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ABSTRACTH-NS family proteins encoded on the chromosome of Pseudomonas putidaKT2440 (TurA and TurB) and the IncP-7 plasmid pCAR1 (Pmr) commonly have an N-terminal dimerization/oligomerization domain constituted by a central and a terminal dimerization sites. To clarify the dimerization manner at the central dimerization sites of the three homologs, we performed chemical cross-linking analyses with protein variants inactivated at the terminal dimerization site. Comparison of the hetero-dimer ratios among them suggested stronger affinities between the central dimerization sites of TurA and TurB monomers than between TurA and Pmr or TurB and Pmr. Furthermore, analyses of the interaction between truncated TurB containing only a functional terminal dimerization site and full-length proteins suggested that TurB exhibited higher affinities for oligomer complex formation with TurB itself and TurA but not Pmr. Altogether, we revealed stronger interaction between the N-terminal domains of TurA and TurB than between either of them and Pmr.

Ferriperoxin is a novel peroxidase essential for aerobiosis of Hydrogenobacter thermophilus. Although the ferriperoxin-deficient mutant (Δfpx) was unable to grow aerobically, a suppressor mutant capable of aerobic growth was obtained after long aerobic cultivation. The alkyl hydroperoxide reductase gene was significantly upregulated in the suppressor mutant, indicating that the enzyme counteracts oxidative stress in the absence of ferriperoxin.

Bioelectrochemistry system (BES) provides a new strategy for the improvement of low temperature anaerobic digestion (AD) for methane production. In this study, the BES with carbon brushes as electrodes was operated at 15 °C to analyze the changes of microbial abundance and functional genes. The microbial community structure showed that the application of voltage had no effect to type and relative abundance of bacteria based on 16S-rRNA sequencing. Metatranscriptomic analyses revealed that significantly enhanced the abundance of fermentation bacteria in the fluid activated sludge, and the relative abundance of biosynthesis of other secondary metabolites (61.56%) was also highly expressed. More than that, the abundance of electricigens (Pseudomonas, Syntrophomonas) was also enriched in the BES system. These electricigens could interact with methanogens to promote electron transfer by direct interspecies electron transfer (DIET). The expression level of functional enzyme related to electron transport was increased at the anode (30.60%), but decreased at the cathode (−33.84%). Analysis of methanogens found that the application of voltage decreased the relative abundance of Methanosarcinaand promoted the enrichment of Methanothrix. At 15 ℃, acetoclastic was the mainly methanogenic pathway. What’s more, the application of voltage could enhance the acetoclastic and inhibited the hydrogenotrophic and the methylotrophic pathway. Further, the acetoclastic pathway was changed from acetate kinase (EC: 2.7.2.1) and phosphate acetyltransferase (EC: 2.3.1.8) to acetate-CoA ligase (EC: 6.2.1.1) by applied voltage. These results are conducive to the further development of low-temperature AD treatment technology.

Sugarcane bagasse is an important lignocellulosic by-product with potential for conversion to biofuels and chemicals in biorefinery. As a step towards an understanding of microbial diversity and the processes existing in bagasse collection sites, the microbial community in industrial bagasse feedstock piles was investigated. Molecular biodiversity analysis of 16S rDNA sequences revealed the presence of a complex bacterial community. A diverse group of mainly aerobic and facultative anaerobic bacteria was identified reflecting the aerobic and high temperature microenvironmental conditions under the pile surface. The major bacterial taxa present were identified as Firmicutes, Alpha- and Gammaproteobacteria, Acidobacteria, Bacteroidetes, and Actinobacteria. Analysis of the eukaryotic microbial assemblage based on an internal transcribed spacer revealed the predominance of diverse cellulolytic and hemicellulolytic ascomycota. A microbial interaction model is proposed, focusing on lignocellulose degradation and methane metabolism. The insights into the microbial community in this study provide a basis for efficient utilization of bagasse in lignocellulosic biomass-based industries.

The goal of this research was to investigate the effects of oxygen concentration on the composting process in China. The compost system was composed of cattle manure, chicken manure, and wheat straw combined at a ratio 1:3:6 for a period of 60 days. Microaerobic treatment was attained by manual turning, while aerobic treatment was performed by forced air plus turning. The fermentation parameters such as C/N ratio, composting temperature, lginocellulose content and O2concentration were determined. Results showed that oxygen concentration of compost in the microaerobic treatment was always lower than 1.5% during composting periods except for the first four hours and oxygen concentration was more than 4.0% in the aerobic treatment at all times. The duration of the thermophilic phase above 50°C under microaerobic treatment was 23 days, compared to 15 days under the aerobic treatment. However, the composting temperature at the later phases declined more slowly under aerobic conditions than under microaerobic conditions. The hemicellulose content decreased from 13.9% to 8.0% in the microaerobic treatment, compared to 8.5% in the aerobic treatment. The cellulose content decreased from 21.5% to 16.1% in the microaerobic treatment, compared to 18.0% in the aerobic treatment at the end of composting process. When C/N ratio, NO−3content and composting temperature were used as indexes to evaluate compost maturity, the compost matured in 35 days in the microaerobic treatment, compared with 45-50 days in the aerobic treatment. Laboratory culture experiment with compost samples taken at the thermophilic phase corroborated that cellulose degradation was more rapid under microaerobic conditions.

The genes for a nitric oxide reductase-like cytochrome bccomplex were cloned from a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilusTK-6. The structural genes norCand norB, which encode cytochrome cand cytochrome bsubunits of the complex respectively, are probably transcribed as a tricistronic operon with a following gene encoding a putative membrane protein. NorC has, unusually, two hydrophobic transmembrane spans in its N-terminus. Immunoblot analysis showed that expression of NorC was induced by nitrate, nitrite, or sodium nitropurusside, suggesting that the norCBgene product is a denitrification enzyme, nitric oxide reductase. The consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide-sensing regulators of proteobacteria were not found in the norCpromoter region, but consensus −35 and −10 sequences were found in this region. These results indicate that strain TK-6 has a nitrogen oxide-sensing regulatory system that differs from proteobacterial systems.

A thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilusTK-6, assimilates carbon dioxide viathe reductive tricarboxylic acid cycle. A gene encoding a ferredoxin involved in this cycle as an electron donor (HtFd1) was cloned and sequenced. Interestingly, another ferredoxin gene (encoding HtFd2) was found in tandem with the HtFd1 gene. These two ferredoxin genes overlapped by four bp, and transcriptional analysis revealed that they are co-transcribed as an operon. The deduced amino acid sequences of HtFd1 and HtFd2 were 42.9% identical and each contained four cysteine residues that serve as probable ligands to an iron-sulfur cluster. Spectroscopic analyses of the purified recombinant ferredoxins heterologously expressed in Escherichia coliindicated that each ferredoxin contains a single [4Fe–4S]2+⁄1+cluster.

Pseudomonas aeruginosastrains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1mutant. These results indicated that cytochrome bc1complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.

Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Δ(ΔG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (ΔCP), resulting in higher ΔG values over a wide temperature range (0–100°C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.

Three c-type cytochromes, NirM, NirC, and NirN, are encoded in the nirSMCFDLGHJEN gene cluster for cytochrome cd1-type nitrite reductase (NIR) of Pseudomonas aeruginosa. nirS is the structural gene for NIR. NirM (cytochrome c551) is reported to be a physiological electron donor for nitrite reductase. The respective functions of NirC and NirN have remained unclear. In this study, we produced recombinant NirC and NirN in P. aeruginosa, and purified them from the periplasmic fraction. N-terminal amino acid sequences of the purified proteins showed that the N-terminal 31 and 18 residues of NirC and NirN precursors were cleaved, respectively, indicating that cleaved peptides act as signals for membrane translocation. In addition, the ability of NirC for electron donation to nitrite reductase was investigated. NirC, as well as NirM, was able to mediate the electron donation from the membrane electron pathway to NIR, suggesting that the structural gene for NIR is followed by the genes for two electron donors for NIR.

The cbbRencoding the LysR‐type transcriptional regulator is located downstream of cbbLSQOYAand this gene is located upstream of cbbFPTin divergent transcription. The two promoter regions with LysR‐binding sites are located in the cbbLupstream region and in the cbbR–cbbFintergenic region. Electrophoretic mobility shift assays using a cell extract of Escherichia coliharboring a plasmid containing cbbRand the DNA fragments of promoter regions indicated that CbbR binds in both regions. NADPH caused differences in the complex of CbbR and DNA.

2-Oxoglutarate: ferredoxin oxidoreductase (OGOR) of a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, is involved in carbon dioxide fixation via a reductive TCA cycle. The enzyme is a heterodimer comprising subunits of 70 and 35 kDa. The structural genes for the subunits (korAB) were cloned with primers designed from N-terminal sequences of the purified enzyme. The korAB genes were followed by two open reading frames of unknown function (orf3 and orf4). KorA carried a binding motif for coenzyme A, and KorB carried binding motifs for Fe-S cluster and thiamine pyrophosphate. Active recombinant enzyme from korAB was produced in E. coli under the control of the lac promoter. orf3 and orf4 were not necessary for the expression of active enzyme in E. coli. The recombinant enzymes had high substrate specificity toward 2-oxoglutarate as in the case of the native enzyme purified from strain TK-6.

Mesophilic cytochromec551of Pseudomonas aeruginosa(PAc551) became as stable as its thermophilic counterpart, Hydrogenobacter thermophiluscytochromec552(HT c552), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HTc552through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c551could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c552, there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.

Four genes, cbbO, cbbY, cbbA, and the pyruvate kinase gene (pyk), were found downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus(formerly Pseudomonas hydrogenothermophila). cbbOwas similar to norDin the denitrification gene cluster, and cbbYwas similar to cbbYfrom other autotrophic bacteria. cbbAencoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins. When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE. However, the cell extract had slightly higher activity than a cell extract of E. coliwithout cbbA. Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H. thermoluteoluswas in class IIA. Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions. The activities of the two enzymes were regulated independently.

A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilusstrain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with α2β2structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent Kmof around 300 μMand with a turnover number around 2900 (min-1). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilusstrain TK-6.

The activity of the promoters involved in transcription of the genes (nirS, nirQand norC) required for anaerobic reduction of nitrite and nitric oxide was investigated in NIR‐ and NOR‐deficient mutants of Pseudomonas aeruginosa. The transcriptional activity of these three promoters was induced by nitrite in a wild‐type strain and the activity was low in an nirSmutant. In norCBDand nirQOPmutants, which were expected to accumulate nitric oxide because of a lack of nitric oxide reductase activity, the norCand nirQpromoters showed significantly enhanced activity in promoting transcription relative to the parental strain, even at low nitrite concentrations. These results suggest that the nirQand norCpromoters are regulated by the concentration of endogenous nitric oxide rather than that of nitrite.

Hydrogenovibrio marinusstrain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, possesses three sets of the genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): namely, two form I type (cbbLS-1and cbbLS-2) and one form II type (cbbM) enzymes. The cbbQ-mgene was located downstream of cbbM,and showed high similarity to other cbbQgenes and nirQ/norQgenes in denitrifying bacteria. Phylogenetic analysis of CbbQ and NirQ/NorQ indicated that CbbQ-m from Hv. marinusclosely resembled CbbQ from Thiobacillus intermediusand Thiobacillus neapolitannusand less closely resembled NirQ and NorQ. The cbbQ-mgene has been shown to activate the form II RubisCO in E. colicells, and the cbbQ-tfrom Hydrogenophilus thermoluteoluscould also activate the form II RubisCO. Both cbbQgenes have also been shown to activate the form I RubisCO from Hp. thermoluteolusin E. colicells. However, the activation levels of two form I RubisCOs from Hv. marinuswere smaller than that of form I RubisCOs from Hp. thermoluteolus.Form II RubisCO activated by CbbQ-m (QM) was purified from E. colicells. The result of the 8-anilino-1-naphthalenesulfonate binding assay and the circular dichroism spectra indicated that QM was conformationally different from Form II RubisCO that was not activated by CbbQ.

A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for quantitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was developed. Microtiter plate wells were precoated and blocked simultaneously with epitope-bound bovine caseins as matrix proteins, and aldehyde-modified proteins were quantitated by a competition assay with a monoclonal antibody specific for acrolein-modified lysine or HNE-modified histidine epitopes. Minimal reaction times required for the coating/blocking; first monoclonal antibody and the peroxidase-conjugated second antibody binding steps were 3, 3, and 7 min, respectively, the former two steps being found to be or akin to diffusion-rate-limiting reactions. The convenient ELISA should find an application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may also be advantageous for the quantitation of various other biologically important haptenic molecules.

The nirQOPoperon, which is located between the genes for nitrite reductase and nitric oxide reductase in Pseudomonas aeruginosa, encodes a putative ATP-binding protein and two putative transmembrane proteins. Phylogenetic analysis showed that NirO belongs to the family of subunit III of cytochrome oxidases but is distantly related to the other bacterial or mitochondrial proteins. P. aeruginosastrains that lacked the nirOPgenes had all enzyme activities for denitrification and could grow under anaerobic conditions with nitrate or nitrite as an electron acceptor. However, the energy conservation efficiency of anaerobic respiration was lower in these strains than in strains harboring the nirOPgene.

Two genes coding for endoglucanase activity in Clostridium cellulolyticum were cloned and expressed in Escherichia coli by using plasmid pUC18. The sizes of two fragments harbouring endoglucanase genes are 4.4 kb and 2.0 kb, respectively. The 2.0-kb fragment was identical with a reported DNA fragment encoding an endoglucanase of C. cellulolyticum. The 4.4-kb fragment was obtained first in this study. Deletion analysis showed that a 1.3-kb portion of the 4.4-kb fragment is necessary for the endoglucanase expression by its own promoter. The 4.4-kb fragment hybridized with several different fragments of the genomic DNA in C. cellulolyticum.

Expression of the enzymes for anaerobic nitrate respiration of Pseudomonas aeruginosarequires two CRP/FNR‐related transcriptional regulators, ANR and DNR. Activity of the consensus CRP‐ or FNR‐dependent promoter in the anrand dnrdeficient mutants was investigated. The CRP‐dependent promoter was active in the mutant strains. Both regulators could activate the promoter with a consensus FNR‐binding motif. DNR‐dependent activation was nitrite‐dependent, whereas activation by ANR was not, suggesting that only DNR is involved in sensing nitrogen oxides.

The membrane-bound hydrogenase from a marine hydrogen-oxidizing bacteriumHydrogenovibrio marinuswas characterized as highly oxygen-tolerant, extremely thermophilic and thermostable in its membrane-bound form. The optimum temperatures for H2oxidation and H2evolution were 90 and 80°C, respectively. The enzyme retained 90% of its activity after heating at 70°C for 50 min under air and retained full activity at 90°C for 80 min under hydrogen. The optimum pH values were 5.5 (H2evolution) and 9.4 (H2oxidation), and the activity ratio (H2evolution/H2oxidation) was high (50.3) at pH 5.5. The hydrogen evolution from reduced methyl viologen continued at high temperatures and acidic pH with the continuous addition of sodium dithionite.

Enzymatic reactions involving pyruvate: ferredoxin oxidoreductase and 2-oxoglutarate: ferredoxin oxidoreductase from a thermophilic, aerobic, chemolithoautotrophic, and hydrogen-oxidizing bacterium, Hydrogenobacter thermophilusTK-6, were investigated as the CO2exchange reaction and CO2fixation reaction using ferredoxin isolated from the same organism as a reductant.The reduced ferredoxin was required in the pyruvate synthetic reaction and the 2-oxoglutarate synthetic reaction by a cell extract that had been treated with a PD-10 column to remove the low molecular weight substances. [14C]Pyruvate and [14C]2-oxoglutarate were detected as products of pyruvate synthetic and 2-oxoglutarate synthetic reactions, respectively. Further evidence for the operation of pyruvate: ferredoxin oxidoreductase and 2-oxoglutarate: ferredoxin oxidoreductase was obtained from experiments on CO2exchange reactions using the purified enzymes.

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from a seaside saline hot spring in Izu Peninsula, Japan. The isolate was a Gram-negative, non-motile, non-spore-forming rod cell measuring 0.3 to 0.5 by 1.0 to 2.5 µm. The optimal temperature for growth was around 70°C, and no growth was observed at 40°C or 80°C. Elemental sulfur or thiosulfate could be an alternative to molecular hydrogen as the sole energy source. The DNA base composition of the isolate was 46.0 mol% G+C. 2-Methylthio-3-VI,VII-tetrahydromultiprenyl7-1,4-naphthoquinone (methionaquinone) was the major component of the quinone system. C18:0, C18:1 and C20:1 were the major components of the cellular fatty acids. These properties clearly indicate that the isolate belongs to genus Hydrogenobacter, but differed from H. thermophilus in some respects. Specifically, the isolate was a halophile which grew optimally at around 0.3–0.5 M NaCl, while H. thermophilus could not grow at such NaCl concentration levels. A new species name H. halophilus is proposed for this new halophilic isolate.

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from seawater of the Shonan Coast, Kanagawa Pref., Japan. The isolate was a Gram-negative, comma-shaped rod cell measuring 0.2 to 0.5 by 1 to 2 µm. The cells occurred singly and were motile by a polar flagellum. The deoxyribonucleic acid base composition of the isolate was 44.1 mol% guanine plus cytosine. The optimal temperature for autotrophic growth on H2-O2-CO2 was around 37°C, and no growth was observed at 5° C or 45° C. The optimal pH for growth was around 6.5. NaCl was required for growth with an optimum of 0.5 M. Elemental sulfur, thiosulfate or tetrathionate was utilized, as well as molecular hydrogen, as the sole energy source. No heterotrophic growth was observed on organic media tested. To our knowledge, this is the first report of the isolation of a marine, aerobic hydrogen-oxidizing bacterium, and of an obligately chemolithoautotrophic, mesophilic and aerobic hydrogen-oxidizing bacterium.

A gene, designated dnr, was identified in the vicinity of the structural genes for nitrite reductase ( nirS) and nitric oxide reductase ( norCB), and the gene for activation of the reductases ( nirQ) from Pseudomonas aeruginosa. It encodes a protein of 227 amino acids homologous with the CRP/FNR-family transcriptional regulators. Promoter activities for nirS, nirQand norCBwere considerably reduced in the dnrmutant as well as in the mutant of anr, the other fnr-like regulatory gene from P. aeruginosa. This is the first finding that two CRP/FNR-related regulators are involved in denitrification in one strain.

The incorporation of 14CO2 by the cell suspensions of an extremely thermophilic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus was studied. After short time incubation of the cell suspensions with 14CO2, the radiactivity was initially present in aspartate, glutamate, succinate, phosphorylated compounds, citrate, malate and fumarate. All of these compounds except phosphorylated compounds were related to the members of the tricarboxylic acid cycle. The proportion of labelled aspartate onglutamate in total radioactivity on each chromatogram decreased with incubation time, while the percentage of the radioactivity incorporated in phosphorylated compounds increased with time up to 10 s. These indicated that aspartate and glutamate is derived from primary products of CO2 fixation.

The autotrophic CO2 fixation pathway inAcidianus brierleyi, a facultatively anaerobic thermoacidophilic archaebacterium, was investigated by measuring enzymatic activities from autotrophic, mixotrophic, and heterotrophic cultures. Contrary to the published report that the reductive tricarboxylic acid cycle operates inA. brierleyi, the enzymatic activity of ATP:citrate lyase, the key enzyme of the cycle, was not detected. Instead, activities of acetyl-CoA carboxylase and propionyl-CoA carboxylase, key enzymes of the 3-hydroxypropionate cycle, were detected only whenA. brierleyi was growing autotrophically. We conclude that a modified 3-hydroxypropionate pathway operates inA. brierleyi.

The endoglucanase gene, celCCD, of Clostridium cellulolyticum has been expressed in Escherichia coli. Multiple active polypeptides were detected in the E. coli cells. The relative molecular mass (Mr) of two major active polypeptides were 56 000 (D56) and 38 000 (D38), which were smaller than the deduced Mr of the mature protein (63 401). D56 and D38 were purified from the periplasmic fraction. The N-terminal sequences of the two purified polypeptides were identical to that of the mature endoglucanase (Ala-Ile-Asn-Ser-Gln-Asp-Met-Val---) deduced from the nucleotide sequence. These data indicated that these polypeptides were produced by processing the original mature protein in the C-terminal region. The enzymatic properties of these two polypeptides were very similar, except that the specific activity of D38 was 2–3.5-fold higher than that of D56, and D38 was more heat stable than D56.

ThecbbQandcbbOgenes are located downstream from the RubisCO genes (cbbLS) in the thermophilic hydrogen-oxidizing bacterium,Pseudomonas hydrogenothermophila.Recombinant RubisCO enzymes were purified fromE. colicells which were transformed with plasmids expressingcbbLS, cbbLSQ, cbbLSO,orcbbLSQO.Co-expression ofcbbQand/orcbbOwithcbbLSmade the maximal rates of carboxylation (Vmax) of the recombinant RubisCOs about two-fold higher than that of the enzyme derived from onlycbbLS.The RubisCOs with highVmax also had a high stability when undergoing ultrasonic treatment. The results of the circular dichroism spectra and the 8-anilino-1-naphthalenesulfonate binding assay indicated that these recombinant RubisCOs were conformationally different to each other.

The authors reported four cases of horizontal homonymous sectoranopia (HHS) caused by damage in a more proximal visual pathway than LGB and they speculated upon the mechanism of HHS. (1) Visual field defects of all cases showed a rather high congruity. (2) Neither sectorial optic atrophies nor nerve fiber bundle defects were seen in any of the cases. (3) (1) and (2) are very important clues to differentiate the site of a lesion between the lateral geniculate body and the proximal part of the visual pathway.

The thermo-neutrophilic hydrogen-oxidizing bacterium Hydrogenobacter thermophilusconstitutively oxidizes thiosulfate. Soluble extracts of it showed temperature-dependent thiosulfate oxidation activity with an optimum at 60 °C. A gene cluster for sox(sulfur oxidation) is presumably responsible for this activity. Among the cluster, two tandemly coded soxAgenes were uniquely found. These results suggest that the Sox system is widespread even in thermo-neutrophilic environments.

The nitrite reductase gene ( denA) and the cytochrome c-551 gene ( denB) are located only 50 bp apart from each other in the Pseudomonas aeruginosachromosome. We report evidence that these two genes are co-transcribed as an operon only under anaerobic (denitrifying) conditions. The nucleotide sequence of the promoter (regulatory) region of the operon is highly AT-rich and contains a sequence closely resembling the consensus FNR binding site in E. coli.

The cytochrome c-551 gene from Pseudomonas aeruginosawas cloned by using two oligonucleotide probes, which had been synthesized based on the known primary structure of the protein. The restriction map of the cloned DNA and sequence analysis showed that the cytochrome c-551 gene is located 50 bp downstream of the nitrite reductase gene, which has recently been cloned and sequenced. DNA sequence analysis also indicated that cytochrome c-551 is synthesized in vivo as a precursor having an amino-terminal signal sequence consisting of 22 amino acid residues.

The transcription of the Pseudomonas aeruginosa denABoperon, which consists of the nitrite reductase and cytochrome c‐551 genes, is induced under anaerobic conditions. However, under anaerobic non‐denitrifying conditions (anaerobic growth on arginine), the promoter activity of the operon was approximately one‐fifth of that under anaerobic denitrifying conditions (anaerobic growth in the presence of nitrite or nitrate). This result clearly demonstrates that the presence of nitrite or nitrate activates the transcription of P. aeruginosa denABoperon under anaerobic conditions.

Species belonging to the order Aquificales are believed to be an early branching lineage within the Bacteria. However, the branching order of this group in single-gene phylogenetic trees is highly variable; for example, it has also been proposed that the Aquificales should be grouped with ε-proteobacteria. To investigate the phylogenetic position of Aquificales at the whole-genome level, here we reconstructed the phylogenetic trees of 18 bacteria including six Aquificales species based on the concatenated data of proteins shared by these bacteria. In the phylogenetic tree based on the whole-genome information, Aquificales was more closely related to Thermotogales than to Proteobacteria, suggesting that the Aquificales is a relatively early branching lineage within the Bacteria. Moreover, we classified the phylogenetic tree of each conserved orthologous protein by its topology. As a result, in the most major type of the phylogenetic trees, Aquificales was closely related to the Thermotogales. However, Aquificales was closely related to ε-proteobacteria in 21.0% of all phylogenetic trees, suggesting that many proteins phylogenetically related to the ε-proteobacteria may be encoded in the genomes of the members of the Aquificales. This unique feature may be responsible for the high variability in the branching order of Aquificales in single-gene phylogenetic trees.

The cbbR encoding the LysR-type transcriptional regulator is located downstream of cbbLSQOYA and this gene is located upstream of cbbFPT in divergent transcription. The two promoter regions with LysR-binding sites are located in the cbbL upstream region and in the cbbR –cbbF intergenic region. Electrophoretic mobility shift assays using a cell extract of Escherichia coli harboring a plasmid containing cbbR and the DNA fragments of promoter regions indicated that CbbR binds in both regions. NADPH caused differences in the complex of CbbR and DNA.

The activity of the promoters involved in transcription of the genes (nirS, nirQ and norC) required for anaerobic reduction of nitrite and nitric oxide was investigated in NIR- and NOR-deficient mutants of Pseudomonas aeruginosa. The transcriptional activity of these three promoters was induced by nitrite in a wild-type strain and the activity was low in an nirS mutant. In norCBD and nirQOP mutants, which were expected to accumulate nitric oxide because of a lack of nitric oxide reductase activity, the norC and nirQ promoters showed significantly enhanced activity in promoting transcription relative to the parental strain, even at low nitrite concentrations. These results suggest that the nirQ and norC promoters are regulated by the concentration of endogenous nitric oxide rather than that of nitrite.

Hydrogenobacter thermophilus is an extremely thermophilic and obligately autotrophic hydrogen-oxidising bacterium with various unusual properties and believed to occupy a unique taxonomic position. Inhibitory patterns of various antibiotics on the cell growth of H. thermophilus strain TK-6 clearly showed that the bacterium possessed prokaryote-type systems of DNA, RNA and protein syntheses. Effect of ionophore antibiotics supported that the bacterium was a Gram-negative bacterium, but high sensitivities against macrolide and some other antibiotics and insensitivity against polymyxin B were unusual as a Gram-negative eubacterium. Growth inhibition by cell wall synthesis inhibitors revealed the existence of peptidoglycan on the surface of H. thermophilus, but ineffectiveness of cell wall lytic enzymes (lysozyme and lysostaphin) on intact cells and purified cell wall strongly suggested the uniqueness of the cell wall structure of the bacterium.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) was purified from an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus MH-110. The protein has a Mr value of approximately 110 000, and is composed of two identical subunits of 55 000. To our knowledge, the existence of L2-form RubisCO in a chemolithoautotrophic bacterium is first reported in this paper. The N-terminal amino acid sequence determination of the purified enzyme showed high homology with those of the L2-form RubisCO of Rhodospirillum rubrum and the Lx-form RubisCO from Rhodobacter sphaeroides.