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1.
A transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test. P. denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c-type cytochromes and accumulated periplasmic apocytochrome C550, but the membrane-bound holocytochromes c1 and C552 were present at 5-10% of the levels observed in wild-type ceils, DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P. denitrificans wild-type DNA library. A hybridizing 3.05 kb Bam HI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced. One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum. Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected. Significant differences between the phenotypes of P. denitrificans DPI08 and the B. japonicum cycH mutant C0X3, especially with respect to cytochrome c1 synthesis, suggest that the cycH gene product may be an assembly factor.  相似文献   

2.
We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3 end of cycH were termed cycJ, cycK and cycL. A deletion mutant (cycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, where-as cycJ codes for a novel protein of 169 amino acids with an Mr of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.  相似文献   

3.
We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3′ end of cycH were termed cycJ, cycK and cycL. A deletion mutant (ΔcycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, where-as cycJ codes for a novel protein of 169 amino acids with an Mr of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.  相似文献   

4.
The CcmH protein of Escherichia coli is encoded by the last gene of the ccm gene cluster required for cytochrome c maturation. A mutant in which the entire ccmH gene was deleted failed to synthesize both indigenous and foreign c-type cytochromes. However, deletion of the C-terminal hydrophilic domain homologous to CycH of other gram-negative bacteria affected neither the biogenesis of indigenous c-type cytochromes nor that of the Bradyrhizobium japonicum cytochrome c 550. This confirmed that only the N-terminal domain containing a conserved CXXC motif is required in E. coli. PhoA fusion analysis showed that this domain is periplasmic. Site-directed mutagenesis of the cysteines of the CXXC motif revealed that both cysteines are required for cytochrome c maturation during aerobic growth, whereas only the second cysteine is required for cytochrome c maturation during anaerobic growth. The deficiency of the point mutants was complemented when 2-mercapto-ethanesulfonic acid was added to growing cells; other thiol compounds did not stimulate cytochrome c formation in these strains. We propose a model for the reaction sequence in which CcmH keeps the heme binding site of apocytochrome c in a reduced form for subsequent heme ligation. Received: 7 September 1998 / Accepted: 15 November 1998  相似文献   

5.
The microaerophilic food‐borne pathogen Campylobacter jejuni uses complex cytochrome‐rich respiratory chains for growth and host colonisation. Cytochrome c biogenesis requires haem ligation to reduced apocytochrome cysteines, catalysed by the cytochrome c synthase, CcsBA. While ccsBA could not be deleted, we showed that the thiol reductase DsbD and the CcsX homologue Cj1207 are involved in, but not essential for, cytochromes c biogenesis. Mutant phenotypic analyses and biochemical studies with purified proteins revealed that the mono‐haem c‐type cytochromes Cj1153 (CccA) and Cj1020 (CccB) and the di‐haem Cj0037 (CccC) are electron donors to the cb‐oxidase (CcoNOQP), with CccC being more efficient than CccA. Remarkably, cccA deletion or site‐directed mutagenesis resulted in an almost complete loss of all other c‐type cytochromes. Cytochrome c structural and biogenesis genes were still transcribed in the cccA deletion mutant and the quinol oxidase genes (cioAB) were up‐regulated. Cytochrome c production could be rescued in this mutant by growth with exogenous dithiothreitol or L‐cysteine, suggesting that in the absence of CccA, apocytochrome c haem binding motifs become oxidised, preventing haem attachment. Our results identify CccA, the most abundant periplasmic c‐type cytochrome in C. jejuni, as a novel and unexpected protein required for cytochrome c biogenesis in this pathogen.  相似文献   

6.
Summary We have isolated twenty-six nuclear, singlegene cytochrome-deficient mutants of Neurospora crassa as an initial step toward the study of the structural components and regulatory mechanisms involved in the biogenesis of the mitochondrial cytochrome system. These mutants, together with two previously described mutants, cyt-1 and cyt-2, have been classified into six distinct groups on the basis of cytochrome phenotype: a) cytochrome aa 3 deficiency (due to mutations affecting loci designated cya); b) cytochrome b deficiency (cyb-1 locus); c) cytochrome b deficiency with a partial deficiency of cytochrome aa 3 (cyb-2 locus); d) deficiency of both cytochromes aa 3 and b (cyt loci); e) deficiency of both cytochromes aa 3 and c (cyt-2 locus); and f) partial deficiency of cytochromes aa 3 and c (cyt-12 locus).Four of seven mutations affecting cya loci have been mapped and are located on linkage groups I, II, V, and VI. It is not yet known whether these genes code for structural components of cytochrome oxidase or have a regulatory function that affects synthesis or assembly of the enzyme. The cyb-1 and cyb-2 genes are located on linkage groups V and VI, respectively, and appear to code for regulatory elements that control the biogenesis of cytochromes b and aa 3 . The positions of the cyt mutations that cause a simultaneous deficiency of cytochromes aa 3 and b are dispersed throughout the genome, except for two gene clusters on the left arm of linkage group I. Some of these mutants may be deficient in mitochondrial protein synthesis. Two mutations, cyt-2 and cyt-12, are located on linkage groups VI and II, respectively, and appear to affect genes that code for components of a regulatory system that controls the biogenesis of cytochromes aa 3 and c.  相似文献   

7.
We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix?) and “respiratory” nitrate reduction (Rnr?). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr? and Fix? phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multi-subunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.  相似文献   

8.
The biosynthesis of bacterial and plastidic c-type cytochromes includes several steps that occur post-translationally. In the case of bacterial cytochromes, the cytosolically synthesized pre-proteins are translocated across the cytoplasmic membrane, the pre-proteins are cleaved to their mature forms and heme is ligated to the processed apoprotein. Although heme attachment has not been studied extensively at the biochemical level, molecular genetic approaches suggest that the reaction generally occurs after translocation of the apoprotein to the periplasm. Recent studies with Bradyrhizobium japonicum and Rhodobacter capsulatus indicate that the process of heme attachment requires the function of a large number of genes. Mutation of these genes generates a pleiotropic deficiency in all c-type cytochromes, suggesting that the gene products participate in processes required for the biosynthesis of all c-type cytochromes. In eukaryotic cells, the biosynthesis of photosynthetic c-type cytochromes is somewhat more complex owing to the additional level of compartmentation. Nevertheless, the basic features of the pathway appear to be conserved. For instance, as is the case in bacteria, translocation and processing of the pre-proteins is not dependent on heme attachment. Genetic analysis suggests that the nuclear as well as the plastid genomes encode functions required for heme attachment, and that these genes function in the biosynthesis of the membrane-associated as well as the soluble c-type cytochrome of chloroplasts. A feature of cytochromes c biogenesis that appears to be conserved between chloroplasts and mitochondria is the sub-cellular location of the heme attachment reaction (p-side of the energy transducing membrane). Continued investigation of all three experimental systems (bacteria, chloroplasts, mitochondria) is likely to lead to a greater understanding of the biochemistry of cytochrome maturation as well as the more general problem of cofactor-protein association during the assembly of an energy transducing membrane.Abbreviations CCHL cytochrome c/heme lyase - CC1HL cytochrome cl/heme lyase - cyt cytochrome - EMS ethyl methane sulphonate - n-side electrochemically negative side of an energy transducing membrane - p-side electrochemically positive side of an energy transducing membrane - PhoA alkaline phosphatase (encoded by the phoA locus)  相似文献   

9.
A Rhizobium phaseoli cytochrome mutant, unable to oxidize N,N,N′,N′ -tetramethyl-p-phenylend(amine (TMPD), was isolated after Mu-dl (Kan lac) mutagenesis of the wild-type strain CE-3. Mutant strain CFN4202 had sixfold less haem-c but similar levels of b type, o and aa3 cytochromes than the wild-type strain. CFN402 strain also showed reduced NADH- and TMPD-oxidase activity than the wild-type strain. Succinate-oxidase activities were very similar. Western blot experiments, using antiserum against bovine c1 and c cytochromes, revealed that both proteins were present in CFN4202 membranes, suggesting a defect of haem binding to cytochrome c. Nodules formed by this strain in Phaseolus vulgaris did not contain bacteroids. These data suggest that the cytochrome c-aa3 chain or some other respiratory chain, containing c-type cytochromes in R. phaseoli, is essential for bacterial division during the early steps of the symbiotic interaction with the legume-host.  相似文献   

10.
    
We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix) and respiratory nitrate reduction (Rnr). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr and Fix phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multi-subunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.  相似文献   

11.
Cytochrome c heme lyases encoded by the Sinorhizobium meliloti cycHJKL operon are responsible for generating the covalent bond between the heme prosthetic group and apocytochromes c. The CycH protein with its presumably membrane-associated N-terminal and periplasmic C-terminal parts is thought to be responsible for binding apocytochrome and presenting it to the heme ligation machinery. We propose that these two modules of CycH play roles in different functions of the protein. The N-terminal 96 amino acids represent an active subdomain of the protein, which is able to complement the protoporphyrin IX (PPIX) accumulation phenotype of the cycH mutant strain AT342, suggesting that it is involved in the final steps of heme C biosynthesis. Furthermore, three tetratricopeptide (TPR) domains have been identified in the C-terminal periplasmic region of the CycH protein. TPR domains are known to mediate protein-protein interactions. Each of these CycH domains is absolutely required for protein function, since plasmid constructs carrying cycH genes with in-frame TPR deletions were not able to complement cycH mutants for their nitrate reductase (Rnr) and nitrogen-fixing (Fix) phenotypes. We also found that the 309-amino acid N-terminal portion of the CycH, which includes all the TPR domains, is able to mediate the assembly of the c-type cytochromes required for the Rnr+ phenotype. In contrast, only the full-length protein confers the ability to fix nitrogen.Communicated by A. Kondorosi  相似文献   

12.
Resonance Raman (RR) spectroscopy was used to investigate conformational characteristics of the hemes of several ferricytochromes of the cytochrome c 3 family, electron transfer proteins isolated from the periplasm and membranes of sulfate-reducing bacteria. Our analysis concentrated on the low-frequency region of the RR spectra, a fingerprint region that includes vibrations for heme-protein C–S bonds [ν(CaS)]. It has been proposed that these bonds are directly involved in the electron transfer process. The three groups of tetraheme cytochrome c 3 analyzed, namely Type I cytochrome c 3 (TpIc 3s), Type II cytochrome c 3 (TpIIc 3s) and Desulfomicrobium cytochromes c 3, display different frequency separations for the two ν(CaS) lines that are similar among members of each group. These spectral differences correlate with differences in protein structure observed among the three groups of cytochromes c 3. Two larger cytochromes of the cytochrome c 3 family display RR spectral characteristics for the ν(CaS) lines that are closer to TpIIc 3 than to TpIc 3. Two other multiheme cytochromes from Desulfovibrio that do not belong to the cytochrome c 3 family display ν(CaS) lines with reverse relative areas in comparison with the latter family. This RR study shows that the small differences in protein structure observed among these cytochrome c 3 correlate to differences on the heme–protein bonds, which are likely to have an impact upon the protein function, making RR spectroscopy a sensitive and useful tool for characterizing these cytochromes.  相似文献   

13.
An apo form of cytochrome C550 can be detected by immunoblotting cell-free extracts of a mutant of Paracoccus denitrificans that is deficient in c-type cytochromes. This apoprotein is found predominantly in the periplasm, the location of the holocytochrome in the wild-type organism, indicating that translocation of the polypeptide occurs in the absence of haem attachment. The polypeptide molecular weight, as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, is indistinguishable from that of the holoprotein and the chemically prepared apoprotein; this suggests that the N-terminal signal sequence is removed in the mutant as in the wild-type organism. In the presence of levulinic acid, an inhibitor of haem biosynthesis, apocytochrome c550 and aponitrite reductase (cytochrome cd1) accumulated in the periplasm of wild-type cells. Synthesis of these apoproteins was blocked by chloramphenicol. Thus in P. denitrificans the synthesis of these polypeptides is neither autoregulated nor regulated by the availability of haem. That the apoproteins appear in the periplasm argues against the possibility of polypeptide/haem co-transport from cytoplasm to periplasm. These observations are related to, and contrasted with, the biosynthesis of c-type cytochromes in eukaryotic cells.  相似文献   

14.
Artemisinin and its derivatives kill malaria parasites and inhibit the proliferation of cancer cells. In both processes, heme was shown to play a key role in artemisinin bioactivation. We found that artemisinin and clinical artemisinin derivatives are able to compensate for a mutation in the yeast Bcs1 protein, a key chaperon involved in biogenesis of the mitochondrial respiratory complex III. The equivalent Bcs1 variant causes an encephalopathy in human by affecting complex III assembly. We show that artemisinin derivatives decrease the content of mitochondrial cytochromes and disturb the maturation of the complex III cytochrome c1. This last effect is likely responsible for the compensation by decreasing the detrimental over-accumulation of the inactive pre-complex III observed in the bcs1 mutant. We further show that a fluorescent dihydroartemisinin probe rapidly accumulates in the mitochondrial network and targets cytochromes c and c1 in yeast, human cells and isolated mitochondria. In vitro this probe interacts with purified cytochrome c only under reducing conditions and we detect cytochrome c-dihydroartemisinin covalent adducts by mass spectrometry analyses. We propose that reduced mitochondrial c-type cytochromes act as both targets and mediators of artemisinin bioactivation in yeast and human cells.  相似文献   

15.
An Escherichia coli K-12 mutant, isolated on the basis of its inability to catalyze formate-dependent nitrite reduction, was characterized. The mutant was defective in the synthesis of all known c-type cytochromes during anaerobic growth. The mutation was localized by conjugation, transduction, and Southern blotting experiments to the dsbA gene at minute 87 on the E. coli chromosome and was complemented by the wild-type allele. Both DsbA and the recently described DipZ protein were shown to be essential for cytochrome c synthesis, suggesting that they act sequentially in a pathway for cytochrome c assembly in the E. coli periplasm. Received: 18 April 1995 / Accepted: 25 July 1995  相似文献   

16.
It has been found that the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21T produces a larger amount of soluble c-type cytochromes at pH 10.0 under air-limited condition than at pH 7.0 under high aeration. Cytochrome c-552 was confirmed as the major c-type cytochrome among three soluble c-type cytochromes in the strain. To understand the physiological function of cytochrome c-552, a P. alcaliphila AL15-21T cytochrome c-552 gene deletion mutant without a marker gene was constructed by electrotransformation adjusted in this study for the strain. The maximum specific growth rate and maximum cell turbidity of cells grown at pHs 7.0 and 10.0 under the high-aeration condition did not differ significantly between the wild-type and cytochrome c-552 deletion mutant strains. In the mutant grown at pH 10.0 under low-aeration condition, marked decreases in the maximum specific growth rate (40%) and maximum cell turbidity (25%) compared with the wild type were observed. On the other hand, the oxygen consumption rates of cell suspensions of the mutant obtained by the growth at pH 10 under low-aeration condition were slightly higher than that of the wild type. Considering the high electron-retaining ability of cytochrome c-552, the above observations could be accounted for by cytochrome c-552 acting as an electron sink in the periplasmic space. This may facilitate terminal oxidation in the respiratory system at high pH under air-limited conditions.  相似文献   

17.
18.
We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.  相似文献   

19.
In a spontaneous mutant (PYM1) of Bacillus cereus impaired in the synthesis of haem A, no haem-A-containing cytochromes were detected spectroscopically. The haem A deficiency was compensated by high levels of haem O and a CO-reactive cytochrome o in membranes; no other oxidases were detected. In contrast, the wild-type strain had considerable amounts of haem A and negligible levels of haem O. The mutant PYM1 exhibited normal colony morphology, growth, and sporulation in nonfermentable media, whereas on fermentable media, the mutant overproduced acid, which led to poor growth and inhibition of sporulation. External control of the pH of the medium in fermentable media allowed close-to-normal growth and massive sporulation of the mutant. The presence of membrane-bound cytochrome caa 3 -OII and aa 3 -II subunits in strain PYM1 was confirmed by Western blots and haem C staining (COII subunit). Western blotting also revealed that in contrast to the wild-type – strain PYM1 contained the membrane-bound subunits caa 3 -COI and aa 3 -I, but in low amounts. The effect of several respiratory inhibitors on the respiratory system of strain PYM1 suggested that the terminal oxidase is highly resistant to KCN and CO and that a c-type cytochrome might be involved in the electron transfer sequence to the putative cytochrome bo. Received: 21 June 1996 / Accepted: 9 October 1996  相似文献   

20.
A part of the gene encoding cbb 3-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c 4 and c 5. Thus, the A. vinelandii respiratory chain is shown to contain cbb 3-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.  相似文献   

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