RNAseq analysis of Xcc strain B100 in minimum media
ABSTRACT: The dataset is a total transcriptome. RNA samples were obtained from fermentation of the Xcc strain B100 in minimal media named XMD (Schatschneider et al., 2013) with 30 g/L glucose as a sole carbon source and 0.4 g/L KNO3 as the sole nitrogen source . the RNA probes were harvested in the middle of the growth phase at an OD = 1 and the library was prepared following the protcol published by Pfeifer-Sancar et al., 2013.
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:To detect Transcription Start sites (TSS) in Staphylococcus aureus wildtype a cDNA library enriched for primary 5′-transcripts according to Peifer-Sancar et al. (2013) was prepared and sequenced on Illumina MiSeq system to retrieve 5′-ends of primary transcripts. Cultivation was performed in Luria Bertani (LB) medium in triplicate at 37°C until cells have reached an optical density at 540 nm of 2.0. Cells were harvested by centrifugation, washed with Belitsky minimal medium (BMM) and adapted to BMM for one hour. S. aureus cells of 3 replicate experiments were harvested and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. After precipitation with 3 M sodium acetate and isopropanol, the total RNA was washed with cold ethanol and the pellet was solved in sterile water. Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. The method from Peifer-Sancar et al. (2013) was used to prepare a cDNA library enriched for primary 5′-transcript. The resulting cDNAs were sequenced paired end on an Illumina MiSeq system (San Diego, CA, USA) using 75 bp read length.
Project description:Transcriptome analyses using a wild-type strain of Saccharomyces cerevisiae were performed to assess the overall pattern of gene expression during the transition from glucose-based fermentative to glycerol-based respiratory growth. These experiments revealed a complex suite of metabolic and structural changes associated with the adaptation process. Alterations in gene expression leading to remodeling of various membrane transport systems and the cortical actin cytoskeleton were observed. Transition to respiratory growth was accompanied by alterations in transcript patterns demonstrating not only a general stress response, as seen in earlier studies, but also the oxidative and osmotic stress responses. In some contrast to earlier studies, these experiments identified modulation of expression for many genes specifying transcription factors during the transition to glycerol-based growth. Importantly and unexpectedly, an ordered series of changes was seen in transcript levels from genes encoding components of the TFIID, SAGA (Spt-Ada-Gcn5-Acetyltransferase), and SLIK (Saga LIKe) complexes and all three RNA polymerases, suggesting a modulation of structure for the basal transcriptional machinery during adaptation to respiratory growth. In concert with data given in earlier studies, the results presented here highlight important aspects of metabolic and other adaptations to respiratory growth in yeast that are common to utilization of multiple carbon sources. Importantly, they also identify aspects specific to adaptation of this organism to growth on glycerol as sole carbon source. A time-series illustrating the transition from fermentation using glucose (dextrose) as sole carbon source to respiration using glycerol as sole carbon source. Time points of 15 minutes, 30 minutes and 60 minutes as well as growth on dextrose, glycerol or ethanol as sole carbon source from a starter culture. Three biological replicates of the 30 minute time point and growth on glycerol from starter culture.
Project description:Rsf1p is a putative transcription factor required for efficient growth using glycerol as sole carbon source but not for growth on the alternative respiratory carbon source ethanol. We use microarrays to determine the differences in the transcriptional program between the delta-rsf1 mutant and the wild type during respiratory growth on glycerol as well as the transition to growth on glycerol as sole carbon source. Experiment Overall Design: delta-rsf1or the isogenic parent strain were grown to early log (A600=0.6) in YPD and then washed twice in prewarmed YPG (30 C) and returned to the air shaker in YPG for 15, 30 or 60 minutes. "Limit" conditions were provided by harvesting cells grown in YPD to early log phase without shifting to YPG and by harvesting cells grown in YPG to early log phase. Since the delta-rsf1 mutant and its isogenic parent strain grow equally well on the respiratory carbon source ethanol, cells were also harvested after being grown in ethanol to early log phase.
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:Background: Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Methylotrophy in this organism depends on the endogenous plasmid pBM19 although all ribulose monophosphate cycle enzymes are also encoded on the chromosome. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising and valuable candidate for future biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5’‑ends. Results: Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5’ UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. The first step of the analysis was the correction of 14 wrongly annotated translation start sites (TLSs) on the basis of the primary transcriptome data. Further investigation of the identified 5’ UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis regulatory RNA elements. In addition to this, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts. Conclusion: The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism. Pooled samples from 16 different cultivation conditions are analysed in whole transcriptome and 5'end enriched transcriptome protocols.
Project description:The four-carbon sugar erythritol is an important component of B. melitensis pathogenesis. To determine the transcriptional response to erythritol, B. melitensis strain 16M was grown in the presence of either glucose or erythritol as a sole carbon source. Two control samples (glucose) and two experimental samples (erythritol) were analyzed on Nimblegen B. melitensis microarray chips.
Project description:L. oligofermentans was grown microaerobically on modified (without malic acid, cellulose and bromocresol green) MLD medium (Cavin JF et al., Appl Environ Microbiol 1989), containing either glucose, ribose or xylose as a sole carbon source (50mM) in three replicates. Samples were taken at three time points 20 h, 24 h and 30 h. RNA extraction and RNA sequencing libraries construction were done as described in Andreevskaya M et al., Appl Environ Microbiol 2015. Libraries were sequenced in two runs with six lanes overall using SOLiD 5500XL to produce 75 bp single-end reads. Obtained .xsq files were converted into .fastq files.
Project description:Among the three major genetic lineages of L. monocytogenes (i.e. LI, LII, and LIII), LI and LII are predominantly associated with foodborne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor lmo0753 that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains including a DNA-binding domain with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, we constructed a lmo0753 deletion and complementation mutants of the fully sequenced L. monocytogenes LII strain EGDe. We found that deletion of lmo0753 led to the loss of L-rhamnose utilization in EGDe. Transcriptomic comparison of the EGDe lmo0753 deletion mutant and the wild type incubated in phenol-red medium containing L-rhamnose as the sole carbon source revealed 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome that were up- and down-regulated for more than 2-fold, respectively. Genes involved in biotin biosynthesis, general stress response and rhamnose metabolism were shown to be differentially regulated by Lmo0753. Findings from this study may partially explain why LIII of L. monocytogenes is underrepresented in the environment and rarely associated with human listeriosis outbreaks due to the inability of rhamnose utilization. We report the transcriptomic profile of L. monocytogenes Δlmo0753 LII strain (EGDe) in broth media with L-rhamnose as the sole carbon source. Examination of deletion of Lmo0753 on L-rhamnose utilization in L. monocytogenes. Two biological replicates per WT and Δlmo0753.
Project description:A putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website. Real time RT-PCR assays showed that expression of YJR127C/ZMS1 was glucose-repressible, and a deletion mutant for the gene showed a growth defect on glycerol-based but not on glucose- or ethanol-based medium. Real time RT-PCR analyses identified severely attenuated transcript levels from GUT1 and GUT2 to be the source of that growth defect, the products of GUT1 and GUT2 are required for glycerol utilization. mRNA levels from a large group of mitochondria- and respiration-related nuclear genes also were shown to be attenuated in the deletion mutant. Importantly, transcript levels from the mitochondrial OLI1 gene, which has an associated organellar UAS, were attenuated in the DeltaYJR127C mutant during glycerol-based growth, but those from COX3 (OXI2), which lacks an associated mitochondrial UAS, were not. Transcriptome analysis of the glycerol-grown deletion mutant showed that genes in several metabolic and other categories are affected by loss of this gene product, including protein transport, signal transduction, and others. Thus, the product of YJR127C/ZMS1 is involved in transcriptional control for genes in both cellular genetic compartments, many of which specify products required for glycerol-based growth, respiration, and other functions. S cerevisiae strain BY4741 and the BY4741 Δrsf2* mutant were grown in medium containing glycerol as the sole carbon source. mRNA was prepared at A600=0.6. Three biological replicates of the wild-type and two of the mutant were prepared. *this locus has also been designated YJR127C and ZMS1