Project description:SwrA activates flagellar gene expression in Bacillus subtilis to increase the frequency of motile cells in liquid and further increase flagellar density to swarm over solid surfaces. Here we perform ChIP-seq and demonstrate that SwrA interacts with many sites on the chromosome indirectly through the response regulator DegU. We identify a DegU-specific inverted repeat and show that SwrA increased DegU DNA binding affinity in parallel to phosphorylation. Moreover, we show that SwrA increased the size of the DegU footprint expanding the region of protection towards the promoter. The remote location of the DegU inverted repeat was critical such that relocating the binding site by full turns of DNA impaired activation by SwrA/DegU and moving the binding site closer to the promoter severely impaired transcription. We conclude that SwrA/DegU forms a heteromeric complex that enables both remote binding and activator-RNA polymerase interaction in the context of an interceding UP element. We speculate that multimeric activators that resolve similar cis-element spatial conflicts are common in bacteria such as the diverse master activators of flagellar biosynthesis and those that activate long operons of other multi-subunit complexes.
Project description:First whole transcriptome assessment of a Bacillus megaterium strain. The B. megaterium DegU regulon was assessed for LB batch cultures with artificially induced degU expression. DegU is a pleiotropic regulator in B. subtilis governing adaptive responses such as secretory enzyme production.
Project description:The Streptomyces coelicolor two genes operon SCO5784-SCO5785 encodes a two-component system which functions in a similar manner to that of the Bacillus subtilis DegS-DegU system. Propagation of the regulatory gene in high copy number results in the overproduction of several extracellular enzymes, among them the major extracellular protease, as well as in a higher level of synthesis of the antibiotic actinorhodin. This two-component system seems to control various processes characterised by the transition from primary to secondary metabolism in S. coelicolor, as determined by proteomic and transcriptomic analices. The presence of the regulatory gene in high copy number in S. coelicolor additionally seems to elicit a stringent response in the bacterial cell. Therefore, we propose renaming S. coelicolor genes SCO5784 and SCO5785 as degS and degU, respectively. All microarray analyses were performed with RNA samples obtained from three independent cultures grown under identical conditions. Hybridisation assays were carried out with cDNA obtained from RNA extracted at the late exponential phase of growth (24h). The transcriptional profile of wild type (S. coelicolor M145) cells carrying the multicopy plasmid pIJ487 was compared with that of the same strain carrying the degU gene cloned in the same plasmid under the control of its own promoter (S. coelicolor M28). And the transcriptional profile of wild type (S, coelicolor M145) cells was compared to that of the DegU deficient strain (S. coelicolor I32).
Project description:Purpose: Tracheal epithelial brush cells are rare chemosensory cells defined by their expression of elements of the bitter taste transduction system, and known to activate the cholinergic nervous system in the murine lung. Similar chemosensory cells in the intestine can generate lipid mediators and pro-inflammatory cytokines but whether brush cell can contribute to airway inflammation is unknown. Furthermore, despite the advances in understanding chemosensory cell effector functions, the receptors that mediate chemosensory cell activation and expansion beyond taste receptors in any compartment remain largely unknown. Methods: In this study, we isolated tracheal brush cells by FACS from naïve ChATBAC-eGFP mice with knockin of eGFP within a BAC spanning the acetylcholine transferase locus, marking brush cells in the epithelium and performed transcriptome profiling using low input RNA sequencing. We compared tracheal brush cells to EpCAM+ epithelial cells and CD45+ hematopoetic cells in naive mice. Results: When compared to EpCAM+ EpCs and to CD45+ cells in the airway, principal component analysis demonstrated that brush cells grouped quite distinctly. This brush cell distinction relative to EpCAM+ cells, was further reflected in the striking number of highly differentially expressed genes. This included 1305 genes expressed at 4-fold or higher levels in EpCAM+eGFP+ cells (brush cells), of which 418 genes were expressed at 32-fold or higher levels in brush cells. Conclusions: Our study represents the first detailed analysis of the transcriptome of tracheal brush cells and identifies a unique set of genes that are primarily expressed in brush cells including the bitter taste transduction system, synthenic machinery for several pro-inflammatory lipid mediators and HoxA2 transciptional factors.
Project description:First whole transcriptome assessment of a Bacillus megaterium strain. The B. megaterium DegU regulon was assessed for LB batch cultures with artificially induced degU expression. DegU is a pleiotropic regulator in B. subtilis governing adaptive responses such as secretory enzyme production. 8 x 15 K customer made microarrays for gene expression analysis of B. megaterium were obtained from Agilent (Agilent Technologies, USA) with up to three probes per open reading frame of the B. megaterium DSM319 genome. Finally, the hybridization and final washing steps of the microarrays occurred as described in the Agilent manual for two color microarrays. The microarrays were scanned with the help of the Agilent C Scanner (Agilent Technologies, USA). For scanning and feature extraction the software Agilent Scan Control 8.4.1 and Feature Extraction 10.7.3.1 (Agilent Technologies, USA), respectively, was used according to the instruction. The analysis of the raw data occurred with the programming language R and Bioconductor as described in Yang and Paquet (2005). Finally, in consequence of the microarray design three different probes belonging to one gene were matched performing mean and median summarization of the logarithmic fold changes (logFC). Nevertheless, the p-values and also the logFC values were given for each probe separately, to account for different hybridization behavior of the different probes (Bunk, 2010). Only genes with a p-value < 0.01 in all replicates and an absolute |logFC| > 1 were considered as to be differentially expressed. Samples from degU expressing cells (xylose induction) of a degSU deletion mutant were compared to samples obtained from the likewise induced empty vector control strain, two time points, biological replicates: 3-5
Project description:This study examined large renal tubule extracellular vesicles (LRT-EVs) isolated from urine of a 5/6 nephrectomy (5/6Nx) rat model that recapitulates many elements of human chronic kidney disease (CKD). Within weeks of 5/6Nx rats spontaneously exhibit proximal tubular damage including brush boarder shedding and the production of LRT-EVs. Analysis of the LRT-EV size and a lack of TUNEL staining in 5/6Nx rats, suggested LRT-EVs to be distinct from exosomes, microvesicles, and apoptotic bodies.
Project description:This data series describes expression data for eight paired, control and treated cell cultures obtained on independent occasions. NG108 rat neuronal cell cultures were exposed to either 0.25% DMSO (control) or 4400 ng/ml mefloquine (treated) for two hours. Validation: Modulation of the following transcripts by mefloquine was confirmed by semi-quantitative RT-PCR: U30186, X63594cds_g_at, X63594cds_at, X17163cds_s_at, rc_AI175959 and rc_AA945867 (unpaired, unequal variance, one tailed t-test, p < 0.05). Keywords: other
Project description:The Streptomyces coelicolor two genes operon SCO5784-SCO5785 encodes a two-component system which functions in a similar manner to that of the Bacillus subtilis DegS-DegU system. Propagation of the regulatory gene in high copy number results in the overproduction of several extracellular enzymes, among them the major extracellular protease, as well as in a higher level of synthesis of the antibiotic actinorhodin. This two-component system seems to control various processes characterised by the transition from primary to secondary metabolism in S. coelicolor, as determined by proteomic and transcriptomic analices. The presence of the regulatory gene in high copy number in S. coelicolor additionally seems to elicit a stringent response in the bacterial cell. Therefore, we propose renaming S. coelicolor genes SCO5784 and SCO5785 as degS and degU, respectively.
Project description:This data series describes expression data for eight paired, control and treated cell cultures obtained on independent occasions. NG108 rat neuronal cell cultures were exposed to either 0.25% DMSO (control) or 4400 ng/ml mefloquine (treated) for two hours. Validation: Modulation of the following transcripts by mefloquine was confirmed by semi-quantitative RT-PCR: U30186, X63594cds_g_at, X63594cds_at, X17163cds_s_at, rc_AI175959 and rc_AA945867 (unpaired, unequal variance, one tailed t-test, p < 0.05).