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Journal of Bacteriology, August 2009, p. 4896-4904, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00087-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification and Gene Disruption of Small Noncoding RNAs in Streptomyces griseus{triangledown} ,{dagger}

Takeaki Tezuka, Hirofumi Hara,{ddagger} Yasuo Ohnishi, and Sueharu Horinouchi*

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

Received 23 January 2009/ Accepted 14 May 2009

Small noncoding RNAs (sRNAs) have been shown to control diverse cellular processes in prokaryotes. To identify and characterize novel bacterial sRNAs, a gram-positive, soil-inhabiting, filamentous bacterium, Streptomyces griseus, was examined, on the assumption that Streptomyces should express sRNAs as important regulators of morphological and physiological differentiation. By bioinformatics investigation, 54 sRNA candidates, which were encoded on intergenic regions of the S. griseus chromosome and were highly conserved in those of both Streptomyces coelicolor A3(2) and Streptomyces avermitilis, were selected. Of these 54 sRNA candidates, 17 transcripts were detected by Northern blot analysis of the total RNAs isolated from cells grown on solid medium. Then, the direction of transcription of each sRNA candidate gene was determined by S1 nuclease mapping, followed by exclusion of four sRNA candidates that were considered riboswitches of their downstream open reading frames (ORFs). Finally, a further sRNA candidate was excluded because it was cotranscribed with the upstream ORF determined by reverse transcription-PCR. Thus, 12 sRNAs ranging in size from 40 to 300 nucleotides were identified in S. griseus. Seven of them were apparently transcribed in a growth phase-dependent manner. Furthermore, of the 12 sRNAs, the expression profiles of 7 were significantly influenced by a mutation of adpA, which encodes the central transcriptional regulator of the A-factor regulatory cascade involved in both morphological differentiation and secondary metabolism in S. griseus. However, disruption of all 12 sRNA genes showed no detectable phenotypic changes; all the disruptants grew and formed aerial mycelium and spores with the same time course as the wild-type strain on various media and produced streptomycin similarly to the wild-type strain.

* Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81 3 5841-5123. Fax: 81 3 5841-8021. E-mail: asuhori{at}mail.ecc.u-tokyo.ac.jp

{triangledown} Published ahead of print on 22 May 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

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

Journal of Bacteriology, August 2009, p. 4896-4904, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00087-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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