Identification and Gene Disruption of Small Noncoding RNAs in Streptomyces griseus
Identification and Gene Disruption of Small Noncoding RNAs in Streptomyces griseus
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DOI：
10.1128/jb.00087-09
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发表时间：
2009-08-01
影响因子：
3.2
通讯作者：
Horinouchi, Sueharu
中科院分区：
文献类型：
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作者：
Tezuka, Takeaki;Hara, Hirofumi;Horinouchi, Sueharu
小非编码 RNA (sRNA) 已被证明可以控制原核生物中的多种细胞过程。为了鉴定和表征新型细菌 sRNA，我们对革兰氏阳性、土壤栖息的丝状细菌灰色链霉菌进行了检查，假设链霉菌应该表达 sRNA 作为形态和生理分化的重要调节因子。通过生物信息学研究，筛选出54个候选sRNA，它们编码在灰色链霉菌染色体的基因间区域，并且在天蓝色链霉菌A3(2)和阿维链霉菌中高度保守。在这 54 个候选 sRNA 中，通过对从固体培养基上生长的细胞中分离的总 RNA 进行 Northern 印迹分析，检测到 17 个转录本。然后，通过S1核酸酶图谱确定每个sRNA候选基因的转录方向，然后排除被认为是其下游开放阅读框（ORF）核糖开关的四个sRNA候选基因。最后，进一步排除了 sRNA 候选物，因为它与逆转录 PCR 测定的上游 ORF 共转录。因此，在 S. griseus 中鉴定出了 12 个大小从 40 到 300 个核苷酸不等的 sRNA。其中七个显然是以生长阶段依赖的方式转录的。此外，在 12 种 sRNA 中，7 种的表达谱受到 adpA 突变的显着影响，adpA 编码 A 因子调控级联的中央转录调节因子，参与灰色链霉菌的形态分化和次级代谢。然而，所有 12 个 sRNA 基因的破坏并未显示出可检测到的表型变化；所有破坏株均在各种培养基上以与野生型菌株相同的时间生长并形成气生菌丝体和孢子，并与野生型菌株类似地产生链霉素。
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.