Aerobic Hydrogen Bacteria Research Articles

The giant linear plasmid pHG207 from Rhodococcus sp. encoding hydrogen autotrophy: characterization of the plasmid and its termini.

As described previously, in Rhodococcus sp. (formerly Nocardia opaca) strains MR11 and MR22, the ability to grow as an aerobic hydrogen bacterium (the Aut character) is located on giant conjugative linear plasmids--on pHG201 (270 kb) in strain MR11 and on pHG205 (280 kb) in strain MR22. In an autotrophic transconjugant originating from MR22 a smaller plasmid, pHG207 (225 kb), was detected and shown to be a recombination product of the wild-type plasmids pHG204 and pHG205. A donor carrying pHG207 as the sole plasmid transferred the Aut marker at a 1000-fold frequency compared to the wild-type plasmid pHG205. Analysis of the plasmid ends revealed that plasmid pHG207 carries proteins at both ends; the proteins are linked to the 5' ends of the strands. The cloned end fragments of about 2 kb were sequenced and found to contain highly homologous sequences within the terminal 583 bp (left end part) and 560 bp (right end part). Several potential reading frames were detected, but database searching gave no indication about possible functions.

Recent advances in the knowledge of the metabolism of bacterial polyhydroxyalkanoic acids and potential impacts on the production of biodegradable thermoplastics

AbstractBacteria are able to synthesize a wide variety of different thermoplastic polyhydroxyalkanoic acids (PHA). The physiology of PHA metabolism has been studied in most detail in the aerobic hydrogen bacterium Alcaligenes eutrophus. Strains of this bacterium are already used for the production of biodegradable polyesters on a technological scale. The current knowledge on the pathways for PHA synthesis, on the genes essential for PHA biosynthesis and on genes affecting the accumulation of PHA in this bacterium will be reviewed.

Immunological comparison of subunits isolated from various hydrogenases of aerobic hydrogen bacteria

Polyclonal, monospecific antibodies were produced against the two subunits ( M r 62 000, and M r 31 000), isolated from the membrane-bound hydrogenase of Alcaligenes eutrophus H16. The antibodies (IgG fractions) were purified from crude sera by Protein A-Sepharose CL-4B chromatography. By double immunodif usion assays and tandem-crossed immunoelectrophoresis the large and the small subunit were demonstrated not to be immunologically related. Immunological comparison of these subunits with the four non-identical subunits ( M r 63 000, 56 000, 30 000 and 26 000) of the NAD-linked, soluble hydrogenase from A. eutrophus H16 showed that the subunits of the membrane-bound hydrogenase did not cross-react with any of the antibodies raised against the four subunits of the NAD-linked enzyme and that, vice versa, none of these four subunits cross-reacted with antibodies raised against the two subunits of the membrane-bound hydrogenase. This means that A. eutrophus H16 contains altogether six non-identical immunologically unrelated hydrogenase polypeptides. The membrane-bound hydrogenases were isolated and purified from various aerobic H 2-oxidizing bacteria: A. eutrophus H16, A. eutrophus type strain, A. eutrophus CH34, A. eutrophus Z1, A. hydrogenophilus, Paracoccus denitrificans and strain Cd2/01. All These proteins resembled each other and each consisted of two non-identical polypeptides. A complete separation of these subunits was achieved at high-yield by preparative FPLC gel filtration of three Superose 12 columns connected in series, using SDS and DTT-containing sodium phosphate buffer (pH 7.0). The small subunits of these enzymes turned out to be immunologically closely related to each other; they were either identical or almost identical. The large subunits were also related, but less pronounced. Only the large subunits from Z1 and type strain reacted fully identical with the H16 subunit. Of the two isolated, homogeneous subunits of the membrane-bound hydrogenase from A. eutrophus H16, the amino acid compositions and the NH 2-terminal sequences have been determined. The results confirmed the diversity of the large and the small subunit. Furthermore, for comparison also the NH 2-terminal sequences of the two subunits from the hydrogenase of A. eutrophus CH34 have been analysed.

Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.

ESR properties of membrane-bound hydrogenases from aerobic hydrogen bacteria

Analysis of the membrane-bound hydrogenase of Alcaligenes eutrophus, strain H16, indicated a content of 7–9 Fe, 7–9 labile sulphide and 0.6–0.7 nickel atoms per molecule of 98 kDa. The protein, as prepared, gave a complex electron-spin resonance (ESR) spectrum in the oxidized state at low temperatures (below 30 K), with average g value greater than 2.0. This was interpreted as being due to the interaction of [3Fe-χS] or [4Fe-4S] 3+ cluster and another paramagnetic centre. At higher temperatures a rhombic spectrum, attributred to Ni(III), was observed. On treatment with mercaptoethanol, or reduction to a redox potential below +160 mV, the low-temperature spectrum changed into a narrow signal at g = 2.02, with increased amplitude. The g = 2.02 signal was reduced reversibly with a midpoint potential of 40 ± 30 mV. The complex spectrum could be restored if the enzyme was rapidly reduced and reoxidized, but not under the conditions of the redox titration, when a broader signal appeared instead. The reductive titration was accompanied by a 3–7-fold increase in enzymic activity. On further reduction of A. eutrophus H16 hydrogenase a complex spectrum with average g value less than 2.0 was observed. This indicate the presence of a [4Fe-45] 1+ cluster, interacting with either another such cluster or another paramagnetic centre. This cluster or clusters were estimated to have a midpoint potential of −90 ± 30 mV. The ESR spectra of the hydrogenases of A. eutrophus, type strain, and Pseudomonas pseudoflava, in the oxidized and reduced states, were similar to those of the A. eutrophus H16 enzyme, indicating that they have a similar arrangement of redox centres. The characteristic spectra of the reduced e indicate that they represent a new type of membrane-bound hydrogenase.

Production and Consumption of Hydrogen in a Eutrophic Lake

The vertical distribution of hydrogen was measured in the Loclat, a eutrophic and holomictic lake near Neuchâtel, Switzerland, before and during summer stratification. H(2) concentrations decreased with depth in the anaerobic hypolimnion and were often below the detection limit (2.5 nl of H(2) liter) in the water adjacent to the lake sediment. H(2) was apparently not released from the lake sediment. The highest H(2) concentrations (>4 mul of H(2) liter) were observed in the aerobic water of the epilimnion and metalimnion. There, the H(2) concentrations changed with time, indicating a turnover of H(2). The H(2) production processes could not be studied in the laboratory since incubation of water samples in light or darkness did not result in H(2) production but rather always in H(2) consumption. The possible role of cyanobacteria and algae for H(2) production is discussed. Aerobic or anaerobic H(2) consumption activities were observed at all depths of the water column, with highest activities in the hypolimnion. Aerobic H(2) consumption activity was insensitive to azide inhibition, but sensitive to heat, mercuric chloride, or cyanide. It was restricted to a particle fraction of 0.2 to 3.0 mum in size, so that it must be due to single bacterial cells. Aerobic hydrogen bacteria, on the other hand, occurred in clusters of >3.0 mum. Therefore, the hydrogen bacteria could not have caused the H(2) consumption in lake water. The aerobic H(2) consumption activity followed Michaelis-Menten kinetics, with a K(m) of 67 nM H(2). This is an exceptionally low value compared with K(m) values of hydrogenases in hydrogen bacteria and other species, but is similar to that for H(2)-decomposing abiontic soil hydrogenases.

Isolation of a hydrogenase-cytochromebcomplex from cytoplasmic membranes ofXanthobacter autotrophicusGZ 29

The aerobic nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus strain GZ29 [1] can grow autotrophically with hydrogen, oxygen, carbon dioxide, and molecular nitrogen as sole sources of electrons, energy, carbon, and nitrogen. Carbon dioxide is fixed via the Calvin cycle, and for activation of hydrogen only a membrane-bound hydrogenase activity was detected which does not reduce NAD [2]. This is true also for many other aerobic hydrogen bacteria [2,3], and it is assumed that in these organisms the membrane-bound hydrogenase has to provide electrons for NAD reduction in a reversed electron transport as well as for energy conservation. In X. autotrophicus the hydrogenase beyond it has to supply electrons for reduction of nitrogen by nitrogenase at the redox potential of ferredoxin (around 4 2 0 mV). For this reason the hydrogenase of X. autotrophicus should differ from the type of membrane-bound hydrogenase found in Alcaligenes eutrophus which reduces electron acceptors only at the redox potential of quinones [4]. It was also considered that even two different hydrogenases might be present, one reducing ferredoxin and one providing electrons for the respiratory chain. The involvement of ferredoxin in the nitrogen fixation process of X. autotrophicus GZ29 has recently been shown by electron transfer and regulation studies [5]. In the present study the membrane-bound hydrogenase system of S. autotrophicus is shown to be associated with a cytochrome b complex, and catalytic as well as molecular properties are described.

Physiological characterization of the hydrogen bacterium Aquaspirillum autotrophicum

Aquaspirillum autrotrophicum, an aerobic hydrogen bacterium recently isolated from an eutrophic freshwater lake, was characterized physiologically. It grew autotrophically in a fermenter with a doubling time of 4 h. Heterotrophic growth was faster. pH-Optimum ranged from 5.0–7.5, temperature optimum was about 28° C. During autotrophic growth about 10 moles hydrogen were consumed per 1 mole carbon dioxide fixed.

Localization and stability of hydrogenases from aerobic hydrogen bacteria.

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction. While the second enzyme was found to be particulate and unable to react with NAD. All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gently methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis. Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 micronmoles H2 oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme for A. eutrophus H 16 which was shown to be more stable under aerobic conditions.

Micromorphology of Gram-negative hydrogen bacteria

The fine structure of the cell envelope, of membrane systems and of cytoplasmic inclusions of Gram-negative aerobic hydrogen bacteria has been studied. The results have been tabulated, and three main groups could be recognized: Group 1: Alcaligenes eutrophus, A.paradoxus, A.ruhlandii, Pseudomonas facilis, P.flava, P.pseudoflava, P.palleronii, and Aquaspirillum autotrophicum; Group 2: "Corynebacterium" autotrophicum and strains MA 2 and SA 35; Group 3: Paracoccus denitrificans. Special structures related to the chemoautotrophic way of life of the hydrogen bacteria were not observed.

Micromorphology of Gram-negative hydrogen bacteria

The cell morphology, the arrangement and fine structure of flagella and the piliation of the following Gram-negative aerobic hydrogen bacteria have been studied: Alcaligenes eutrophus, Alcaligenes paradoxus, Alcaligenes ruhlandii, Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas facilis, Aquaspirillum autotrophicum, Paracoccus denitrificans, Corynebacterium autotrophicum, and strains MA 2 and SA 35. The identity of the bacteria was examined by their substrate spectra and type of flagellation. Three types of flagellar fine structure were differentiated. The presence of pili was noted in strains of Alcaligenes paradoxus, Pseudomonas flava, P.pseudoflava, P.palleronii, and P.facilis.