RNA-seq of Streptomyces coelicolor and Streptomyces davawensis including a dodecin gene deletion variant in response to plumbagin treatment
ABSTRACT: Genes encoding dodecin proteins are present in almost 20% of archaeal and in more than 50% of bacterial genomes. Archaeal dodecins bind riboflavin (vitamin B2), are thought to play a role in flavin homeostasis and possibly also help to protect cells from radical or oxygenic stress. Bacterial dodecins were found to bind riboflavin-5’-phosphate (also called flavin mononucleotide or FMN) and coenzyme A, but their physiological function remained unknown. In this study, we set out to investigate the relevance of dodecins for flavin metabolism and oxidative stress management in the phylogenetically related bacteria Streptomyces coelicolor and Streptomyces davawensis. Therfore we have treated Streptomyces coelicolor wildtype, Streptomyces davawensis wildytype and Streptomyces davawensis dodecin deletion strain with plumbagin, a compound, which induces oxidative stress in exponential and stationary growth phase and analysed the transcriptome via RNA-seq (Illumina TruSeq stranded mRNA libraries sequenced on Illumina HiSeq rapid mode 2 x 70nt PE).
Project description:SigE is a sigma factor found in Streptomyces species and is the key regulator of cell envelope stress response in Streptomyces coelicolor. To determine the regulon of SigE under conditions of cell envelope stress, 10 ug / ml vancomycin was used to induce cell envelope stress in the wild type and a sigE deletion strain of S. coelicolor. Samples were taken 0, 30, 60 and 90 minutes after addition of vancomycin and subjected to transcriptional profiling using Affymetrix arrays. Note – equivalent data for wild type Streptomyces coelicolor M600 is submitted as part of E-MEXP-3032. The data for both M600 (E-MEXP-3032) and J2130 (this submission) were generated at the same time employing the same protocols.
Project description:We identified genome-wide binding regions of NdgR in Streptomyces coelicolor using chromatin immunoprecipitation sequencing (ChIP-seq). We constructed 6×myc-tagged NdgR strain using homologous recombination with myc-tagging vector. Analysis of the sequencing data aligned to Streptomyces coelicolor genome database (NC_003888). Overall design: 6x-myc tagged Streptomyces coelicolor M145 was cultured on solid minimal media supplemented with N-acetylglucosamine and asparagine during 36hrs. Anti-myc antibody (9E10) was used for immunoprecipitation (IP). IP-DNA was sequenced using Illumina Genome Analyzer Ⅱx
Project description:This study compared the genome of Streptomyces rimosus rimosus against that of Streptomyces coelicolor. It also compared 4 strains with changes in oxytetracycline production and derived from G7, the type strain, against G7. Keywords: Comparative genomic hybridization Overall design: Duplicate samples, each replicate may be found in the corresponding supplementary file for the Sample
Project description:RNA-Seq of roseoflavin producer Streptomyces davaonensis reveals transcription of unusual high number of riboflavin biosynthetic genes (rib genes) in exponential and stationary growth phase. In this study mRNA from three biological replicates of Streptomyces davaonensis harvested in exp. (after 18 h of growth) and stationary growth phase (after 48 h of growth) were sequenced after Illumina Ribozero treatment via Illumina TruSeq stranded mRNA libs on Illumina HiSeq1500. Moreover primary 5′-end-enriched cDNA libraries were prepared according to Peifer-Sancar et al. (2013) and sequenced on Illumina MiSeq system.
Project description:We have integrated nucleotide resolution genome-scale measurements of the transcriptome and translatome of the Streptomyces coelicolor A3(2), the model antibiotic-producing actinomycete. Our systematic study determined 3,473 transcription start sites, leading to discovery of a high proportion (~21%) of leaderless mRNAs and 230 non-coding RNAs; this enabled deduction of promoter architecture on a genome-scale. Ribosome profiling analysis revealed that the translation efficiency was negatively correlated for secondary metabolic genes. These results provide novel fundamental insights into translational regulation of secondary metabolism that enables rational synthetic biology approaches to awaken such ‘silent’ secondary metabolic pathways. Profiles of primary transcripts, whole transcripts, and ribosome protected fragments (RPFs) of Streptomyces coelicolor were generated by deep sequencing using Illumina Miseq.
Project description:Comparative analyses of the transcriptomes of the Streptomyces coelicolor A3(2) wild-type and the generated hbpSc-senSc-senRc (hsr) mutant under native and oxidative-stressing conditions allowed to identify differentially expressed genes, whose products may enhance the anti-oxidative defense of the bacterium. To obtain well-grown mycelia, spores of S. coelicolor (WT and hsr mutant) were inoculated in 10 ml R2 medium and grown as standing culture at 30C for 16 h and afterwards on a rotary shaker for 16 h after the addition of 90 ml R2 medium. The cultures were washed four times in minimal medium without supplement. The mycelia were suspended in 50 ml R2 medium and divided in two 25 ml-portions, one of which contained H2O2 (0.15 mM). Cultivation was continued at 30 C on a rotary shaker for two hours. Mycelia were harvested by centrifugation and the mycelia pellets were store at -80 C. Total RNA was isolated from two biological replicates. cDNA libraries of RNA from of S. coelicolor (WT and hsr mutant) were constructed using the TruSeq Stranded mRNA Library Prep Kit (Illumina,San Diego, CA, USA), and subsequently sequenced paired-end on an Illumina MiSeq system (San Diego, CA, USA) using 75 bp read length.
Project description:The Streptomyces coelicolor two genes operon SCO5784-SCO5785 encodes a two-component system which functions in a similar manner to that of the Bacillus subtilis DegS-DegU system. Propagation of the regulatory gene in high copy number results in the overproduction of several extracellular enzymes, among them the major extracellular protease, as well as in a higher level of synthesis of the antibiotic actinorhodin. This two-component system seems to control various processes characterised by the transition from primary to secondary metabolism in S. coelicolor, as determined by proteomic and transcriptomic analices. The presence of the regulatory gene in high copy number in S. coelicolor additionally seems to elicit a stringent response in the bacterial cell. Therefore, we propose renaming S. coelicolor genes SCO5784 and SCO5785 as degS and degU, respectively. All microarray analyses were performed with RNA samples obtained from three independent cultures grown under identical conditions. Hybridisation assays were carried out with cDNA obtained from RNA extracted at the late exponential phase of growth (24h). The transcriptional profile of wild type (S. coelicolor M145) cells carrying the multicopy plasmid pIJ487 was compared with that of the same strain carrying the degU gene cloned in the same plasmid under the control of its own promoter (S. coelicolor M28). And the transcriptional profile of wild type (S, coelicolor M145) cells was compared to that of the DegU deficient strain (S. coelicolor I32).