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Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle.

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and [NAD.sup.+]) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and [NAD.sup.+] were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.

Glutamate synthases are classified according to their specificities for electron donors. Ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have NADPHdependent glutamate synthases. In this study, Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. This is the first observation, to our knowledge, of a ferredoxin-dependent glutamate synthase in a nonphotosynthetic organism. The purified enzyme from H. thermophilus was shown to be a monomer of a 168-kDa polypeptide homologous to ferredoxin-dependent glutamate synthases from phototrophs. In contrast to known ferredoxin-dependent glutamate synthases, the H. thermophUus glutamate synthase exhibited glutaminase activity. Furthermore, this glutamate synthase did not react with a plant-type ferredoxin (Fd3 from this bacterium) containing a [2Fe-2S] cluster but did react with bacterial ferredoxins (Fdl and Fd2 from this bacterium) containing [4Fel4S] clusters. Interestingly, the H. thermophilus glutamate synthase was activated by some of the organic acids in the reductive tricarboxylic acid cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other glutamate synthases, and this property may enable the control of nitrogen assimilation by carbon metabolism.

The assimilation of acetate generated in the anoxic rice field during the degradation of organic matter by microorganisms is studied using isotopes of carbon. The bacteria were found to utilize acetate under methanogenic conditions where carbon dioxide is the predominant electron acceptor on the polymerase chain reaction analysis of RNA isolated from the bacterial cells.

Hydrogenovibrio marinus MH-110 possesses three different sets of genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): two form I (cbbLS-1 and cbbLS-2) and one form II (cbbM). We have previously shown that the expression of these RubisCO genes is dependent on the ambient C[O.sub.2] concentration. LysR-type transcriptional regulators, designated CbbR1 and CbbRm, are encoded upstream of the cbbLS-1 and cbbM genes, respectively. In this study, we revealed by gel shift assay that CbbR1 and CbbRm bind with higher affinity to the promoter regions of cbbLS-1 and cbbM, respectively, and with lower affinity to the other RubisCO gene promoters. The expression patterns of the three RubisCOs in the cbbR1 and the cbbRm gene mutants showed that CbbR1 and CbbRm were required to activate the expression of cbbLS-1 and cbbM, respectively, and that neither CbbR1 nor CbbRm was required for the expression of cbbLS-2. The expression of cbbLS-1 was significantly enhanced under high-C[O.sub.2] conditions in the cbbRm mutant, in which the expression of cbbM was decreased. Although cbbLS-2 was not expressed under high-C[O.sub.2] conditions in the wild-type strain or the single cbbR mutants, the expression of cbbLS-2 was observed in the cbbR1 cbbRm double mutant, in which the expression of both cbbLS-1 and cbbM was decreased. These results indicate that there is an interactive regulation among the three RubisCO genes.

The regulatory, gene for [[sigma].sup.54]-dependent-type transcriptional regulator, fhpR, is located upstream of the fhp gene for flavohemoglobin in Pseudomonas aeruginosa. Transcription of fhp was induced by nitrate, nitrite, nitric oxide (NO), and NO-generating reagents. Analysis of the fhp promoter activity in mutant strains deficient in the denitrification enzymes indicated that the promoter was regulated by NO or related reactive nitrogen species. The NO-responsive regulation was operative in a mutant strain deficient in DNR (dissimilatory nitrate respiration regulator), which is the NO-responsive regulator required for expression of the denitrification genes. A binding motif for [[sigma].sup.54] was found in the promoter region of fhp, but an FNR (fumarate nitrate reductase regulator) box was not. The fhp promoter was inactive in the fhpR or rpoN mutant strain, suggesting that the NO-sensing regulation of the fhp promoter was mediated by FhpR. The DNR-dependent denitrification promoters (nirS, norC, and nosR) were active in the fhpR or rpoN mutants. These results indicated that P. aeruginosa has at least two independent NO-responsive regulatory systems. The fhp or fhpR mutant strains showed sensitivity to NO-generating reagents under aerobic conditions but not under anaerobic conditions. These mutants also showed significantly low aerobic NO consumption activity, indicating that the physiological role of flavohemoglobin in P. aeruginosa is detoxification of NO under aerobic conditions.

Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, fixes C[O.sub.2] by the Calvin-Benson-Bassham cycle. Strain MH-110 possesses three different sets of genes for Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO): CbbLS-1 and CbbLS-2, which belong to form I ([L.sub.8][S.sub.8]), and CbbM, which belongs to form II ([L.sub.x]). In this paper, we report that the genes for CbbLS-1 (cbbLS-1) and CbbM (cbbM) are both followed by the cbbQO genes and preceded by the cbbR genes encoding LysR-type regulators. In contrast, the gene for CbbLS-2 (cbbLS-2) is followed by genes encoding carboxysome shell peptides. We also characterized the three RubisCOs in vivo by examining their expression profiles in environments with different C[O.sub.2] availabilities. Immunoblot analyses revealed that when strain MH-110 was cultivated in 15% C[O.sub.2], only the form II RubisCO, CbbM, was expressed. When strain MH-110 was cultivated in 2% C[O.sub.2], CbbLS-1 was expressed in addition to CbbM. In the 0.15% C[O.sub.2] culture, the expression of CbbM decreased and that of CbbLS-1 disappeared, and CbbLS-2 was expressed. In the atmospheric C[O.sub.2] concentration of approximately 0.03%, all three RubisCOs were expressed. Transcriptional analyses of mRNA by reverse transcription-PCR showed that the regulation was at the transcriptional level. Electron microscopic observation of MH-110 cells revealed the formation of carboxysomes in the 0.15% C[O.sub.2] concentration. The results obtained here indicate that strain MH-110 adapts well to various C[O.sub.2] concentrations by using different types of RubisCO enzymes.

Research has been conducted on genes for nitrous oxide reduction which are clustered on Pseudomonas aeruginosa chromosomes. Results demonstrate that nitrous oxide is the inducer molecule for the nos gene transcription.

A three-dimensional solution structure of thermostable cytochrome c-552 from Hydrogenobacter thermophilus was determined by proton nuclear magnetic resonance spectroscopy. Twenty structures were calculated and it was found that the main chain folding of the cytochrome c-552 was similar to that of Pseudomonas aeruginosa (PA) cytochrome c-551. The structures of HT c-552 and PA c-551 were compared and structural differences, which may lead to differences in the thermostability between the two proteins, were discovered.

A locus of the denitrification gene cluster from Pseudomonas aeruginosa was analyzed to identify the genes involved in the biosynthesis of heme d1. Ten genes located downstream of the nirS gene were characterized as mediators of heme d1 biosynthesis in Pseudomonas aeruginosa. All of the nirSMCFDLGHJEN genes of an nir gene cluster were also transcribed in a similar direction by ANR under denitrifying conditions. Furthermore, a gene designated as NirN encoded a typical N-terminal signal sequence for membrane translocation.

The thermophilic, aerobic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, synthesizes 2-oxoglutarate:ferrodoxin oxidoreductase, a key enzyme in the reductive tricarboxylic acid cycle. Sequence analysis shows it has a molecular mass of about 105 kilodaltons. Enzymatic analysis shows it is thermostable, has an optimum pH from 7.6-7.8 and undergoes oxidation at temperatures above 80 degrees Celsius.

The presence of autoantibodies in sera of 8 of 14 patients with multiple sclerosis (MS) to brain and retinal soluble antigens was described. The autoantibodies were identified as beta-arrestin 1 and arrestin, respectively. In two patients with MS, serum antibody titers during relapse were higher than found during remission. The recognition sites for antibodies in sera of MS patients was located at a dominant immunogenic site on arrestin located in the C-terminal region of the molecule.

Research has been conducted on the Hydrogenobacter thermophilus strain TK-6. The phospholipid composition of this strain has been analyzed and the results suggest that the phospholipids detected from the strain are phosphatidylinositol and phosphatidylglycerol.

The behavior of antibiotic-sensitive bacteria within biofilms containing antibiotic-producing bacteria is examined. Findings reveal that the survival of sensitive bacteria in biofilm is possible by covering them with a layer of resistant bacteria.

The construction of a cellulose-degrading, defined mixed culture consisting of five bacterial strains that are dominant bacteria in the original microflora is described. The characteristics of each of the members in the pure cultures are investigated and the roles and relationships among the members of the community in situ are evaluated based on these results.

An investigation of the relationship between the dominant members of the microbial community and their roles in the decomposition of biopolymers is made. It is also shown that these dominant members play a pivotal role as extracellular gelatinase and amylase producers during the composting process.

Research has been conducted on acyl coenzyme A carboxylase. The results of molecular characterization and purification of this carboxylase from Acidianus brierleyi are reported.

Hydrogenobacter thermophilus is a thermophilic, obligately chemolithoautotrophic and aerobic hydrogenoxidizing bacterium. It is unique in its ability to fix carbon dioxide via the reductive tricarboxylic acid cycle under aerobic conditions. It utilizes molecular hydrogen, elemental sulfur, or thiosulfate as the sole energy source. Here, we report the complete genome sequence of H. thermophilus TK-6. doi: 10.1128/JB.00158-10

Electrochemical measurements taken at 25 degree Celsius using temperature dependent cyclic voltammetry were utilized for studying the relationship between redox function and protein stability of cytochromes c. The findings suggest that the overall protein stability, Fe-S bond stability, and redox property of cytochrome c were mutually related.

The results of paramagnetic (Super1) H NMR and optical studies on a homologous cytochrome c(cyt c) series, which demonstrate that interactions among amino acid side chains in distal regions influence heme axial coordinations in cyt c is presented. The conclusion states that alteration in interaction among acid side chains in the protein interior influence the stability of Fe-methionine coordination at the heme active site, which in turn affects thermostability of the protein.