles; SM, solubilized membrane proteins; FT, flowthrough after three passages through the anti-FLAG agarose column; W, column wash; E1 to E4, elutions 1 to 4 from the column) were subjected to SDS-PAGE (100 µg and 10 µg of proteins in lanes 2 to 4 and 5 to 9, respectively), and immunoblot analyses were carried out using polyclonal antibodies against the FLAG epitope, as described in Materials and Methods. Lane 1 corresponds to 100 µg of intracytoplasmic membrane vesicles from a wild-type strain and thus does not contain a FLAG epitope (CM wild type). Molecular mass markers (in kDa) are shown on the left.

Simultaneous overproduction of CcmI-1 and CcmG also overcomes completely the holocyt c formation defects due to the absence of CcmI. As the CcmI-1 segment and CcmF-CcmH_Rc couple as well as the CcmI-2 segment and CcmG appeared functionally equivalent for cyt c maturation, we surmised that simultaneous overproduction of CcmI-1 and CcmG might also bypass completely the need for CcmI to produce adequate amounts of cyts c under all growth conditions. Indeed, plasmid pCS1589 (Fig. 2, row 15), producing a CcmI-1::FLAG derivative as in pCB1 and an active CcmG as in pCS1540 (45), mediated Nadi⁺/Ps⁺ phenotypes for appropriate null mutants of CcmI and CcmG (MT-SRP1 or MT-SRP1.r1 and MD11), indicating that simultaneous overproduction of the CcmI-1 segment and CcmG, similarly to that of the CcmF-CcmH_Rc couple and CcmG, substitutes completely for the CcmI function during cyt c maturation. The data therefore suggest that the membrane-spanning segment of CcmI or CcmF together with CcmH_Rc on the one hand and the periplasmic segment of CcmI or CcmG on the other act cooperatively during cyt c biogenesis.

Notably, additional plasmids, like pCB5 and pCS1590 (Fig. 2, rows 13 and 14), carrying a ccmI1 allele fused in frame to a ccmG derivative without its corresponding signal peptide and membrane anchor sequence at its 5' end (ccmI1::FLAG-ccmG::His₆) and expressing the fusion product from different promoters were not able to restore the Nadi⁺/Ps⁺ phenotypes of a CcmG-null mutant (Table 1, MD11), although they readily complemented a CcmI-null mutant (MT-SRP1) for Nadi⁺ on all media and for Ps⁺ only on minimal medium. These data point out that the fusion proteins produced by pCB5 or pCS1590 were functional for only their CcmI-1, but not their CcmG, moieties, suggesting that the membrane anchor of CcmG is important for its function, as has been described similarly for its E. coli homologue (1).

Overexpression of cycA encoding cyt c₂ can also substitute for the function of CcmI-2 during cyt c maturation. Previously, we reported that overexpression of cycA encoding cyt c₂ complemented a CycA-null mutant (FJM13) carrying an additional point mutation in CcmI-2 for Ps⁺ growth on minimal and enriched media (36). Consistent with this result, plasmid pHM14 harboring cycA also conferred the Ps⁺ phenotype on both media to a null mutant of CcmI-2 (SEL12) and a null CcmI-2 and cyt c₂ double mutant (SEL22). While pHM14 (Fig. 2, row 16) could not complement the CcmI-null mutant MT-SRP1, it could fully complement MT-SRP1.r1 (CcmI^– CcmF^up CcmH_Rc^up) on both media. Thus, overproduction of apocyt c₂ could substitute for the role of CcmI-2 under appropriate conditions, provided that either the CcmI-1 segment is produced or the CcmF-CcmH_Rc couple is overproduced during cyt c biogenesis. This effect is seemingly specific to cycA, as cycY encoding cyt c_y (pFJ63), petABC encoding the cyt bc₁ complex (pMT0-404), and ccoNOQP encoding the cyt cbb₃ oxidase (pOX15) (Table 1) could not mediate similar phenotypes to appropriate strains. Furthermore, the cyt c profiles of appropriate MT-SRP1.r1 transconjugants grown on enriched medium under Res conditions indicated that the amounts of soluble cyts c₂ and of membrane-bound cyts c_p, c₁, and c_o were significantly increased in the derivatives containing pHM14 (Fig. 6, upper and lower panels).

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FIG. 6. Cytochrome c profiles of a CcmI mutant overproducing the CcmF-CcmHRc couple and various c-type cytochromes. Intracytoplasmic membrane vesicles of appropriate R. capsulatus strains grown in enriched medium under Res conditions were subjected to SDS-PAGE using approximately 75 µg of proteins per lane. Membrane-integral (upper panel) and soluble (lower panel) holocyts c were detected via their endogenous peroxidase activities by use of TMBZ and H2O2, as described in Materials and Methods. Lanes 1 to 5 correspond to intracytoplasmic membrane vesicles of the wild type (MT1131/pRK415), ccmI ccmFHRcup (MT-SRP1.r1/pRK415), cycA (MT-SRP1.r1/pHM14), cycY (MT-SRP1.r1/pFJ63), and petABC (MT-SRP1.r1/pMT0-404) (Table 1), respectively. Molecular mass markers (in kDa) are shown on the left.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In most gram-negative bacteria, including R. capsulatus, CcmI is an essential cyt c maturation component thought to act as a membrane-bound chaperone that directs apocyts c after their translocation across the cytoplasmic membrane to the heme ligation sites (36). CcmI has a bipartite topology with an N-terminal membrane-spanning segment (CcmI-1) formed of two transmembrane helices encompassing a cytoplasmic loop carrying a leucine zipper-like motif and a C-terminal periplasmic segment (CcmI-2) that contains four TPR-like motifs (36, 45). Both of these motifs are considered to facilitate protein-protein interactions between CcmI and its putative partners (6, 14, 35, 40). Furthermore, our earlier work indicated that two distinct events are required to fully bypass the role of CcmI during cyt c biogenesis and that CcmI might interact closely with the CcmF-CcmH_Rc couple to form a heme ligation complex (17, 45). Here, we have dissected these distinct segments and their protein-protein interaction motifs to better define the specific roles of the membrane-spanning and periplasmic segments of CcmI during cyt c maturation in R. capsulatus.

Our work indicates that production of CcmI derivatives that contain (i) only the first transmembrane helix with or without any portion of its cytoplasmic loop or (ii) the CcmI-2 segment preceded by the second transmembrane helix of CcmI (Fig. 1) is insufficient to support any holocyt c production or to eliminate the associated heme precursor excretion phenotype. Moreover, deletion of the cytoplasmic loop of CcmI decreases, but does not abolish completely, cyt c maturation, suggesting that this portion of CcmI with its leucine zipper-like motif is also important for its function. Thus, an intact CcmI-1 segment composed of its two transmembrane helices encompassing the leucine zipper-like domain is the minimum topological unit required for the formation of at least some c-type holocyts. Interestingly, overproduction of the CcmI-1 segment can further bypass the need for CcmI during cyt c maturation in a growth medium-dependent manner, similar to that seen previously with the overproduction of the CcmF-CcmH_Rc couple (17). Although CcmI-1 and the CcmF-CcmH_Rc couple behave similarly in the presence of cyt c₂, strains lacking CcmI-2 and cyt c₂, like SEL22, are complemented only with an intact CcmI-1 for adequate production of membrane-anchored cyts c₁ and c_y required for Ps growth. Together, these findings provide evidence that CcmI, CcmF, and CcmH_Rc act cooperatively during cyt c maturation (Fig. 7) and further support earlier proposals by us and others (42, 55) that they might form a core complex catalyzing heme ligation to apocytochromes. If such a multisubunit protein core complex indeed exists, then the membrane-integral segment of CcmI appears to be a critical factor for its stability or activity (Fig. 7).

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FIG. 7. Roles of the membrane-integral and periplasmic segments of CcmI during cytochrome c biogenesis in R. capsulatus. The different roles of the two CcmI segments during cyt c biogenesis are depicted schematically. We propose that the periplasmic CcmI-2 segment acts cooperatively with CcmG to render apocyt c competent for heme ligation ("apoprotein substrate preparation") and that this step is then catalyzed by a protein complex ("core ligase") functionally comprising the membrane-integral CcmI-1 segment and the CcmF-CcmHRc couple. CcmE-heme is the holoform of the periplasmic heme chaperone.

Interestingly, different threshold levels of CcmI-1 and of the CcmF-CcmH_Rc couple are needed to restore to different extents the Nadi⁺/Ps⁺ phenotypes of CcmI-null mutants during growth in different media. When these amounts are below the required threshold levels, then additional production of CcmG, CcmI-2, or even the apocyt c₂ acts cooperatively to fully bypass the role of CcmI during cyt c maturation. Thus, like CcmI-1 and the CcmF-CcmH_Rc couple, CcmI-2 and CcmG also play similar roles, as indicated by their functional cooperation upon their coproduction, albeit the activities currently attributed to the two sets of proteins during cyt c maturation are distinct. CcmG is a component of the thio-reductive pathway involved in keeping the thiol groups of the apocyt c heme binding sites reduced, while the CcmF-CcmH_Rc pair is needed for the apocyt c heme ligation process itself (38, 45). Thus, it is tempting to conclude that the two segments of CcmI bring together two separate steps, namely, the preparation of catalytically competent apoprotein substrates and the subsequent heme attachment (Fig. 7), rationalizing why R. capsulatus cells might need two additive mutations to fully bypass the absence of CcmI for efficient production of all c-type cyts under all growth conditions.

Cytoplasmic accumulation of heme precursors, as seen with S. meliloti or Rhizobium etli (9, 54), or its extracellular excretion, like in R. capsulatus (16, 23), is a notable phenotype often associated with cyt c production defects in bacteria. The molecular basis of this phenotype is unclear, and to what extent it reflects other physiological roles of cyt c maturation components, like siderophore synthesis or iron acquisition (8), is unknown. S. meliloti or R. capsulatus mutants that lack the entire CcmI, but not those that miss only the CcmI-2 segment, accumulate or excrete porphyrin derivatives. Expression of the N-terminal 96 amino acids of CcmI containing its first transmembrane helix and the adjacent cytoplasmic loop, but lacking its second transmembrane helix, alleviates porphyrin accumulation in CcmI-null mutants of S. meliloti (9), but a similar derivative (CcmI-A96::FLAG) does not suppress porphyrin excretion in similar R. capsulatus mutants. Physiological conditions that affect accumulation or excretion of heme precursors in different species need additional studies that are beyond the scope of this work.

How at the molecular level the functions of CcmG and CcmI-2 are related to each other during cyt c biogenesis and how their activities are interconnected to those of CcmF, CcmH_Rc, and CcmI-1 are unknown, and these issues await biochemical studies. Overexpression of cycA, presumably leading to the overproduction of apocyt c₂ and acting like the CcmI-2 segment, suggests that this portion of CcmI might facilitate direction of the catalytically active apocyts c to the heme ligation sites involving CcmI-1, CcmF, and CcmH_Rc (Fig. 7). If this is the case, whether the TPR-like motifs in the CcmI-2 segment facilitate these functions needs to be tested.

Currently, CcmG is considered to be a component of the periplasmic apocyt c thioreduction pathway using its thioreduction function to keep the thiol groups of the apocyt c heme binding sites reduced (38). However, it has been shown recently that the reduced form of ResA, which is the Bacillus subtilis homologue of CcmG in Ccm system II, can recognize apocyt c and release it in its reduced form to the system II-specific heme ligation sites formed by ResB and ResC (10, 12). Aside from being a thioredoxin-like protein, if CcmG also plays a similar role in Ccm system I at least for some apocyts c (e.g., apocyt c₂) prior to heme ligation, then its ability to enhance holocyt c production in the absence of the CcmI-2 segment becomes less puzzling. In this respect, it is important to point out that no mixed disulfide bond formation has been detected so far between CcmG and CcmH_Rc in any bacteria to demonstrate electron transfer between these proteins. Furthermore, it is now known that the thioreduction function of CcmG and ResA in Ccm systems I and II, respectively, is required for cyt c maturation only in the presence of an active thio-oxidative pathway formed by DsbA and DsbB (19; S. Turkarslan et al., unpublished data). Our ongoing works indicate that R. capsulatus CcmG has an additional function(s) distinct from its thioredoxin activity associated with cyt c production and that CcmG binds apocyt c in vitro (C. Sanders et al., unpublished data). Finally, it is noteworthy that the levels of CcmG are much higher in CcmI-null mutants (Fig. 3C, D, and E, lanes 2, 5, and 6), as if CcmG is somehow regulated by the absence of holocyts c.

In summary, the membrane-integral and the periplasmic segments of CcmI clearly play distinct roles that are related to those of the CcmF-CcmH_Rc couple and CcmG, respectively. Our overall findings led us to propose that CcmI coordinates with its bipartite topology the thio-oxidoreduction and chaperoning of apocyts c with the covalent and stereospecific heme attachment steps during cyt c maturation (Fig. 7). Hopefully, future biochemical studies will further define the branched interaction networks between the Ccm system I components and better elucidate the molecular mechanisms underlying cyt c biogenesis processes.

 
This work was supported by grants from the DOE (91ER20052) and the NIH (GM38237) to F.D.

We thank Neal Jaffe and Meenal Deshmukh for constructing the plasmids pNJ1 and pNJ2 and Majisha Doleyres and Janice Gunther for their editorial comments. C.B. was a visiting student from the Département Biosciences, Institut Nationale des Sciences Appliquees de Lyon, and UMR CNRS 5557 Ecologie Microbienne, Université Claude Bernard Lyon I, 69622 Villeurbanne, France.

 
* Corresponding author. Mailing address: Department of Biology, Plant Science Institute, University of Pennsylvania, 103B Lynch Building, Philadelphia, PA 19104. Phone: (215) 898-4394. Fax: (215) 898-8780. E-mail: fdaldal{at}sas.upenn.edu.

Published ahead of print on 22 November 2006.

Present address: Centre d'études de Saclay, Direction des Sciences du Vivant, Département de Biologie Joliot-Curie, Service de Bioénergétique, 91191 Gif-sur-Yvette, France.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, February 2007, p. 789-800, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01441-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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