| 1. |
- Ahuja, Umesh, et al.
(författare)
-
Haem-delivery proteins in cytochrome c maturation System II
- 2009
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 73:6, s. 1058-1071
-
Tidskriftsartikel (refereegranskat)abstract
- P>Cytochromes of the c-type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacter CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.
|
|
| 2. |
- Liu, Yiming, et al.
(författare)
-
Penicillin-binding protein SpoVD disulfide is a target for StoA in Bacillus subtilis forespores.
- 2010
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 75:1, s. 46-60
-
Tidskriftsartikel (refereegranskat)abstract
- Summary The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulfide bonds in hitherto unknown proteins in the inter-membrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high molecular weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae, confirmed that a disulfide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD, stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulfide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.
|
|
| 3. |
- Fridén, H, et al.
(författare)
-
Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate:quinone oxidoreductase (complex II)
- 1990
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 4:6, s. 1045-1056
-
Tidskriftsartikel (refereegranskat)abstract
- Cytochrome 5558 in the cytoplasmic membrane ofBacilius subtiiis constitutes the anchor and electronacceptor to the flavoprotein (Fp) and iron-sulphurprotein (Ip) in succinate:quinone oxidoreductase, andseemingly contains two haem groups. EPR and MCDspectroscopic data indicate bis-imidazole ligation ofthe haem. Apo-cytochrome was found in the mem-brane fraction of haem-deficient B. subtilis, suggest-ing that during biogenesis of the oxidoreductase thecytochrome b558 polypeptide is embedded into themembrane prior to the incorporation of haem andsubsequent binding of Fp and Ip. The six His residuesin cytochrome b558 were individually changed to Tyrto attempt identification of residues serving as haemaxial ligands and to analyse the role of His residues forassembly and function of the oxidoreductase. Fromthe properties of the mutants, His-47 can be excludedas a haem ligand. The remaining His residues (atpositions 13,28,70,113 and 155) are located in or closeto four predicted transmembrane segments. TheTyr-28 and Tyr-70 mutant proteins appeared to lackone of the two haems. Only the Tyr-13 and Tyr-47mutant cytochromes were found to function asanchors for Fp and Ip, but the Tyr-13 mutant cyto-chrome assembles into an enzymatically defectivesuccinate:quinone oxidoreductase. It is concludedfrom a combination of the experimental findings,sequence comparisons and membrane topology datathat His-28, His-70 and His-155 are probably haemaxial ligands in a dihaem cytochrome 6558. His-70 andHis-155 may be tigands to the same haem.
|
|
| 4. |
- Le Brun, Nick E., et al.
(författare)
-
Genes required for cytochrome c synthesis in Bacillus subtilis
- 2000
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 36, s. 638-650
-
Tidskriftsartikel (refereegranskat)abstract
- Cytochromes of c-type contain covalently bound haem and in bacteria are located on the periplasmic side of the cytoplasmic membrane. More than eight different gene products have been identified as being specifically required for the synthesis of cytochromes c in Gram-negative bacteria. Corresponding genes are not found in the genome sequences of Gram-positive bacteria. Using two random mutagenesis approaches, we have searched for cytochrome c biogenesis genes in the Gram-positive bacterium Bacillus subtilis. Three genes, resB, resC and ccdA, were identified. CcdA has been found previously and is required for a late step in cytochrome c synthesis and also plays a role in spore synthesis. No function has previously been assigned for ResB and ResC but these predicted membrane proteins show sequence similarity to proteins required for cytochrome c synthesis in chloroplasts. Attempts to inactivate resB and resC in B. subtilis have indicated that these genes are essential for growth. We demonstrate that various nonsense mutations in resB or resC can block synthesis of cytochromes c with no effect on other types of cytochromes and little effect on sporulation and growth. The results strongly support the recent proposal that Gram-positive bacteria, cyanobacteria, ε-proteobacteria, and chloroplasts have a similar type of machinery for cytochrome c synthesis (System II), which is very different from those of most Gram-negative bacteria (System I) and mitochondria (System III).
|
|
| 5. |
- Svensson, Birgitta, et al.
(författare)
-
Bacillus subtilis CtaA and CtaB function in haem A biosynthesis
- 1993
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 10:1, s. 193-201
-
Tidskriftsartikel (refereegranskat)abstract
- Haem A, a prosthetic group of many respiratory oxidases, is probably synthesized from haem B (protohaem IX) in a pathway in which haem 0 is an intermediate. Possible roles of the Bacillus subtilis ctaA and ctaB gene products in haem 0 and haem A synthesis were studied. Escherichia coli does not contain haem A. The ctaA gene on plasmids in E. coli resulted in haem A accumulation in membranes. The presence of ctaB together with ctaA increased the amount of haem A found in E. coli. Haem 0 was not detected in wild-type B. subtilis strains. A previously isolated B. subtilis ctaA deletion mutant was found to contain haem B and haem 0, but not haem A. B. subtilis ctaB deletion mutants were constructed and found to lack both haem A and haem 0. The results with E. coli and B. subtilis strongly suggest that the B. subtilis CtaA protein functions in haem A synthesis. It is tentatively suggested that it functions in the oxygenation/oxidation of the methyl side group of carbon 8 of haem 0. B. subtilis CtaB, which is homologous to Saccharomyces cerevisiae COX10 and E. coli CyoE, also has a role in haem A synthesis and seems to be required for both cytochrome a and cytochrome o synthesis.
|
|
| 6. |
- van der Oost, John, et al.
(författare)
-
Bacillus subtilis cytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa3-type oxidases
- 1991
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 5:8, s. 2063-2072
-
Tidskriftsartikel (refereegranskat)abstract
- The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127-degrees on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker. The mutant was unable to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from ctaC, was found to express neither of these enzymes. Radioactive haem-labelling was used to identify subunit 11, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B. subtilis.
|
|
| 7. |
- Hederstedt, Lars, et al.
(författare)
-
Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA
- 2017
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 105, s. 98-114
-
Tidskriftsartikel (refereegranskat)abstract
- Endospore cortex peptidoglycan synthesis is notrequired for bacterial growth but essential for endo-spore heat resistance. It therefore constitutes anamenable system for research on peptidoglycan bio-genesis. The Bacillus subtilis sporulation-specificclass B penicillin-binding protein (PBP) SpoVD andmany homologous PBPs contain two conserved cys-teine residues of unknown function in the transpepti-dase domain – one as residue x in the SxN catalyticsite motif and the other in a flexible loop near the cat-alytic site. A disulfide bond between these residuesblocks the function of SpoVD in cortex synthesis.With a combination of experiments with purified pro-teins and B. subtilis mutant cells, it was shown thatin active SpoVD the two cysteine residues most prob-ably interact by hydrogen bonding and that this isimportant for peptidoglycan synthesis in vivo. It wasfurthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reducesSpoVD and that requirement of StoA for cortex syn-thesis can be suppressed by two completely differenttypes of structural alterations in SpoVD. It is con-cluded that StoA plays a critical role mainly duringmaturation of SpoVD in the forespore outer mem-brane. The findings advance our understanding ofessential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells.
|
|
| 8. |
- von Wachenfeldt, Claes, et al.
(författare)
-
YtkA (CtaK) and YozB (CtaM) function in the biogenesis of cytochrome c oxidase in Bacillus subtilis
- 2021
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 116:1, s. 184-199
-
Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase in the respiratory chain of bacteria and mitochondria couples the reduction of molecular oxygen to form water with the generation of a transmembrane proton gradient. Bacillus subtilis has two heme A-containing heme–copper oxidases: the menaquinol oxidase cytochrome aa3 and the cytochrome c oxidase cytochrome caa3. By screening three collections of mutants for defective cytochrome c oxidase, we found the genes for two, new membrane-bound assembly factors in B. subtilis: ytkA and yozB (renamed ctaK and ctaM, respectively). CtaK is a lipoprotein without sequence similarity to any protein of known function. We show that CtaK functions together with Sco1 (YpmQ) in a pathway, leading to the assembly of the CuA center in cytochrome caa3 and seems to be a functional analogue to proteins of the periplasmic CuA chaperone family (PCuAC). CtaM is required for the activity of both cytochrome caa3 and cytochrome aa3 and dispensable for the insertion of heme A into these oxidases. The orthologous Bacillus anthracis protein and the distantly related Staphylococcus aureus CtaM complemented CtaM deficiency in B. subtilis, establishing a common function of CtaM in these bacteria. As the overall result of our work, 12 different proteins are known to function in the biosynthesis of cytochrome c oxidase in B. subtilis.
|
|
