Physiological function of soluble cytochrome <Emphasis Type="Italic">c</Emphasis>-552 from alkaliphilic <Emphasis Type="Italic">Pseudomonas alcaliphila</Emphasis> AL15-21<Superscript>T</Superscript>

It has been found that the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21^T 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 AL15-21^T 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.     

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb ₃-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb ₃-type oxidase is the major terminal oxidase under aerobic conditions while both cbb ₃-type and bd-type oxidases are involved in respiration at low-O₂ tensions. On the contrary, the low O₂-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa ₃-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa ₃-type oxidase in S. oneidensis MR-1 are discussed.     

Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12

 The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated. Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes. The Nrf⁺ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain. Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis. In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport. Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu⁺⁺ ions at concentrations above 5 mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected. These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E. coli periplasm. However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD). Received: 28 February 1996 / Accepted: 5 June 1996     

Characterization of the Escherichia coli CcmH protein reveals new insights into the redox pathway required for cytochrome c maturation

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     

High Yield of Methylophilus methylotrophus Cytochrome c″ by Coexpression with Cytochrome c Maturation Gene Cluster from Escherichia coli

Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c″ from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c″ isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS–PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and ¹H-NMR spectroscopy. The success in expressing the mature cytochrome c″ in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.     

Formation of several bacterial c-type cytochromes requires a novel membrane-anchored protein that faces the periplasm

We report here the discovery of a novel bacterial gene (cycH) whose product is involved in the biogenesis of most of the cellular cytochromes c. The cycH gene was detected in the course of characterizing a cytochrome oxidase-deficient Bradyrhizobium japonicum Tn5 mutant (strain CO×3) in which the transposon insertion disrupted cycH. Ali of the c-type cytochromes detectable in aerobically grown B. Japonicum wild-type cells were absent in the C0X3 mutant, with the exception of cytochrome c₁. A secondary phenotypic effect was the spectroscopic absence of the aa₃-type cytochrome c oxidase. The nucleotide sequence of the cloned wild-type cycH gene predicted a membrane-bound 369-amino-acid protein with an M_r of 39727. Results from studies on its membrane topology suggested that approximately 110 N-terminal amino acids are involved in anchoring the protein in the membrane, whereas the remaining two-thirds of the protein are exposed to the periplasm. We postulate that the CycH protein plays an essential role in an as yet unidentified periplasmic step in the biogenesis of holocytochromes c, except that of cytochrome c₁.     

Control of the syntheses of bacteriochlorophyll,c- and b-type cytochromes,and coproporphyrin III in Rhodopseudomonas sphaeroides mutant H5

Rhodopseudomonas sphaerodes mutant H5 lacking 5-aminolevulinic acid synthase was grown phototrophically in chemostat cultures limited by malate. Tetrapyrrole formation was limited by 5-aminolevulinic acid. With variation of dilution rates the cultures exhibited two regions of almost constant cell protein, dry weight and bacteriochlorophyll levels suggesting the formation of two physiological modifications of the strain. These modifications were further characterized by differences in the rates of 5-aminolevulinic acid consumption, the production of reserve material, the stoichiometries of 5-aminolevulinic acid consumption and bacteriochlorophyll or cytochrome production, specific bacteriochlorophyll and cytochrome contents as well as the ratio of bacteriochlorophyll protein complexes. In contrast, cellular levels of coproporphyrin II stayed almost constant over the entire range of dilution rates employed. Bacteriochlorophyll and b-type cytochrome cellular levels exhibited hyperbolic dependencies on the specific rate of 5-aminolevulinic acid consumption, and c-type cytochrome levels a signmoidal dependency. Bacteriochlorophyll cellular levels showed a biphasic dependency with half maximal saturations at 2.6 and 15.4 nmol of 5-aminolevulinic acid consumed per mg of protein and h, and maximal levels of 15.2 and 21 nmol bacteriochlorophyll per mg of protein. Cellular levels of c- and b-type cytochromes were half maximally saturated at 19.5 and 14.5 nmol 5-aminolevulinic acid consumed per mg protein and h while maximal levels were reached at 0.5 and 0.17 nmol of c- and b-type cytochromes, respectively, per mg of protein.The data suggest that within the cell bacteriochlorophyll as well as c- and b-type cytochrome units are assembled according to a defined pattern of kinetics characteristic of each group of compounds. Under otherwise constant external conditions the expression of the pattern is controlled by the rate of 5-aminolevulinic acid supply.     

Artefacts induced on <Emphasis Type="Italic">c</Emphasis>-type haem proteins by electrode surfaces

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met–His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c ₃ from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.     

Reactivity of the co-type and baa 3-type cytochrome c oxidases from Pseudomonas aeruginosa with different endogenous cytochromes c

The reactivity between different cytochromes c purified from Pseudomonas aeruginosa cells grown aerobically in the absence of nitrate and isolated cytochromes co and baa ₃ was determined. The P. aeruginosa cytochrome co reacted most rapidly with the membrane-bound cytochrome c-551 among three c-type cytochromes analyzed, whereas the cytochrome baa ₃ reacted best with the membrane-bound cytochrome c-555. The results indicated that two terminal electron transfer systems are present in aerobic P. aeruginosa: one contains the cytochrome c-551 and cytochrome co, and the other contains the cytochrome c-555 and cytochrome baa ₃.     

Expression of a Tetraheme Protein, Desulfovibrio vulgaris Miyazaki F Cytochrome c3, in Shewanella oneidensis MR-1

Cytochrome c₃ from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome c₃ gene from D. vulgaris Miyazaki F was used for transformation of S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine residue at the N terminus.     

Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b ₅₅₄ (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b ₅₅₆ (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth. Received: 21 August 1996 / Accepted: 24 October 1996     

Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12

The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated. Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes. The Nrf⁺ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain. Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis. In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport. Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu⁺⁺ ions at concentrations above 5?mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected. These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E. coli periplasm. However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD).     

The Two CcdA Proteins of Bacillus anthracis Differentially Affect Virulence Gene Expression and Sporulation

The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation, while the closely related organism Bacillus subtilis carries only one copy of ccdA. To investigate the roles of the two ccdA gene copies in B. anthracis, strains were constructed without each ccdA gene, and one strain was constructed without both copies simultaneously. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. Complementation and expression analyses indicate that ccdA2 encodes the primary CcdA in B. anthracis, active in all three pathways. While CcdA1 retains activity in cytochrome c maturation and virulence control, it has completely lost its activity in the sporulation pathway. In support of this finding, expression of ccdA1 is strongly reduced when cells are grown under sporulation-inducing conditions. When the activities of CcdA1 and CcdA2 were analyzed in B. subtilis, neither protein retained activity in cytochrome c maturation, but CcdA2 could still function in sporulation. These observations reveal the complexities of thiol-disulfide oxidoreductase function in pathways relevant to virulence and physiology.     

The biosynthesis of bacterial and plastidic c-type cytochromes

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)     

The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum. 1. A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells

Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined. Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with E _m,7 of +180, +72 and –5 mV (561–575 nm), and +244 and +27 mV (551–540 nm), respectively. Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with E _m,7 of +60 and –45 mV and +290, +250, +135, –20 and –105 mV, respectively. In addition to haems of the b- and c-type, two haems of the a-type (E _m,7 of +325 and +175 mV) were present only in cells grown in the dark. Four soluble cytochromes of the c type, but not cytochrome c ₂, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically. Inhibitory studies showed that 85–90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively). These results taken together were interpreted to show that the oxidative electron transport chain of Rsp. salinarum is linear, leading to a membrane-bound oxidase of the aa ₃ type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes. These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c ₂ or a branched respiratory chain. Received: 25 February 1997 / Accepted: 20 May 1997     

Cloning and sequence analysis of cycH gene from Paracoccus denitrificans: the cycH gene product n required for assembly of all c-type cytochromes, including cytochrome c1

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.