Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Comparative microarrays of Actinoplanes sp. SE50/110 ΔmalT with the Actinoplanes sp. SE50/110 wildtype

ABSTRACT: 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.

INSTRUMENT(S): Agilent Microarray Scanner, Agilent Hybridization Oven

ORGANISM(S): Actinoplanes sp. SE50/110

SUBMITTER: Julian Droste

PROVIDER: E-MTAB-8815 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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).

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Comparative microarrays of Actinoplanes sp. SE50/110 ΔmalT with the Actinoplanes sp. SE50/110 wildtype

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