Ortwin Meyer

Reisolation of the carbon monoxide utilizing hydrogen bacterium Pseudomonas carboxydovorans (Kistner) comb. nov.

From enrichment cultures four carbon monoxide utilizing bacteria were isolated; strain OM5 isolated from waste water was studied in detail. The cells are Gram-negative, slightly curved rods, motile by a single subpolarly inserted flagellum. The colonies are smooth, translucent and not slimy.The cells are able to grow autotrophically in mineral medium under an atmosphere of 40% CO, 5% O2 and 55% N2 at a doubling time of 20h (30°C) or of 85% H2, 5% O2 and 10% CO2 at a doubling time of 7h. Heterotrophic growth occurrd on organic acids such as acetate (td=8h), pyruvate (td=8h), lactate, crotonate, malate, succinate (td=8h), formate (td=35h) and glyoxylate as substrates.The enzyme system for carbon monoxide utilization is formed only during growth on CO; hydrogenase is present in cells grown on CO or on H2+CO2 as well as grown on pyruvate. The rate of oxygen reduction by intact CO-grown cells is 3.7-fold higher in the presence of hydrogen than in the presence of carbon monoxide. During growth the stoichiometry of gas uptake was 6.1 CO+2.8 O2+H2O → 〈CH2O〉+5.1 CO2. For the new isolate the name Pseudomonas carboxydovorans (Kistner) comb. nov. has been proposed.

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification, characterisation and distinction from a single reductase for trimethylamine-N-oxide, dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR+ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.

Thermophilic Bacilli growing with carbon monoxide

Four strains of obligately thermophilic Bacilli capable of growing with carbon monoxide as a sole carbon and energy source were isolated from settling ponds of a sugar factory. Most of them could be identified as strains of Bacillus schlegelii on the basis of cell wall composition, DNA homology menaquinone and DNA base content. Growth with CO was very fast (td=3 h) and was optimal at 65°C. No growth occurred below 50°C. As with the mesophilic carboxydotrophs, hydrogen plus carbon dioxide could also serve as autotrophic substrates. Growth of the isolates with CO depended on the presence of molybdenum in the growth medium. This suggested CO oxidase in the newly isolated Bacilli being a molybdenum hydroxylase similar to the enzymes from the mesophilic carboxydotrophs. Some data characterizing the CO-oxidizing activity in extracts of the thermophilic isolates are also provided.

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (“carboxydebacteria”) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H2-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H2 with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD+, FMN+, and NAD(P)+ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H2O→CO2+2H++2e- as previously shown for Pseudomonas carboxydovorans.

R-Bodies in newly isolated free-living hydrogen-oxidizing bacteria

R-Bodies have been found in a recently isolated pseudomonas-like free-living hydrogen oxidizing bacterium. Their isolation, fine structure and chemical composition are described and compared with the R-bodies from the kappa particles (Caedobacter), obligate endosymbionts of Paramecium aurelia. The 2K 1 R-bodies exhibited essential characteristics of the kappa R-bodies; however, their size and some other structural aspects proved that they represent a new type of R-bodies. The presence of phage tail-like particles in cells induced with Mitomycin C is in favour of the hypothesis that the R-bodies might be coded by defective prophages, or by extrachromosomal elements.

Enzymes Oxidizing Carbon Monoxide

Carbon monoxide is oxidized by bacteria from different physiological groupings, including both, aerobes and anaerobes. The groups of CO-oxidizing bacteria are carboxydotrophs, methanotrophs, dinitrogen-fixers, acetogens, methanogens, phototrophs and sulfate- reducers. Some can use CO as sole carbon and energy source for growth (utilitarian CO-oxidizers) whereas the gas is only co-oxidized by others (non-utilitarian CO-oxidizers). Enzymes that Catalyze the oxidation of CO to CO2 may be subdivided according to whether they contain molybdenum or nickel as an integral component of their active center. To date molybdenum-containing CO dehydrogenases have been identified in Pseudomonas carboxydovorans, Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava and Bacillus schlegelii. In addition, these enzymes contain the molybdopterin of the molybdenum cofactor, flavin and two different iron-sulfur centers of the (2Fe-2S) type. Nickel-containing CO dehydrogenases have been found in Clostridium thermoaceticum, Acetobacterium woodii, Methanosarcina barkeri, and Desulfovibrio desulfuricans; most of them contain (4Fe-4S) centers, no flavin, and they are extremely sensitive to oxygen. The cofactor composition of CO dehydrogenases from aerobic bacteria appears to be much more complex than that of most enzymes from anaerobes. It is obvious, that CO dehydrogenases of aerobes are molybdenum iron-sulfur flavoproteins (molybdenum hydroxylases), whereas those of anaerobes are nickel iron-sulfur enzymes. The divergent cofactor composition of CO dehydrogenases as well as significant differences in the affinity for CO reflect the quite different functions that are fulfilled by these enzymes in the bacterial groupings mentioned.

CO2 is the first species formed upon CO oxidation by CO dehydrogenase from Pseudomonas carboxydovorans

The active species of “CO2”, i.e. CO2 or HCO3-, formed in the CO dehydrogenase reaction was determined using the pure enzyme from the carboxydotrophic bacterium Pseudomonas carboxydovorans. Employing an assay system similar to that used to test for carbonic anhydrase, data were obtained which are quite compatible with those expected if CO2 is the first species formed. In addition, carbonic anhydrase activity was not detected in P. carboxydovorans.

The cytochrome composition of carboxydotrophic bacteria

Spectroscopy at room and liquid nitrogen temperatures with extracts of the carbon monoxide-oxidizing bacteria Pseudomonas carboxydovorans, P. carboxydohydrogena, P. carboxydoflava, P. compransoris, Alcaligenes carboxydus, and Arthrobacter 11/x revealed the presence of normal electron transport systems, containing b-, c-, and a-type cytochromes at concentrations that compare to those of other aerobic bacteria. CO did not induce the formation of special CO-insensitive terminal oxidases. The gross composition of the respiratory chains was not affected by the type of growth substrate, and cytochrome d(=a2) was not detected. However, certain b-type cytochromes were only found when CO or H2 + CO2 served as growth substrates. All strains contained at least two different b-type cytochromes. Cytochrome b563 formed a weak CO-complex and was identified as a novel cytochrome o. It functions as CO-insensitive, alternative terminal oxidase in carboxydotrophic bacteria. A soluble CO-binding cytochrome c was present in P. carboxydovorans, P. carboxydohydrogena, and P. carboxydoflava. A CO-binding protoheme compound could be identified as catalase in P. compransoris, P. carboxydovorans, P. carboxydohydrogena, A. carboxydus, and Arthrobacter 11/x. The data are consistent with the presence of branched respiratory chains in the carboxydotrophs examined, and suggest the functioning of both, cytochrome a and the novel cytochrome o as terminal oxidases.

Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H2) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H2) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N′-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H2, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.