E-MTAB-7715
The tremendous effect of the rational ribosomal engineering on the transcriptome in Streptomyces albus nalyzed by RNA-seq of coding RNA.
To elucidate the effect of the rational ribosomal engineering on the changes in the expression of the endogenious biosynthetic gene clusters the transcriptome analysis was performed. The Streptomyces albus strains carrying mutations in rpsL gene (encode for ribosomal protein S12) and the deletion of the rsmG gene (16S rRNA methyltransferase G), as well as their combination were used for the experiment. The list of the strains with mutations is next: S. albus K88E, S. albus GI92, S. albus K88E-GI92, S. albus P91S, S. albus K88E-P91S, S. albus del rsmG, S. albus K88E-GI92 del rsmG, S. albus K88E-P91S del rsmG. Abovementioned strains along with S. albus native strain were grown in NL-19 production medium. Samples were harvested by centrifugation after 48 and 72 hours of cultivation. For total RNA isolation, S. albus cells were grown in SG medium (for ara expression) or NL19 medium (for indigenous BGC expression). Then, 2 ml of 2-day and 3-day cultures was spun down for 20 s at 14,000 × rpm, and the pellets were immediately frozen in liquid nitrogen and stored at ?80 °C. Total RNA extraction was performed using an RNeasy Kit (Qiagen, Hilden, Germany) as previously described (Huser et al. 2003). An RNase-Free DNase set (Qiagen) was used two times for on-column DNA digestion, and an additional DNase treatment was then performed with a DNase I kit (Roche Diagnostics, Mannheim, Germany) to ensure that all DNA was completely removed. To check the RNA samples for DNA contamination, PCR was performed using oligonucleotides designed to create two different products approximately 150 bp and 500 bp in size. Initially, RNA quality was checked with Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kits on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A Ribo-Zero rRNA Removal Kit for bacteria was obtained from Illumina (San Diego, CA, USA) and used to remove ribosomal RNA molecules from isolated total RNA. rRNA removal was checked using an Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) was used to prepare cDNA libraries (Koepff et al. 2017). The resulting cDNAs were pair-end sequenced on an Illumina HiSeq 1500 system (San Diego, CA, USA) using a 70 bp read length. Short read alignments and differential gene expression were illustrated using ReadXplorer 2.2.0 (Hilker et al. 2014) and DEseq (Anders and Huber 2010), respectively.
To elucidate the effect of the rational ribosomal engineering on the changes in the expression of the endogenious biosynthetic gene clusters the transcriptome analysis was performed. The Streptomyces albus strains carrying mutations in rpsL gene (encode for ribosomal protein S12) and the deletion of the rsmG gene (16S rRNA methyltransferase G), as well as their combination were used for the experiment. The list of the strains with mutations is next: S. albus K88E, S. albus GI92, S. albus K88E-GI92, S. albus P91S, S. albus K88E-P91S, S. albus del rsmG, S. albus K88E-GI92 del rsmG, S. albus K88E-P91S del rsmG. Abovementioned strains along with S. albus native strain were grown in NL-19 production medium. Samples were harvested by centrifugation after 48 and 72 hours of cultivation. For total RNA isolation, S. albus cells were grown in SG medium (for ara expression) or NL19 medium (for indigenous BGC expression). Then, 2 ml of 2-day and 3-day cultures was spun down for 20 s at 14,000 × rpm, and the pellets were immediately frozen in liquid nitrogen and stored at ?80 °C. Total RNA extraction was performed using an RNeasy Kit (Qiagen, Hilden, Germany) as previously described (Huser et al. 2003). An RNase-Free DNase set (Qiagen) was used two times for on-column DNA digestion, and an additional DNase treatment was then performed with a DNase I kit (Roche Diagnostics, Mannheim, Germany) to ensure that all DNA was completely removed. To check the RNA samples for DNA contamination, PCR was performed using oligonucleotides designed to create two different products approximately 150 bp and 500 bp in size. Initially, RNA quality was checked with Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kits on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A Ribo-Zero rRNA Removal Kit for bacteria was obtained from Illumina (San Diego, CA, USA) and used to remove ribosomal RNA molecules from isolated total RNA. rRNA removal was checked using an Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) was used to prepare cDNA libraries (Koepff et al. 2017). The resulting cDNAs were pair-end sequenced on an Illumina HiSeq 1500 system (San Diego, CA, USA) using a 70 bp read length. Short read alignments and differential gene expression were illustrated using ReadXplorer 2.2.0 (Hilker et al. 2014) and DEseq (Anders and Huber 2010), respectively.