Unknown,Transcriptomics,Genomics,Proteomics

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Whole track RNAseq of two different integrative vectors: pSET152::PrpsJ:acbC and pGUS::PrpsJ:gusA. in Actinoplanes sp. SE50/110.

ABSTRACT: 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.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Actinoplanes sp. SE50/110

SUBMITTER: Schaffert, Lena

PROVIDER: E-MTAB-8431 | 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).

Project description:RacA is the main Rho GTPase in Aspergillus niger regulating polarity maintenance via controlling actin dynamics. We have previously shown that both deletion and dominant activation of RacA (RacG18V) provoke an actin localization defect and thereby loss of polarized tip extension. This loss of apical dominance results in frequent dichotomous branching in the racA deletion strain and an apolar growing phenotype for RacG18V. In the current study we investigated the transcriptomics and physiological consequences of these morphological changes and compared the data with our previously established morphogenetic network model for the dichotomous branching mutant ramosa-1. This integrated approach revealed that polar tip growth is most likely orchestrated by the concerted activities of phospholipid signaling, sphingolipid signaling, TORC2 signaling, calcium signaling and CWI signaling pathways. The transcriptomic signatures and the reconstructed network model for all three morphology mutants imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking. We furthermore followed that fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching racA deletion mutant, and provide data demonstrating that hyperbranching does not per se result in increased protein secretion. This data set consists of 14 samples in total and covers the genome-wide transcriptional responses of Aspergillus niger towards deletion of racA and expression of a dominant active racA allele (G18V). The genome-wide transcriptional effect of deleting racA was monitored by comparison with the A. niger wild type. For both, the racA deletion mutant and the wild type, samples consist of biological triplicates (6 in total). The genome-wide transcriptional effect of expressing the dominant active racA mutant allele was monitored by sampling 2 and 4 hours after induction with maltose. For comparison, the racA wild type allele was induced and expressed similarly and samples were taken at corresponding time points. The experimental setup for the dominant active racA expression consisted of biological triplicates (8 in total).

Dataset Information

Whole track RNAseq of two different integrative vectors: pSET152::PrpsJ:acbC and pGUS::PrpsJ:gusA. in Actinoplanes sp. SE50/110.

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