SummaryVirginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a gbutyrolactone autoregulator and the corresponding receptor protein of the Streptomyces species, and has been shown to regulate the production of antibiotic virginiamycin (VM) in S. virginiae. A divergently transcribed barX gene is situated 259 bp upstream of the barA gene, and the BarX protein has been shown to be highly homologous (39.8% identity, 74.6% similarity) to S. griseus AfsA. Although AfsA is thought to be a biosynthetic enzyme for A-factor, another member of the family of g-butyrolactone autoregulators, the in vivo function of S. virginiae BarX was investigated in this study by phenotypic and transcriptional comparison between wild-type S. virginiae and a barX deletion mutant. With the same growth rate as wild-type S. virginiae on both solid and liquid media, the barX mutant showed no apparent changes in its morphological behaviour, indicating that barX does not participate in morphological control in S. virginiae. However, the barX mutant became more sensitive to virginiamycin M 1 than did the wild-type strain (minimum inhibitory concentration, 50 mg ml 21 compared with . 200 mg ml 21 ) and exhibited reduced VB and VM production. The VM production was not restored by exogenous addition of VB, suggesting that BarX per se is not a biosynthetic enzyme of VBs but a pleiotropic regulatory protein controlling VB biosynthesis. DNA sequencing of a 5.6 kbp downstream region of barX revealed the presence of five open reading frames (ORFs): barZ, encoding a BarB-like regulatory protein; orf2, encoding a Streptomyces coelicolor RedDlike pathway specific regulator; varM, encoding a homologue of ATP-dependent transporters for macrolide antibiotics; orf4, encoding a homologue of b-ketoacyl ACP/CoA reductase; and orf5, encoding a homologue of dNDP-glucose dehydratase. Reverse transcription polymerase chain reaction (RT-PCR) analyses of the downstream five genes together with those of the three upstream genes (barA, barB, encoding a regulatory protein; and varS, encoding a virginiamycin S specific transporter) revealed that, in the barX mutant, the transcriptions of barZ, orf2, varM and orf5 were completely repressed and those of barB and varS were derepressed. Because free BarA (BarA in the absence of VB) in wild-type S. virginiae represses the transcription of bicistronic barB±varS operon through binding to a specific DNA sequence (BarA-responsive element, BARE) overlapping the barB transcriptional start site, the derepression of barB±varS transcription in the barX mutant suggested that the in vivo function of BarA was impaired by the lack of BarX protein. Gel-shift assays revealed that BarA easily lost its DNA-binding activity in the absence of BarX but that the defect was restored by the presence of recombinant BarX as a fusion with maltose-binding protein (MBP±BarX), whereas MBP± BarX itself showed no DNA-binding activity, indicating that BarX is likely to be a co-repressor of BarA, enforcing the DNA-bindin...

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Methylotrophs, which can utilize methanol as a sole carbon source, are promising microorganisms to be exploited in a methanol-based bioeconomy, in which a variety of useful compounds are biotechnologically produced from natural gas-derived methanol. Pinkpigmented facultative methylotrophs (PPFMs) are common plant phyllospheric bacteria and are known to enhance seedling growth and total biomass of various plants. However, improvement of crop yield by inoculation of PPFMs at the field level has not been well investigated. We herein describe improvement of crop yield of several rice cultivars by foliar spraying of PPFMs. After selection of PPFM strains and rice cultivars by the in vitro seedling growth test, we further conducted paddy field experiments. The crop yield of the sake-brewing rice Oryza sativa cultivar Hakutsurunishiki was reproducibly improved in a commercial paddy field for over a 5-year period. A one-time foliar spray of PPFM cells (living or killed) or a cell wall polysaccharide fraction, after the heading date, acted in the phyllosphere and effectively improved crop yield. Our results show that the established process with PPFMs is feasible for improvement of food production in the methanol bioeconomy.

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Gas chromatography/mass spectrometry(GC/MS) -based metabolomics technology is a useful tool for comprehensive profiling of low-molecular-weight hydrophilic compounds in various samples. In this study, we examined whether the characteristics of sake according to the type of yeast strain and rice cultivar could be clarified by this technology. We analyzed hydrophilic compounds in 15 Daiginjo-shu samples by GC/MS. As a result, we detected 80 peaks from the chromatogram data of sake, and 51 compounds were identified among them. By multivariate analysis, such as principal component analysis(PCA) and partial least square-discriminant analysis(PLS-DA) , we were able to clarify the differences among component profiles of sake by yeast strain and rice cultivar.Key words： 清酒(sake) ，メタボロミクス(metabolomics) ，ガスクロマトグラフィー／質量分析(gas chromatography/mass spectrometry) ──────────────────────────────────