Evaluation of mtr cluster expression in Shewanella RCRI7 during uranium removal.

In recent years, bioremediation is considered as an efficient method to remove the pollutants from the industrial wastewater. In this study, quantitative gene expressions (Real-time RT-PCR) of mtr gene cluster (mtrA, mtrB, mtrC, mtrD, mtrE, mtrF and omcA) in five different uranium concentrations (0.1, 0.25, 0.5, 1 and 2 mM) were performed with ICP and microscopic live cell counting analysis under anaerobic condition, by Shewanella RCRI7 as a native bacterium. The results indicated that the amount of uranium removal and live-cell counting were decreased in the higher uranium concentrations (1 and 2 mM), due to the uranium toxicity, suggesting 0.5 mM as the optimum uranium concentration for Shewanella RCRI7 resistance. The expression of mtrCED and omcA genes presented increasing trend in the lower uranium concentrations (0.1, 0.25 and 0.5 mM) and a decreasing trend in 1 and 2 mM, while mtrABF, presented an inverse pattern, proving the alternative role of mtrF for mtrC and omcA, as the substantial multiheme cytochromes in Extracellular Electron Transfer (EET) pathway. These data are a proof of these gene vital roles in the EET pathway, proposing them for genetic engineering toward EET optimization, as the certain pathway in heavy metal bioremediation process.

Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive.

COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.

Mitochondrial Diseases: Shortcuts to Therapies and Therapeutic Shortcuts.

In this issue of Molecular Cell, Barrow et al. (2016) use two complementary approaches-one an assessment of a chemical library, and the other a genome-wide CRISPR screen-that both identify bromodomain-containing protein 4 (Brd4) as a therapeutic target for mtDNA diseases affecting complex I.

Bromodomain Inhibitors Correct Bioenergetic Deficiency Caused by Mitochondrial Disease Complex I Mutations.

Mitochondrial diseases comprise a heterogeneous group of genetically inherited disorders that cause failures in energetic and metabolic function. Boosting residual oxidative phosphorylation (OXPHOS) activity can partially correct these failures. Herein, using a high-throughput chemical screen, we identified the bromodomain inhibitor I-BET 525762A as one of the top hits that increases COX5a protein levels in complex I (CI) mutant cybrid cells. In parallel, bromodomain-containing protein 4 (BRD4), a target of I-BET 525762A, was identified using a genome-wide CRISPR screen to search for genes whose loss of function rescues death of CI-impaired cybrids grown under conditions requiring OXPHOS activity for survival. We show that I-BET525762A or loss of BRD4 remodeled the mitochondrial proteome to increase the levels and activity of OXPHOS protein complexes, leading to rescue of the bioenergetic defects and cell death caused by mutations or chemical inhibition of CI. These studies show that BRD4 inhibition may have therapeutic implications for the treatment of mitochondrial diseases.

Thiosulfate dehydrogenase: a widespread unusual acidophilic c-type cytochrome.

In this work we identified the gene for the tetrathionate-forming thiosulfate dehydrogenase (TsdA) from the purple sulfur bacterium Allochromatium vinosum by sequence analysis and reverse genetics. The recombinant protein produced in Escherichia coli is a periplasmic, monomeric 25.8 kDa dihaem cytochrome c with an enzyme activity optimum at pH 4. UV-visible and electron paramagnetic resonance spectroscopy indicate methionine (strictly conserved M(222) or M(236)) and cysteine (C(123) ) as probable sixth distal axial ligands of the two haem irons in TsdA. These results place TsdA in the group of c-type cytochromes with an unusual axial histidine-cysteine coordination of the haem iron. These proteins appear to play a pivotal role in sulfur-based energy metabolism. Exchange of C(123) to glycine rendered thiosulfate dehydrogenase inactive, proving the importance of this residue for catalysis. TsdA homologues are present in α-, ß-, δ-, γ- and ε-Proteobacteria. Three of these were produced in E. coli and exhibited the expected enzymatic activity. The widespread occurrence of tsdA agrees with reports of tetrathionate formation not only by specialized sulfur oxidizers but also by many chemoorganoheterotrophs that use thiosulfate as a supplemental but not as the sole energy source.

Identification and characterization of UndAHRCR-6, an outer membrane endecaheme c-type cytochrome of Shewanella sp. strain HRCR-6.

UndA(HRCR-6) was identified from the metal-reducing bacterium Shewanella sp. strain HRCR-6. Both in vivo and in vitro characterization results indicate that UndA(HRCR-6) is an outer membrane endecaheme c-type cytochrome and probably has a key functional role in the extracellular reduction of iron [Fe(III)] oxides and uranium [U(VI)] by Shewanella sp. HRCR-6.

Importance of c-Type cytochromes for U(VI) reduction by Geobacter sulfurreducens.

BACKGROUND: In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated. RESULTS: The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50-60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24-30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III). CONCLUSION: This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.

Interaction between uranium and the cytochrome c3 of Desulfovibrio desulfuricans strain G20.

Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide.

Uranium reduction by Desulfovibrio desulfuricans strain G20 and a cytochrome c3 mutant.

Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.

Cytochrome complex essential for photosynthetic oxidation of both thiosulfate and sulfide in Rhodovulum sulfidophilum.

Many photosynthetic bacteria use inorganic sulfur compounds as electron donors for carbon dioxide fixation. A thiosulfate-induced cytochrome c has been purified from the photosynthetic alpha-proteobacterium Rhodovulum sulfidophilum. This cytochrome c(551) is a heterodimer of a diheme 30-kDa SoxA subunit and a monoheme 15-kDa SoxX subunit. The cytochrome c(551) structural genes are part of an 11-gene sox locus. Sequence analysis suggests that the ligands to the heme iron in SoxX are a methionine and a histidine, while both SoxA hemes are predicted to have unusual cysteine-plus-histidine coordination. A soxA mutant strain is unable to grow photoautotrophically on or oxidize either thiosulfate or sulfide. Cytochrome c(551) is thus essential for the metabolism of both these sulfur species. Periplasmic extracts of wild-type R. sulfidophilum exhibit thiosulfate:cytochrome c oxidoreductase activity. However, such activity can only be measured for a soxA mutant strain if the periplasmic extract is supplemented with purified cytochrome c(551). Gene clusters similar to the R. sulfidophilum sox locus can be found in the genome of a green sulfur bacterium and in phylogenetically diverse nonphotosynthetic autotrophs.

Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts.

Physiological and pathological Ca2+ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+ into mitochondria. Mobilization of Ca2+ stores with bradykinin (BK) increased the cytosolic Ca2+ concentration ([Ca2+]c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca2(+)-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca2+ concentration ([Ca2+]mt) of 2.0 +/- 0.2 microM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca2+]mt increase, although it did not affect the [Ca2+]c transient. Basal [Ca2+]c and [Ca2+]mt in control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 +/- 1% in normal cells to 19 +/- 2% in the PDH-deficient fibroblasts. BK-elicited [Ca2+]mt transients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca2+. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca2+.

The Paracoccus denitrificans ccmA, B and C genes: cloning and sequencing, and analysis of the potential of their products to form a haem or apo- c-type cytochrome transporter.

Two c-type cytochrome deficient mutants of Paracoccus denitrificans, HN49 and HN53, were isolated by Tn5 mutagenesis and screening for failure to oxidize dimethylphenylenediamine (the Nadi test). Both were completely deficient in c-type cytochromes. Genomic DNA flanking the site of Tn5 insertion in HN53 was cloned by marked rescue and a 3.1 kb region sequenced. Three of the genes, designated ccmA, ccmB and ccmC, present in this region are proposed to encode the components of a membrane transporter of the ABC-(ATP-binding cassette) superfamily, which is similar to a group of transporters postulated to translocate either haem or apocytochromes c. The Tn5 elements in HN49 and HN53 shown to be inserted in ccmB and ccmA, respectively. Sequence analysis suggested that both CcmB and CcmC have the potential to interact with CcmA and thus that the three gene products probably associate to form a complex with (CcmA)2-CcmB-CcmC stoichiometry; it is also indicated a lack of similarity between CcmB and CcmC and the membrane-integral components of transporters mediating uptake of haem or other iron complexes. Supplementation of growth media with haem did not stimulate c-type cytochrome formation in HN49 or HN53, although it elevated levels of soluble haemoproteins and membrane-bound cytochromes b, suggesting that exogenous haem can traverse both outer and inner membranes of P. denitrificans. HN49 and HN53 accumulated apocytochrome C550 to much lower levels than other c-type cytochrome deficient mutants of P. denitrificans but expression and translocation of an apocytochrome C550-alkaline phosphatase fusion protein and apocytochrome cd1 were unaffected in HN53. The results suggest that the substrate for the putative CcmABC-transporter is probably neither haem nor c-type apocytochromes.

Rhizobium phaseoli cytochrome c-deficient mutant induces empty nodules on Phaseolus vulgaris L.

A Rhizobium phaseoli cytochrome mutant, unable to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine (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. CFN4202 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.

Mapping electrostatic interactions in macromolecular associations.

In the association of electron transfer proteins, electrostatics has been proposed to play a role in maintaining the stability and specificity of the biomolecular complexes formed. An excellent model system is the interaction between mammalian cytochrome b5 and cytochrome c, in which the X-ray structures of the individual components reveal a complementary asymmetry of charges surrounding their respective redox centers. Determining the exact extent of the electrostatic interactions and identifying the specific residues involved in the formation of the electron transfer complex has proved more elusive. We report herein the utilization of high-pressure techniques, together with site-directed mutagenesis, to provide a map of the interaction domains in biomolecular complex formation. The application of high pressure disrupts macromolecular associations since dissociation of the complex results in a decreased volume of the system due to the solvation of charges that had been previously sequestered in the interface region and force solvation of hydrophobic surfaces. Site-directed mutagenesis of a totally synthetic gene for rat liver cytochrome b5, which expresses this mammalian protein in Escherichia coli as a hemecontaining soluble component, was used to selectively alter negatively charged residues of cytochrome b5 to neutral amide side-chains. We have demonstrated that the interaction domain of cytochrome b5 with cytochrome c can be mapped from a comparison of dissociation volumes of these modified cytochrome b5-cytochrome c complexes with the native complex. Using these techniques we can specifically investigate the role of particular residues in the equilibrium association of these two electron transfer proteins. Single-point mutations in the interaction domain give nearly identical effects on the measured dissociation volumes, yet removal of acidic residues outside the recognition surface yield volumes similar to wild-type protein. Multiple mutations in the proposed protein-protein interaction site are found to allow greater solvent-accessibility of the interface as reflected in a diminution in the volume changes on subsequent charge removal. This is indicative that the interprotein salt-bridges in this complex provide a mechanism for a greater exclusion of solvent from the interfacial domain of the complex, resulting in a more stable association.

Isolation and characterization of a complementary DNA clone for an algal pre-apoplastocyanin.

We have isolated a cDNA clone for the Chlamydomonas reinhardtii pre-apoplastocyanin. The sequence contains codons for the complete pre-protein including a two-domain, lumen-targeting transit sequence and the mature apoprotein. The transit sequence (47 amino acids) is the shortest one described for chloroplast lumenal proteins, and like other C. reinhardtii lumen-targeting transit sequences appears to lack an uncharged amino-terminal domain usually present in plant lumen-directing sequences. The mature protein is deduced to be 98 amino acids in length and shows highest primary sequence similarity (74-76% identity) to other unicellular algal plastocyanins. Southern hybridization analysis of C. reinhardtii genomic DNA indicates the presence of a single nuclear gene, as is the case for all other plastocyanin genes characterized to date, although the algal gene might be interrupted. Codon usage in this gene reflects the high GC content of C. reinhardtii nuclear DNA, but is more highly biased than that found in the C. reinhardtii copper-repressible gene for the functionally equivalent pre-apocytochrome c552 (perhaps contributing to the more efficient synthesis in vivo of plastocyanin over cytochrome c552). The deduced physical properties of this plastocyanin are compared to those of the C. reinhardtii plastidic cytochrome c552.

The Cu(II)-repressible plastidic cytochrome c. Cloning and sequence of a complementary DNA for the pre-apoprotein.

We have cloned a complementary DNA for pre-apocytochrome c-552 from Chlamydomonas reinhardtii. The deduced sequence of the mature protein shows high homology to those of cytochromes c-553 from cyanobacteria. Its homology to mitochondrial cytochrome c or bacterial photosynthetic cytochrome c2 is lower and appears to be concentrated in sequences around amino acids involved in the interaction with heme. With respect to primary sequence, the "transit sequence" for cytochrome c-552 appears to show no homology to other transit sequences for nuclear encoded chloroplast proteins. However, based on analogy to transit sequences for other proteins (Daldal, F., Cheng, S., Applebaum, J., Davidson, E., and Prince, R. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2012-2016; Goldschmidt-Clermont, M., and Rahire, M. (1986) J. Mol. Biol. 191, 421-432; Smeekens, S., de Groot, M., van Binsbergen, J., and Weisbeek, P. (1986) Cell 46, 365-375) the transit sequence of cytochrome c-552 can be divided into envelope-traversing and thylakoid-traversing domains. Cytochrome c-552 appears to encoded by a single nuclear gene in C. reinhardtii. The gene is expressed exclusively in Cu(II)-deficient cells.