Anaerobic, Nitrate-Dependent Oxidation of U(IV) Oxide Minerals by the Chemolithoautotrophic Bacterium Thiobacillus denitrificans

Under anaerobic conditions and at circumneutral pH, cells of the widely distributed, obligate chemolithoautotrophic bacterium Thiobacillus denitrificans oxidatively dissolved synthetic and biogenic U(IV) oxides (uraninite) in nitrate-dependent fashion: U(IV) oxidation required the presence of nitrate and was strongly correlated with nitrate consumption. This is the first report of anaerobic U(IV) oxidation by an autotrophic bacterium.     

C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Development of a Genetic System for the Chemolithoautotrophic Bacterium Thiobacillus denitrificans

Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification to sulfur compound oxidation; to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV); and to oxidize mineral electron donors. Recent analysis of its genome sequence also revealed the presence of genes encoding two [NiFe]hydrogenases, whose role in metabolism is unclear, as the sequenced strain does not appear to be able to grow on hydrogen as a sole electron donor under denitrifying conditions. In this study, we report the development of a genetic system for T. denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. The antibiotic sensitivity of T. denitrificans was characterized, and a procedure for transformation with foreign DNA by electroporation was established. Insertion mutations were generated by in vitro transposition, the mutated genes were amplified by the PCR, and the amplicons were introduced into T. denitrificans by electroporation. The IncP plasmid pRR10 was found to be a useful vector for complementation. The effectiveness of the genetic system was demonstrated with the hynL gene, which encodes the large subunit of a [NiFe]hydrogenase. Interruption of hynL in a hynL::kan mutant resulted in a 75% decrease in specific hydrogenase activity relative to the wild type, whereas complementation of the hynL mutation resulted in activity that was 50% greater than that of the wild type. The availability of a genetic system in T. denitrificans will facilitate our understanding of the genetics and biochemistry underlying its unusual metabolism.     

Formation of active nitrite reductase (cytochromecd 1) in the strainParacoccus denitrificans HUUG25

Strain HUUG25 ofParacoccus denitrificans has been frequently thought to be devoid of allc-type cytochromes. We show here by means of enzymological and immunological techniques that the mutant synthesizes active nitrite reductase (cytochromecd ₁) upon prolonged exposure to microoxic conditions. The synthesis occurred faster in the presence of exogenous hemin. The time pattern of 5-aminolevulinate synthase activity was also altered by the mutation. These findings suggest a defective regulation of heme supply to the site of nitrite reductase assembly in the periplasm.     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

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.     

A novel identified Pseudomonas aeruginosa,which exhibited nitrate- and nitrite-dependent methane oxidation abilities,could alleviate the disadvantages caused by nitrate supplementation in rumen fluid fermentation

After the occurrence of nitrate-dependent anaerobic methane oxidation (AMO) in rumen fluid culture was proved, the organisms that perform the denitrifying anaerobic methane oxidizing (DAMO) process in the rumen of dairy goat were investigated by establishing two enrichment culture systems, which were supplied with methane as the sole carbon source and NaNO₃ or NaNO₂ as the electron acceptor. Several Operational Taxonomic Units (OTU) belonging to Proteobacteria became dominant in the two enrichment systems. The identified Pseudomonas aeruginosa, which was isolated from the NaNO₂ enrichment system, could individually perform a whole denitrifying anaerobic methane oxidizing process. Further in vitro rumen fermentation showed that supplementation with the isolated P. aeruginosa could reduce methane emissions, alleviate the nitrite accumulation and prevent the decrease in propionic acid product caused by nitrate supplementation.     

Apo forms of cytochrome C550 and cytochrome cd1 are translocated to the periplasm of Paracoccus denitrificans in the absence of haem incorporation caused by either mutation or inhibition of haem synthesis

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.     

biochemical reaction mechanisms in sulfur oxidation by chemosynthetic bacteria

Summary Aspects of the biochemistry of the oxidation of inorganic sulfur compounds are discussed in thiobacilli but chiefly inThiobacillus denitrificans. Almost all of the thiobacilli (e.g. T. denitrificans, T. neapolitanus, T. novellus, andThiobacillus A ₂) were capable of producing approximately 7.5 moles of sulfuric acid aerobically from 3.75 moles of thiosulfate per gram of cellular protein per hr. By far the most prolific producer of sulfuric acid (or sulfates) from the anaerobic thiosulfate oxidation with nitrates wasT. denitrificans which was capable of producing 15 moles of sulfates from 7.5 moles of thiosulfate with concomitant reduction of 12 moles of nitrate resulting in the evolution of 6 moles of nitrogen gas/g protein/hr. The oxidation of sulfide was mediated by the flavo-protein system and cytochromes ofb, c, o, anda-type. This process was sensitive to flavoprotein inhibitors, antimycin A, and cyanide. The aerobic thiosulfate oxidation on the other hand involved cytochromec : O₂ oxidoreductase region of the electron transport chain and was sensitive to cyanide only. The anaerobic oxidation of thiosulfate byT. denitrificans, however, was severely inhibited by the flavoprotein inhibitors because of the splitting of the thiosulfate molecule into the sulfide and sulfite moieties produced by the thiosulfate-reductase. Accumulation of tetrathionate and to a small extent trithionate and pentathionate occurred during anaerobic growth ofT. denitrificans. These polythionates were subsequently oxidized to sulfate with the concomitant reduction of nitrate to N₂. Intact cell suspensions catalyzed the complete oxidation of sulfide, thiosulfate, tetrathionate, and sulfite to sulfate with the stoichiometric reduction of nitrate, nitrite, nitric oxide, and nitrous oxide to nitrogen gas thus indicating that NO₂ ⁻, NO, and N₂O are the possible intermediates in the denitrification of nitrate. This process was mediated by the cytochrome electron transport chain and was sensitive to the electron transfer inhibitors. The oxidation of sulfite involved cytochrome-linked sulfite oxidase as well as the APS-reductase pathways. The latter was absent inT. novellus andThiobacillus A ₂. In all of the thiobacilli the inner as well as the outer sulfur atoms of thiosulfate were oxidized at approximately the same rate by intact cells. The sulfide oxidation occurred in two stages: (a) a cellular-membrane-associated initial and rapid oxidation reaction which was dependent upon sulfide concentration, and (b) a slower oxidation reaction stage catalyzed by the cellfree extracts, probably involving polysulfides. InT. novellus andT. neapolitanus the oxidation of inorganic sulfur compounds is coupled to energy generation through oxidative phosphorylation, however, the reduction of pyridine nucleotides by sulfur compounds involved an energy-linked reversal of electron transfer. Paper read at the Symposium on the Sulphur Cycle, Wageningen, May 1974. Summary already inserted on p. 189 of the present volume.     

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

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.     

Interaction between uranium and the cytochrome c 3 of Desulfovibrio desulfuricans strain G20

Cytochrome c₃ 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 c₃ 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 c₃ (cycA) to lacZ. Instead, cytochrome c₃ 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 c₃ with U(IV) was interpreted to be non-specific, since pure cytochrome c₃ adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe₂O₃), and commercially available U(IV) oxide.An erratum to this article can be found at     

The role ofc-type cytochromes in the terminal respiratory chain of the methylotrophic bacteriumMethylophilus methylotrophus

Whole cells of the methylotrophic bacteriumMethylophilus methylotrophus cultured under methanol-limited conditions contain approximately equal amounts of two majorc-type cytochromes,c _H andc _L. Virtually all of the cytochromec _H, and over one-third of the cytochromec _L, are loosely attached to the periplasmic surface of the respiratory membrane whence they can be released by sonication or by washing cells in ethylenediaminetetraacetate (EDTA). The latter causes inhibition of methanol oxidase activity and stimulation of ascorbate-N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) oxidase activity, neither of which effects are reversible by divalent metal ions. Kinetic analyses indicate that ascorbate-TMPD is oxidised via two routes, viz. a slow low-affinity pathway involving loosely membrane-boundc-type cytochromes plus cytochrome oxidaseaa ₃, and a faster higher-affinity pathway involving the firmly membrane-bound cytochrome oxidasec _L o complex; the former route predominates in the presence of divalent metal ions, and the latter route after exposure to EDTA. These and other results are discussed in terms of the spatial organisation of the terminal respiratory chain, and of the role ofc-type cytochromes in the oxidation of methanol and ascorbate-TMPD.Abbreviations EDTA Enthylenediaminetetraacetate - PMS Phenazinemethosulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - SDS Sodium dodecylsulphate - I₅₀ Concentration of inhibitor required to give 50% inhibition of enzyme activity - PQQ Pyrroloquinoline quinone     

Spectroscopic and potentiometric characterization of cytochromes in two Sporomusa species and their expression during growth on selected substrates

The homoacetogenic bacteria Sporomusa ovata and Sporomusa sphaeroides were grown on betaine, betaine + formate, and acetoin in the absence of carbon dioxide, and the formation of membrane-bound cytochromes was determined. In S. sphaeroides, the growth substrate had little influence on the expression of cytochromes. In contrast, membranes from betaine-or acetoin-grown S. ovata cells had an 11-or 3-fold higher cytochrome b content than cells grown on betaine + formate. The cytochrome c content was reduced below the detection level after growth on the latter two substrates. The cytochromes in the membranes of S. sphaeroides and S. ovata were characterized by low-temperature difference spectroscopy, hemochrome difference spectroscopy, and redox potentiometry. Membranes of S. ovata were shown to contain two b-type cytochromes with E_m,7=-153±10 mV and E_m,7=-226±14 mV and two c-type cytochromes with E_m,7=-86±6 mV and E_m,7=-265±10 mV. In S. sphaeroides also two b-type cytochromes with E_m,7=-165±7 mV and E_m,7=-241±2 mV and two c-type cytochromes with E_m,7=-101±4 mV and E_{m, 8.5}=-338±9 mV could be distinguished. Cell extracts of S. sphaeroides were shown to contain all the enzymes of the acetyl-CoA (Wood) pathway. The degradation pathways of the substrates tested and the possible role of the cytochromes are discussed.Abbreviations E_m,7 midpoint potential at pH 7 and 25°C - H₄F tetrahydrofolate     

The genome sequence of the obligately chemolithoautotrophic, facultatively anaerobic bacterium Thiobacillus denitrificans

The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, beta-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by beta-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.     

Isolation,sequencing and mutational analysis of a gene cluster involved in nitrite reduction inParacoccus denitrificans

By using the gene encoding the C-terminal part of thecd ₁-type nitrite reductase ofPseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene fromParacoccus denitrificans was isolated. This gene,nirS, codes for a mature protein of 63144 Da having high homology withcd ₁-type nitrite reductases from other bacteria. Directly downstream fromnirS, three othernir genes were found in the ordernirECF. The organization of thenir gene cluster inPa. denitrificans is different from the organization ofnir clusters in some Pseudomonads.nirE has high homology with a S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (uro'gen III methylase). This methylase is most likely involved in the hemed ₁ biosynthesis inPa. denitrificans. The third gene,nirC, codes for a small cytochromec of 9.3 kDa having high homology with cytochromec _55X ofPs. stutzeri ZoBell. The 4th gene,nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity.nirS mutants lack thecd ₁-type nitrite reductase whilenirE, nirC andnirF mutants produce a small amount ofcd ₁-type nitrite reductase, inactive due to the absence of hemed ₁. Upstream from thenirS gene the start of a gene was identified which has limited homology withnosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene andnirS.Abbreviations SDS sodium dodecyl sulfate - NBT nitroblue tetrazolium - PAGE polyacrylamide gel electrophoresis     

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

Impacts of Shewanella oneidensis c‐type cytochromes on aerobic and anaerobic respiration

Shewanella are renowned for their ability to utilize a wide range of electron acceptors (EA) for respiration, which has been partially accredited to the presence of a large number of the c-type cytochromes. To investigate the involvement of c-type cytochrome proteins in aerobic and anaerobic respiration of Shewanella oneidensis Mr -1, 36 in-frame deletion mutants, among possible 41 predicted, c-type cytochrome genes were obtained. The potential involvement of each individual c-type cytochrome in the reduction of a variety of EAs was assessed individually as well as in competition experiments. While results on the well-studied c-type cytochromes CymA(SO4591) and MtrC(SO1778) were consistent with previous findings, collective observations were very interesting: the responses of S. oneidensis Mr -1 to low and highly toxic metals appeared to be significantly different; CcoO, CcoP and PetC, proteins involved in aerobic respiration in various organisms, played critical roles in both aerobic and anaerobic respiration with highly toxic metals as EA. In addition, these studies also suggested that an uncharacterized c-type cytochrome (SO4047) may be important to both aerobiosis and anaerobiosis.     

Identification and analysis of the Shewanella oneidensis major oxygen-independent coproporphyrinogen III oxidase gene

Shewanella oneidenesis MR-1 is a facultative anaerobe that can use a large number of electron acceptors including metal oxides. During anaerobic respiration, S. oneidensis MR-1 synthesizes a large number of c cytochromes that give the organism its characteristic orange color. Using a modified mariner transposon, a number of S. oneidensis mutants deficient in anaerobic respiration were generated. One mutant, BG163, exhibited reduced pigmentation and was deficient in c cytochromes normally synthesized under anaerobic condition. The deficiencies in BG163 were due to insertional inactivation of hemN1, which exhibits a high degree of similarity to genes encoding anaerobic coproporphyrinogen III oxidases that are involved in heme biosynthesis. The ability of BG163 to synthesize c cytochromes under anaerobic conditions, and to grow anaerobically with different electron acceptors was restored by the introduction of hemN1 on a plasmid. Complementation of the mutant was also achieved by the addition of hemin to the growth medium. The genome sequence of S. oneidensis contains three putative anaerobic coproporphyrinogen III oxidase genes. The protein encoded by hemN1 appears to be the major enzyme that is involved in anaerobic heme synthesis of S. oneidensis. The other two putative anaerobic coproporphyrinogen III oxidase genes may play a minor role in this process.     

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)