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DNA microarray analysis of global gene regulation by A-factor in Streptomyces griseus

Hirofumi Hara

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

A-factor (2-isocapryloyl-3R-hydroxymethyl--butyrolactone) is a microbial hormone that triggers morphological differentiation and secondary metabolism in Streptomyces griseus. The effects of A-factor on global gene expression were determined by DNA microarray analysis of transcriptomes obtained with the A-factor-deficient mutant afsA. A-factor was added at a concentration of 25 ng ml^–1 to mutant afsA at the middle of the exponential growth phase, and RNA samples were prepared from the cells grown after A-factor addition for a further 5, 15 and 30 min, and 1, 2, 4, 8 and 12 h. The effects of A-factor on transcription of all protein-coding genes of S. griseus were evaluated by comparison of the transcriptomes with those obtained from cells grown in the absence of A-factor. Analysis of variance among the transcriptomes revealed that 477 genes, which were dispersed throughout the chromosome, were differentially expressed during the 12 h after addition of A-factor, when evaluated by specific criteria. Quality threshold clustering analysis with regard to putative polycistronic transcriptional units and levels of upregulation predicted that 152 genes belonging to 74 transcriptional units were probable A-factor-inducible genes. Competitive electrophoretic mobility shift assays using DNA fragments including putative promoter regions of these 74 transcriptional units suggested that AdpA bound 37 regions to activate 72 genes in total. Many of these A-factor-inducible genes encoded proteins of unknown function, suggesting that the A-factor regulatory cascade of S. griseus affects gene expression at a specific time point more profoundly than expected.

Correspondence
Sueharu Horinouchi
asuhori{at}mail.ecc.u-tokyo.ac.jp

Present address: Department of Biomedical Engineering, Okayama University of Science, Ridaicho, Kita-ku, Okayama 700-0005, Japan.

Details of the microarray design, transcriptome experimental design, and transcriptome data have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE13804.

Twelve supplementary tables, listing PCR primers used in this study, genes differentially expressed at various times after A-factor addition, and genes classified into clusters 1 and 2, are available with the online version of this paper.