Cornelius G. Friedrich

Oxidation of Reduced Inorganic Sulfur Compounds by Bacteria: Emergence of a Common Mechanism?

Biological oxidation of hydrogen sulfide to sulfate is one of the major reactions of the global sulfur cycle. Reduced inorganic sulfur compounds (referred to below as sulfur) are exclusively oxidized by prokaryotes, and sulfate is the major oxidation product. Sulfur oxidation in members of the

Novel Genes of the sox Gene Cluster, Mutagenesis of the Flavoprotein SoxF, and Evidence for a General Sulfur-Oxidizing System in Paracoccus pantotrophus GB17

The novel genes soxFGH were identified, completing the sox gene cluster of Paracoccus pantotrophus coding for enzymes involved in lithotrophic sulfur oxidation. The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction. soxF coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif. SoxF was 37% identical to the flavoprotein FccB of flavocytochrome c sulfide dehydrogenase of Allochromatium vinosum. The mature SoxF (42,832 Da) contained 0.74 mol of flavin adenine dinucleotide per mol. soxG coded for a novel protein of 303 amino acids with a signal peptide containing a twin-arginine motif. The mature SoxG (29,657 Da) contained two zinc binding motifs and 0.90 atom of zinc per subunit of the homodimer. soxH coded for a periplasmic protein of 317 amino acids with a double-arginine signal peptide. The mature SoxH (32,317 Da) contained two metal binding motifs and 0.29 atom of zinc and 0.20 atom of copper per subunit of the homodimer. SoxXA, SoxYZ, SoxB, and SoxCD (C. G. Friedrich, A. Quentmeier, F. Bardischewsky, D. Rother, R. Kraft, S. Kostka, and H. Prinz, J. Bacteriol. 182:4476-4487, 2000) reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome c reduction, and this system is the first described for oxidizing different inorganic sulfur compounds. SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation. From use of a homogenote mutant with an in-frame deletion in soxF and complementation analysis, it was evident that the soxFGH gene products were not required for lithotrophic growth with thiosulfate.

Formation of Enzymes of Autotrophic Metabolism During Heterotrophic Growth of Alcaligenes eutrophus

SUMMARY: Alcaligenes eutrophus strain H16 formed key enzymes of autotrophic metabolism during heterotrophic growth. The formation of the soluble and membrane-bound hydrogenases, ribulose-5-phosphate kinase and ribulosebisphosphate carboxylase were investigated. In addition, selected enzymes shared by autotrophic and heterotrophic carbon metabolism were examined. Key enzymes of autotrophic metabolism were not detected during exponential growth on succinate, pyruvate or acetate, but were found at intermediate activities in cells grown on fructose, gluconate or citrate. Growth with succinate at a suboptimal pH of 7.7 resulted in a decreased growth rate and a marked increase of enzyme activities. Oxygen-limited growth with succinate also led to a derepression of the synthesis of the hydrogenases and the key enzymes of the Calvin cycle. During growth on glycerol or formate, the activities of these enzymes were comparable with those found under autotrophic conditions with H2 and CO2. The results indicate that both the hydrogenases and the key enzymes of the Calvin cycle were formed under conditions of limited availability of energy. Molecular hydrogen was not required for the formation of the hydrogenases. The regulation of the gluconeogenetic enzymes, common to both autotrophic and heterotrophic carbon metabolism, was more balanced. An increase of enzyme activities was observed under autotrophic conditions, in accord with the physiological role of these enzymes under autotrophic and heterotrophic growth conditions.

Formate and Oxalate Metabolism in Alcaligenes eutrophus

Summary: Alcaligenes eutrophus strain H16 when grown on formate or oxalate as the sole source of carbon and energy had doubling times between 3.5 and 4.5 h. The respective molar growth yields (Y m) were 2.35 and 3.9. During growth on formate or oxalate both a soluble and a membrane-bound formate dehydrogenase were formed. The key enzymes of autotrophic CO2 fixation, ribulose-5-phosphate kinase and ribulosebisphosphate carboxylase, were formed during growth on formate but not on oxalate. Oxalate induced the synthesis of the enzymes of the glycerate pathway. Mutants impaired in autotrophic CO2 fixation but unaffected in the synthesis of the formate dehydrogenases lost their ability to grow on formate but not to grow on oxalate, giving further evidence that formate was assimilated via CO2.

Evidence for two pathways of thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus)

Abstract. The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt. Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism. Here we report the cloning and sequencing of the soxBCD genes from S. novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus. These findings suggest two distinct pathways for thiosulfate oxidation in S. novella. The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis. The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose. In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present. The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB.

Unusual FTIR and EPR properties of the H2-activating site of the cytoplasmic NAD-reducing hydrogenase from Ralstonia eutropha.

Soluble NAD‐reducing [NiFe]‐hydrogenase (SH) from Ralstonia eutropha (formerly Alcaligenes eutrophus) has an infrared spectrum with one strong band at 1956 cm−1 and four weak bands at 2098, 2088, 2081 and 2071 cm−1 in the 2150–1850 cm−1 spectral region. Other [NiFe]‐hydrogenases only show one strong and two weak bands in this region, attributable to the NiFe(CN)2(CO) active site. The position of these three bands is highly sensitive to redox changes of the active site. In contrast, reduction of the SH resulted in a shift to lower frequencies of the 2098 cm−1 band only. These and other properties prompted us to propose the presence of a Ni(CN)Fe(CN)3(CO) active site.

The cysteine residue of the SoxY protein as the active site of protein‐bound sulfur oxidation of Paracoccus pantotrophus GB17

Four proteins of Paracoccus pantotrophus are required for hydrogen sulfide‐, sulfur‐, thiosulfate‐ and sulfite‐dependent horse heart cytochrome c reduction. The lack of free intermediates suggested a protein‐bound sulfur oxidation mechanism. The SoxY protein has a novel motif containing a cysteine residue. Electrospray ionization and matrix‐assisted laser desorption ionization mass spectrometry of the SoxYZ protein revealed one mass for SoxZ and different masses for SoxY, indicating native SoxY (10 977 Da) and SoxY with additional masses of +32, +80, +112 and +144 Da, suggesting addition of sulfur, sulfite, thiosulfate and thioperoxomonosulfate. Reduction of SoxY removed the additional masses, indicating a thioether or thioester bond. N‐Ethylmaleimide inhibited thiosulfate‐oxidation and the kinetics suggested a turn‐over‐dependent mode of action. These data were evidence that the sulfur atom to be oxidized was covalently linked to the thiol moiety of the cysteine residue of SoxY and the active site of sulfur oxidation.

Nickel transport in Alcaligenes eutrophus

Transport of nickel ions was studied in Alcaligenes eutrophus. Two transport systems for nickel ions exist to satisfy the nickel demand for the lithotrophic hydrogen metabolism. A major nickel transport activity exhibited an apparent affinity constant (Km) of 17 μM nickel chloride. This activity was competitively inhibited by Mg2+, Mn2+, Zn2+, and Co2+. A minor nickel transport activity was determined in the presence of high (0.8 mM) magnesium. This activity was not inhibited by Zn2+ or Mn2+; its K′m was determined to be 0.34 μM nickel chloride. These kinetics suggested a second transport system in A. eutrophus. The membrane potential of A. eutrophus was decreased upon the addition of ammonium ions leading to a decreased nickel transport. This inhibition could be reversed by fructose or by hydrogen indicating an energy dependent nickel transport. Protonophores inhibited the nickel transport. However, inhibitors of ATP synthase like dicyclohexylcabodimide or venturicidin had little or no effect on nickel transport. These data indicated that the transport was coupled to the proton motive force.

Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans

Abstract Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase was located in the 140,000 ×g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in an 85% pure preparation with a specific activity of 6.0 U (mg protein)–1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The optimum of enzyme activity was at pH 9 and at 49° C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme. The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50–100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase.

Interaction between Sox proteins of two physiologically distinct bacteria and a new protein involved in thiosulfate oxidation.

Organisms using the thiosulfate‐oxidizing Sox enzyme system fall into two groups: group 1 forms sulfur globules as intermediates (Allochromatium vinosum), group 2 does not (Paracoccus pantotrophus). While several components of their Sox systems are quite similar, i.e. the proteins SoxXA, SoxYZ and SoxB, they differ by Sox(CD)2 which is absent in sulfur globule‐forming organisms. Still, the respective enzymes are partly exchangeable in vitro: P. pantotrophus Sox enzymes work productively with A. vinosum SoxYZ whereas A. vinosum SoxB does not cooperate with the P. pantotrophus enzymes. Furthermore, A. vinosum SoxL, a rhodanese‐like protein encoded immediately downstream of soxXAK, appears to play an important role in recycling SoxYZ as it increases thiosulfate depletion velocity in vitro without increasing the electron yield.