Comparative microarrays of Actinoplanes sp. SE50/110 ΔacrA with the Actinoplanes sp. SE50/110 wildtype
ABSTRACT: In order to characterize the transcriptional regulator AcrA, comparative genome wide transcriptome analyses were conducted. Therefore, the wild type Actinoplanes sp. SE50/110 and the mutant ΔacrA were each cultivated in triplicates in minimal medium supplemented with maltose or glucose as single carbon source. RNA samples from the biological replicates were taken from the middle of the growth phase of both strains in each maltose and glucose minimal medium, respectively. RNA was isolated and the three replicates were combined for each strain and condition. For each cultivation condition, the data from two arrays (dye swap) were combined to make statistically reliable conclusions.
Project description:In order to characterize the transcriptional regulator MalT, comparative genome wide transcriptome analyses were conducted. Therefore, the wild type Actinoplanes sp. SE50/110 and the mutant ΔmalT were each cultivated in triplicates in minimal medium supplemented with maltose or glucose as single carbon source. RNA samples from the biological replicates were taken from the middle of the growth phase of both strains in each maltose and glucose minimal medium, respectively. RNA was isolated and the three replicates were combined for each strain and condition. For each cultivation condition, the data from two arrays (dye swap) were combined to make statistically reliable conclusions.
Project description:Actinoplanes sp. SE50/110 was grown in three biological replicates in fermenter cultivation in maltose minimal medium. The transcriptomic changes during growth were monitored by sampling every 24 h. RNA-seq of all 7 replicates and a pooled RNA sample of all time points of each fermenter was performed in Paired-End mode (2x 70 nt) using Illumina HiSeq. Mapping to the reference genome (GenBank: LT825010.1) was perfromed using bowtie2 Version 2.3.2.
Project description:The acarviose metabolite acarbose is an a glucosidase inhibitor produced by Actinoplanes sp. SE50/110. It is medically important because it is used in the treatment of type 2 diabetes. In this work a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out. The associated txt and RAW files were used for two different analyses and publications. While one study focused on a comparative analysis of Actinoplanes sp. SE50/110 to elucidate differences in the proteome cultures that were grown with either maltose or glucose, the other study applied spectral counting and analyzed only the maltose-grown cultures to determine the major proteins and their location in the cell. The txt files for the comparative data are labeled as "heavy_light" and of the spectral counting data as "light". Both datasets were derived from the same RAW files.
Project description:The whole coding RNA of Actinoplanes sp. SE50/110 mutants containing two different integrative vectors (pSET152::acbB and pSETT4::acbB) were sequenced. Both vectors are integrated via a phiC31 integrase (Bierman et al. 1992) into the genetic locus ACSP50_6589 (former: acpl_6602) (Gren et al. 2016). The novel expression vector pSETT4 is excelled by an easy cloning mechanism allowing the integration of different promoters. By this, the system can be quickly adapted to further species of the order Actinomycetales. Additionally, T4-terminators were introduced before and after the expression cassette, since they are able to block the transcription efficiently and prevent antisense formation and read-through from the integrase gene into the gene of interest.
Project description:The whole coding RNA of Actinoplanes sp. SE50/110 mutants containing two different integrative vectors (pSET152::PrpsJ:acbC and pGUS::PrpsJ:gusA) were sequenced. Both vectors are integrated via a phiC31 integrase (Bierman et al. 1992) into the genetic locus ACSP50_6589 (former: acpl_6602) (Gren et al. 2016). For expression of the gene of interest (either gusA or acbC) the native promoter of the ribosomal gene rpsJ (ACSP50_0690) was used. The pGUS and pSET152 backbone mainly differ by the integration of a spectinomycin resistance cassette aadA in the pGUS-vector. The cassette is flanked by two T4-terminators to block the transcription of the goi from upstream promoters (Myronovskyi et al. 2011). As shown by this experiment, the two T4-terminators block transcription and prevent read-through efficiently in pGUS, whereas continuous tracks can be observed in pSET152, which extent from int to acbC.
Project description:The promoter structure influences binding and clearance of RNA polymerase and therefore substantially influences expression of a gene. A promoter usually consists of a -10 and a -35-region, an extended -10-motif and A+T-rich upstream promoter elements. Most of these elements are optional, whereas the -10-region is essential (Albersmeier et al. 2017). Knowledge about the transcription start sites (TSS) of genes allows genome-wide localization and determination of the promoter regions. In our group, a special protocol for the amplification of primary transcripts was developed, including the capture of primary transcripts, rewriting them into cDNA (complementary DNA) and amplification in the further course of the protocol (Pfeifer-Sancar et al. 2013). Here, TSS were manually determined with special regard to the heterologous promoters. For each construct, at least one and up to three different TSS were found, leading to the identification of one or several -10-core-hexamers. These were located mostly 6 to 7 nucleotides upstream of each TSS, which corresponds to the average distance of 6.4 nt described for Actinoplanes sp. SE50/110 by Schwientek et al. (2014).
Project description:Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. A PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), is localized upstream of the maltase gene amlE (ACSP50_2474), which is organized in an operon with and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. A targeted gene deletion mutant of amlR was constructed by use of CRISPR/Cas9 technology. The transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ∆amlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ∆amlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes, which was shown by RNAseq. We used a pooled library for RNAseq for global pre-screening and validated these results for a total of 25 genes by RT-qPCR. RNA of triplicates of the wildtype and the deletion mutant was isolated from growing cultures and pooled equimolar separately for the wildtype and the deletion mutant (total amount of 2.5 µg RNA each in 26 µL RNase-free water). rRNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina, San Diego, USA). Successful depletion of rRNA was verified by an Agilent RNA 6000 Pico chip in the Bioanalyzer (Agilent, Böblingen, Germany). cDNA libraries were prepared following protocols from Pfeifer-Sancar et al. (2013) and Irla et al. (2015) by use of the TruSeq stranded mRNA kit (Illumina, San Diego, CA, USA). The libraries were quantified by a DNA High Sensitivity Assay chip in the Bioanalyzer (Agilent, Böblingen, Germany) and sequenced on a 2 x 75 nt HiSeq 1500 run (Illumina, San Diego, CA, USA). Sequencing yielded about 10.7 million read pairs for the wildtype library and 10.2 million read pairs for the deletion mutant library, respectively. Raw reads were quality-trimmed using Trimmomatic v0.3.5 (Bolger et al., 2014). Trimmed reads were mapped to the respective reference sequence (GenBank: LT827010.1) using bowtie2 in the paired-end mode (Langmead and Salzberg, 2012), resulting in 20.7 and 19.5 million mappings. ReadXplorer was used for visualization and differential gene expression analysis (Hilker et al., 2016).