Frontiers MediaActa Medica Okayama1664-462X152024Illumina-based transcriptomic analysis of the fast-growing leguminous tree Acacia crassicarpa: functional gene annotation and identification of novel SSR-markers1339958ENShougoIshioTsukuba Research Institute, Sumitomo Forestry Co. Ltd.KazutakaKusunokiTsukuba Research Institute, Sumitomo Forestry Co. Ltd.MichikoNemotoGraduate School of Environment, Life, Natural Science and Technology, Okayama UniversityTadayoshiKanaoGraduate School of Environment, Life, Natural Science and Technology, Okayama UniversityTakashiTamuraInstitute of Global Human Resource Development, Okayama UniversityAcacia crassicarpa is a fast-growing leguminous tree that is widely cultivated in tropical areas such as Indonesia, Malaysia, Australia, and southern China. This tree has versatile utility in timber, furniture, and pulp production. Illumina sequencing of A. crassicarpa was conducted, and the raw data of 124,410,892 reads were filtered and assembled de novo into 93,317 unigenes, with a total of 84,411,793 bases. Blast2GO annotation, Benchmark Universal Single-Copy Ortholog evaluation, and GO-term classification produced a catalogue of unigenes for studying primary metabolism, phytohormone signaling, and transcription factors. Massive transcriptomic analysis has identified microsatellites composed of simple sequence repeat (SSR) loci representing di-, tri-, and tetranucleotide repeat units in the predicted open reading frames. Polymorphism was induced by PCR amplification of microsatellite loci located in several genes encoding auxin response factors and other transcription factors, which successfully distinguished 16 local trees of A. crassicarpa tested, representing potentially exploitable molecular markers for efficient tree breeding for plantation and biomass exploitation.No potential conflict of interest relevant to this article was reported.Frontiers MediaActa Medica Okayama1664-302X152024Tetrathionate hydrolase from the acidophilic microorganisms1338669ENTadayoshiKanaoDepartment of Agricultural and Biological Chemistry, Graduate School of Environment, Life, Natural Science, and Technology, Okayama UniversityTetrathionate hydrolase (TTH) is a unique enzyme found in acidophilic sulfur-oxidizing microorganisms, such as bacteria and archaea. This enzyme catalyzes the hydrolysis of tetrathionate to thiosulfate, elemental sulfur, and sulfate. It is also involved in dissimilatory sulfur oxidation metabolism, the S-4-intermediate pathway. TTHs have been purified and characterized from acidophilic autotrophic sulfur-oxidizing microorganisms. All purified TTHs show an optimum pH in the acidic range, suggesting that they are localized in the periplasmic space or outer membrane. In particular, the gene encoding TTH from Acidithiobacillus ferrooxidans (Af-tth) was identified and recombinantly expressed in Escherichia coli cells. TTH activity could be recovered from the recombinant inclusion bodies by acid refolding treatment for crystallization. The mechanism of tetrathionate hydrolysis was then elucidated by X-ray crystal structure analysis. Af-tth is highly expressed in tetrathionate-grown cells but not in iron-grown cells. These unique structural properties, reaction mechanisms, gene expression, and regulatory mechanisms are discussed in this review.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama2186-77551092020酸性鉱山廃水の 効果的な生物的処理プロセスの開発2936ENKazuoKamimuraGraduate School of Environmental and Life Science, Okayama UniversityTadayoshiKanaoGraduate School of Environmental and Life Science, Okayama University Acid mine drainage (AMD) is acidic and generally enriched with iron, aluminum, sulfate and heavy metals, such as lead and cadmium. AMD is a growing problem of emerging concern that cause detrimental effects to the environment and living organisms. Yanahara mine in Misaki Town, Okayama, Japan, had mainly produced pyrite for sulfuric acid manufacture. Although it was closed in 1991, AMD is being generated from the mine now. A passive treatment based on the biological oxidation of ferrous iron is a promising strategy for AMD remediation. AMD from Yanahara mine is treated in a plant using iron-oxidizing bacteria, Acidithiobacillus ferrooxidans and Ferrovum spp. The AMD generation continues for several centuries with dramatic consequences on the receiving environments. Therefore, the development of sustainable and cost effective treatment process is required. A development of the effective biological treatment process with an iron oxidation reactor operated at pH 3.5 is described in this report. Economic aspects are also discussed.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama2186-77551042015Characterization of a putative chromosome segregation and condensation protein (ScpB) in an acidophilic iron‒oxidizing bacterium Acidithiobacillus ferrooxidans512ENKazuoKamimuraNozomuNagataMeiKikumotoSultanaSharminSatoshiWakaiTadayoshiKanao Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide. This bacterium uses ferrous iron and reduced inorganic sulfur compounds (RISCs) as energy sources. Transcriptions of genes thought to be involved in the oxidation of RISCs have been known to be highly activated in A. ferrooxidans cells grown on RISCs, while transcriptions of genes involved in the iron oxidation were repressed in the cells grown on
RISCs. A gene encoding a putative chromosome segregation and condensation protein (ScpB) with a
helix-turn-helix motif was found in the upstream region of sulfide : quinone oxidoreductase gene, whose expression was up-regulated in cells grown in sulfur and tetrathionate. A semi-quantitative PCR analysis using cDNA prepared from iron-, sulfur-, or tetrathionate-grown cells revealed that the transcription of scpB was up-regulated in cells grown on sulfur or tetrathionate as the energy source. Electrophoretic mobility shift assays were employed to examine whether the ScpB functions as a transcription factor. The result indicated that the recombinant His-tagged ScpB protein was able to nonspecifically bind in
vitro to DNA. This is the first report on a direct association of ScpB with DNA.No potential conflict of interest relevant to this article was reported.Springer JapanActa Medica Okayama1431-06511532011Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans403410ENMohammed AbulManchurMeiKikumotoTadayoshiKanaoJunTakadaKazuoKamimuraAn OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75A degrees C or heated to 100A degrees C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at a parts per thousand currency sign40A degrees C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549512006Discrimination among the Three Acidithiobacillus Species, A. ferrooxidans, A. thiooxidans and A. caldus, Based on Restriction Fragment Length Polymorphism Analysis of the 16S-23S rDNA Intergenic Spacer Region711ENSatoshiWakaiKentaroYamamotoTadayoshiKanaoTsuyoshiSugioKazuoKamimuraThe PCR-amplified 16S-23S rDNA intergenic spacer regions (ISRs) of Acidithiobacillus ferrooxidans, A. thiooxidans, and A. caldus strains were scquenced and evaluated for differentiation and identification of these bacteria. The total length of the 16S-23S ISRs of A. ferrooxidans and A. thiooxidans strains and A.caldus GO-1 were 441, 456, nd 379bp, respectively. Two genes. encoding tRNA and tRNA, and the box A-like sequences were highly conserved in the ISRs of all Acidithiobacillus species. The restriction fragment length polymorphism (RFLP) profiles of the PCR-amplified 16S-23S rDNA ISRs digested by HaeIII and AluI could clearly discriminate A. ferrooxidans from A. thiooxidans and A. caldus. The results indicated that RFLP analysis of the 16S-23S ISRs is an easy and rapid method for discrimination and identification of Acidithiobacillus species.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549412005還元的 ＴＣＡ 回路の鍵酵素 ATP-citrate lyase について3945ENTadayoshiKanaoAlmost all organic compounds which construct life are derived from carbon dioxide (CO2) assimilated by autotrrophic organisms.The reductive tricarboxylic acid (RTCA) cycle functions as a carbon dioxide fixation pathway, distinct from Calvin cycle, in a green sulfur bacterium Chlorobium limicola. ATP-citrate lyase (ACL) is one of the key enzymes of this cycle. The enzyme of C. limicola (Cl-ACL) was encoded in two adjacent open reading frames, aclB(1197bp) and aclA(1827bp), whose products showed signficant similarity to the N- and C-terminal regions of the human enzyme, respectively. Heterologous expression of these genes in Escherichia coli proved that both gene products were essential for ACL activity. Kinetic examination of the enzyme revealed that the enzyme displayed typical Michaelis-Monten kinetics toward ATP with an apparent Km value of 0.19mM. However, strong negative cooperativity was observed with respect to citrate binding. ADP was a competitive inhibitor of ATP with a Kj value of 0.036mM. Together with the feature that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic properties indicated that Cl-ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola.No potential conflict of interest relevant to this article was reported.