論文 - 赤沼 元気
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EliA facilitates the induction of lipase expression by stearyl alcohol in Ralstonia sp. NT80. 査読あり 国際誌
Genki Akanuma, Hayato Ishibashi, Takahiro Miyagawa, Rie Yoshizawa, Satoru Watanabe, Yu Shiwa, Hirofumi Yoshikawa, Kazutoshi Ushio, Morio Ishizuka
FEMS microbiology letters 339 ( 1 ) 48 - 56 2013年02月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
Extracellular lipase activity from Ralstonia sp. NT80 is induced significantly by fatty alcohols such as stearyl alcohol. We found that when lipase expression was induced by stearyl alcohol, a 14-kDa protein (designated EliA) was produced concomitantly and abundantly in the culture supernatant. Cloning and sequence analysis revealed that EliA shared 30% identity with the protein-like activator protein of Pseudomonas aeruginosa, which facilitates oxidation and assimilation of n-hexadecane. Inactivation of the eliA gene caused a significant reduction in the level of induction of lipase expression by stearyl alcohol. Furthermore, turbidity that was caused by the presence of emulsified stearyl alcohol, an insoluble material, remained in the culture supernatant of the ΔeliA mutant during the late stationary phase, whereas the culture supernatant of the wild type at 72 h was comparatively clear. In contrast, when lipase expression was induced by polyoxyethylene (20) oleyl ether, a soluble material, inactivation of eliA did not affect the extracellular lipase activity greatly. These results strongly indicate that EliA facilitates the induction of lipase expression, presumably by promoting the recognition and/or incorporation of the induction signal that is attributed to stearyl alcohol.
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Single mutations introduced in the essential ribosomal proteins L3 and S10 cause a sporulation defect in Bacillus subtilis. 査読あり
Genki Akanuma, Shota Suzuki, Koichi Yano, Hideaki Nanamiya, Yousuke Natori, Eri Namba, Kazuya Watanabe, Kazumi Tagami, Takuya Takeda, Yuka Iizuka, Ako Kobayashi, Morio Ishizuka, Hirofumi Yoshikawa, Fujio Kawamura
The Journal of general and applied microbiology 59 ( 2 ) 105 - 17 2013年
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：MICROBIOL RES FOUNDATION
We introduced single mutations into the rplC and rpsJ genes, which encode the essential ribosomal proteins L3 (RplC) and S10 (RpsJ), respectively, and are located in the S10 gene cluster of the gram-positive, endospore-forming bacterium Bacillus subtilis, and examined whether these mutations affected their growth rate, sporulation, competence development and 70S ribosome formation. Mutant cells harboring the G52D mutation in the L3 ribosomal protein, which is located at the peptidyl transferase center of 50S, accumulated 30S subunit at 45°C, probably due to a defect in 50S formation, and exhibited a reduction in the sporulation frequency at high temperature. On the other hand, mutant cells harboring the H56R mutation in the S10 protein, which is located near the aminoacyl-tRNA site of 30S, showed severe growth defect and deficiency in spore formation, and also exhibited significant delay in competence development.
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Inactivation of ribosomal protein genes in Bacillus subtilis reveals importance of each ribosomal protein for cell proliferation and cell differentiation. 査読あり 国際誌
Genki Akanuma, Hideaki Nanamiya, Yousuke Natori, Koichi Yano, Shota Suzuki, Shuya Omata, Morio Ishizuka, Yasuhiko Sekine, Fujio Kawamura
Journal of bacteriology 194 ( 22 ) 6282 - 91 2012年11月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：AMER SOC MICROBIOLOGY
Among the 57 genes that encode ribosomal proteins in the genome of Bacillus subtilis, a Gram-positive bacterium, 50 genes were targeted by systematic inactivation. Individual deletion mutants of 16 ribosomal proteins (L1, L9, L15, L22, L23, L28, L29, L32, L33.1, L33.2, L34, L35, L36, S6, S20, and S21) were obtained successfully. In conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in B. subtilis, at least for cell proliferation. Although several mutants that harbored a deletion of a ribosomal protein gene did not show any significant differences in any of the phenotypes that were tested, various mutants showed a reduced growth rate and reduced levels of 70S ribosomes compared with the wild type. In addition, severe defects in the sporulation frequency of the ΔrplA (L1) mutant and the motility of the ΔrpsU (S21) mutant were observed. These data provide the first evidence in B. subtilis that L1 and S21 are required for the progression of cellular differentiation.
DOI： 10.1128/JB.01544-12
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Proteomic analysis of the Streptomyces griseus ribosomal fraction. 査読あり 国際誌
Genki Akanuma, Hideaki Nanamiya, Yoshihiro Mouri, Morio Ishizuka, Yasuo Ohnishi
Bioscience, biotechnology, and biochemistry 76 ( 12 ) 2267 - 74 2012年
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：TAYLOR & FRANCIS LTD
The Streptomyces griseus 70S ribosome fraction was analyzed by radical-free and highly reducing two-dimensional (RFHR 2D) gel electrophoresis and mass spectrometry. Among the 60 putative ribosomal proteins that are encoded by the S. griseus genome, 48 were identified in the 70S ribosome fraction prepared from mycelia grown in liquid culture for 12, 36, and 48 h. Ribosomal protein S3 was detected at two different positions on the 2D gel, and the distribution changed completely in the course of the growth, suggesting that it was modified or processed. The SGR3624 protein was also identified in the 70S ribosome fraction, but detailed cellular fractionation analysis indicated that it localizes mainly at the membrane rather than the ribosome. An SGR3624-deleted mutant showed slow growth on solid media, indicating that SGR3624 has an important role in the growth of the substrate mycelium in solid culture.
DOI： 10.1271/bbb.120556
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Control of aerial mycelium formation by the BldK oligopeptide ABC transporter in Streptomyces griseus 査読あり 国際誌
Genki Akanuma, Masayoshi Ueki, Morio Ishizuka, Yasuo Ohnishi, Sueharu Horinouchi
FEMS MICROBIOLOGY LETTERS 315 ( 1 ) 54 - 62 2011年02月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC
An oligopeptide permease family ATP-binding cassette (ABC) transporter encoded by SGR2418-SGR2414 was shown to be essential for aerial mycelium formation on glucose-containing media in Streptomyces griseus. In spite of only weak sequence similarity, the operon was equivalent to the bldK operon of Streptomyces coelicolor A3(2) in terms of chromosomal location and function. Transcription of the operon appeared not to be directly regulated by AdpA, a global regulator of morphological and physiological development in S. griseus, although it was affected by adpA inactivation. This study revealed that an ABC transporter was essential for aerial mycelium formation not only in S. coelicolor A3(2) but also in S. griseus, indicating that extracellular signaling by certain peptides should be conserved among different Streptomyces species.
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Bacillus subtilis mutants harbouring a single copy of the rRNA operon exhibit severe defects in growth and sporulation 査読あり 国際誌
Hideaki Nanamiya, Makiko Sato, Kenta Masuda, Mikiko Sato, Tetsuya Wada, Shota Suzuki, Yousuke Natori, Masato Katano, Genki Akanuma, Fujio Kawamura
MICROBIOLOGY-SGM 156 2944 - 2952 2010年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：SOC GENERAL MICROBIOLOGY
The number of copies of rRNA genes in bacterial genomes differs greatly among bacterial species. It is difficult to determine the functional significance of the heterogeneity of each rRNA operon fully due to the existence of multiple rRNA operons and because the sequence heterogeneity among the rRNA genes is extremely low. To overcome this problem, we sequentially deleted the ten rrn operons of Bacillus subtilis and constructed seven mutant strains that each harboured a single rrn operon (either rrnA, B, D, E, I, J or O) in their genome. The growth rates and sporulation frequencies of these mutants were reduced drastically compared with those of the wild-type strain, and this was probably due to decreased levels of ribosomes in the mutants. Interestingly, the ability to sporulate varied significantly among the mutant strains. These mutants have proved to be invaluable in our initial attempts to reveal the functional significance of the heterogeneity of each rRNA operon.
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Dynamic changes in the extracellular proteome caused by absence of a pleiotropic regulator AdpA in Streptomyces griseus 査読あり 国際誌
Genki Akanuma, Hirofumi Hara, Yasuo Ohnishi, Sueharu Horinouchi
MOLECULAR MICROBIOLOGY 73 ( 5 ) 898 - 912 2009年09月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC
P>In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers morphological development and secondary metabolism by inducing a pleiotropic transcriptional regulator AdpA. Extracellular proteome analysis of the wild-type and Delta adpA strains grown to the end of the exponential phase in liquid minimal medium revealed that 38 secreted proteins, including many catabolic enzymes, such as protease, glycosyl hydrolase and esterase, were produced in an AdpA-dependent manner. Transcriptome analysis showed that almost all of these AdpA-dependent secreted proteins were regulated at the transcriptional level. In vitro AdpA-binding assays and determination of transcriptional start sites led to identification of 11 promoters as novel targets of AdpA. Viability staining revealed that some hyphae lysed during the exponential growth phase, which could explain the detection of 3 and 23 cytoplasmic proteins in the culture media of the wild-type and Delta adpA strains respectively. In the wild-type strain, due to high protease activity in the culture medium, cytoplasmic proteins that leaked from dead cells seemed to be degraded and reused for the further growth. The existence of many AdpA-dependent (i.e. A-factor-inducible) secreted catabolic enzymes, which are likely involved in the assimilation of material that leaked from dead cells, reemphasizes the importance of A-factor in the morphological differentiation of S. griseus.
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Biosynthesis of aliphatic polyketides by type III polyketide synthase and methyltransferase in Bacillus subtilis. 査読あり 国際誌
Nakano C, Ozawa H, Akanuma G, Funa N, Horinouchi S
J Bacteriol 2007年08月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Biosynthesis of aliphatic polyketides by type III polyketide synthase and methyltransferase in Bacillus subtilis. 査読あり 国際誌
Natori Y, Nanamiya H, Akanuma G, Kosono S, Kudo T, Ochi K, Kawamura F
Mol Microbiol 2007年07月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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A fail-safe system for the ribosome under zinc-limiting conditions in Bacillus subtilis. 査読あり 国際誌
Natori Y, Nanamiya H, Akanuma G, Kosono S, Kudo T, Ochi K, Kawamura F
Molecular microbiology 63 ( 1 ) 294 - 307 2007年01月
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Construction and characterization of Bacillus subtilis deletion mutants lacking the prophage 2-trnS region 査読あり 国際誌
G Akanuma, C Habu, Y Natori, R Murayama, H Nanamiya, F Kawamura
FEMS MICROBIOLOGY LETTERS 258 ( 2 ) 220 - 226 2006年05月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：BLACKWELL PUBLISHING
During development of a novel method for constructing a series of deletions in Bacillus subtilis using an isogenic set of gene-disrupted mutants created by integration of pMutin, deletion of the trnS operon, consisting of seven tRNA genes, was found to affect cell growth, development of competence and spore formation. A suppressor (sts1) of the Delta trnS mutant was isolated, sequenced and found to have undergone a single base change, (C) under bar AG to (G) under bar AG, in the first anticodon of tRNA(Leu), in the trnB operon.
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Construction of Bacillus subtilis strains carrying the transcriptional bgaB fusion with the promoter region of each rrn operon and their differential transcription during spore development 査読あり 国際誌
Keiko Koga, Akihiko Ikegami, Kaoru Nakasone, Rikinori Murayama, Genki Akanuma, Yousuke Natori, Hideaki Nanamiya, Fujio Kawamura
JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 52 ( 2 ) 119 - 124 2006年04月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：MICROBIOL RES FOUNDATION
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Liberation of zinc-containing L31 (RpmE) from ribosomes by its paralogous gene product, YtiA, in Bacillus subtilis 査読あり 国際誌
G Akanuma, H Nanamiya, Y Natori, N Nomura, F Kawamura
JOURNAL OF BACTERIOLOGY 188 ( 7 ) 2715 - 2720 2006年04月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：AMER SOC MICROBIOLOGY
We have found that alternative localization of two types of L31 ribosomal protein, RpmE and YtiA, is controlled by the intracellular concentration of zinc in Bacillus subtilis. The detailed mechanisms for the alternation of L31 proteins under zinc-deficient conditions were previously unknown. To obtain further information about this regulatory mechanism, we have studied the stability of RpmE in vivo and the binding affinity of these proteins to ribosomes in vitro, and we have found that liberation of RpmE from ribosomes is triggered by the expression of ytiA, which is induced by the derepression of Zur under zinc-deficient conditions.
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Spontaneous transformation and its use foir genetic mapping in Bacillus subtilis 査読あり 国際誌
R Murayama, G Akanuma, Y Makino, H Nanamiya, F Kawamura
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 ( 8 ) 1672 - 1680 2004年08月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：TAYLOR & FRANCIS LTD
Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.
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Zinc is a key factor in controlling alternation of two types of L31 protein in the Bacillus subtilis ribosome 査読あり 国際誌
H Nanamiya, G Akanuma, Y Natori, R Murayama, S Kosono, T Kudo, K Kobayashi, N Ogasawara, SM Park, K Ochi, F Kawamura
MOLECULAR MICROBIOLOGY 52 ( 1 ) 273 - 283 2004年04月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：BLACKWELL PUBLISHING LTD
We have analysed changes in the composition of ribosomal proteins during cell growth in Bacillus subtilis. Ribosome fractions were prepared from B. subtilis cells at different phases of growth and were separated by radical-free and highly reducing (RFHR) two-dimensional polyacrylamide gel electrophoresis. We identified 50 ribosomal proteins, including two paralogues of L31 protein (RpmE and YtiA). Although the ribosome fraction extracted from exponentially growing cells contained RpmE protein, this protein disappeared during the stationary phase. In contrast, YtiA was detected in the ribosome fraction extracted after the end of exponential growth. Expression of the ytiA gene encoding YtiA was found to be negatively controlled by Zur, a zinc-specific transcriptional repressor that controls zinc transport operons. Analysis by inductively coupled plasma mass spectrometry (ICP-MS) indicated that RpmE contains one zinc ion per molecule of protein. In addition, mutagenesis of the rpmE gene encoding RpmE revealed that Cys-36 and Cys-39, located within a CxxC motif, are required not only for binding zinc but also for the accumulation of RpmE in the cell. Taken together, these results indicate that zinc plays an essential role in the alternation between two types of L31 protein in the ribosome of B. subtilis.