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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.

Abstract Background Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst. Results The two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts. Conclusions Asymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing heterologous alcohol dehydrogenase showed advantages for practical usage relative to the in vitro coupling system. The results suggest a hopeful perspective of the hydrogen-driven bioprocess as an environmentally outstanding method to achieve industrial green innovation. Hydrogen-oxidizing bacteria can be useful hosts for the development of hydrogen-driven microbial cell factories.

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.