Project description:Glycine betaine (GB) is a potent osmoprotectant for salt stressed Bacillus subtilis cells, which possess three high-affinity uptake systems for GB. OpuA is the dominant transporter for this compatible solute. Northern blot analysis, primer extension experiments and opuA-treA reporter gene fusion studies demonstrated that opuA expression is strongly induced at high osmolality from a single SigA-type promoter. Promoter mutations that improve the match of the opuA promoter to the consensus sequence substantially increase basal-level expression but reduce inducibility by high salinity. Expression of opuA is sensitively controlled by GB, which causes significant repression of opuA transcription at low and high salinity. GB influences the kinetics as well as the final level of high salinity induction of opuA in a concentration-dependent fashion. The repressing effect of GB on opuA transcription requires the intracellular presence of GB, regardless whether it is taken up via OpuA or other transporters or synthesized from choline. Genome-wide transcriptional profiling of salt-stressed B. subtilis cells demonstrated that the repressive effect of GB targets only a subset of high-salinity induced genes. This group of GB-responsive genes comprises in essence the full set of genes with demonstrated functions in either the uptake or synthesis of compatible solutes. Four different samples (untreated, GB, NaCl, and GB + NaCl treated) were analyzed and each sample was hybridized in triplicate.
Project description:Glycine betaine (GB) is a potent osmoprotectant for salt stressed Bacillus subtilis cells, which possess three high-affinity uptake systems for GB. OpuA is the dominant transporter for this compatible solute. Northern blot analysis, primer extension experiments and opuA-treA reporter gene fusion studies demonstrated that opuA expression is strongly induced at high osmolality from a single SigA-type promoter. Promoter mutations that improve the match of the opuA promoter to the consensus sequence substantially increase basal-level expression but reduce inducibility by high salinity. Expression of opuA is sensitively controlled by GB, which causes significant repression of opuA transcription at low and high salinity. GB influences the kinetics as well as the final level of high salinity induction of opuA in a concentration-dependent fashion. The repressing effect of GB on opuA transcription requires the intracellular presence of GB, regardless whether it is taken up via OpuA or other transporters or synthesized from choline. Genome-wide transcriptional profiling of salt-stressed B. subtilis cells demonstrated that the repressive effect of GB targets only a subset of high-salinity induced genes. This group of GB-responsive genes comprises in essence the full set of genes with demonstrated functions in either the uptake or synthesis of compatible solutes.
Project description:To investigate whether salt-stress dependent activation of SigB is mitigated when cells are pre-loaded with the compatible solute glycine betaine, we analyzed salt induction of the SigB regulon in the presence of the precursor choline at a genome-wide level. The median expression value of SigB-regulated genes was 1.7-fold higher in cells that received choline immediately before salt stress compared to cells where choline was added 60 minutes prior to the salt stress.
Project description:Here, we investigated for the first time the systems-wide response of B. subtilis to different simultaneous stresses, i.e. nutrient limitation and high osmolarity. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses at the transcriptome, proteome, metabolome and fluxome levels. Our results indicate that the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. The accumulation of the exogenously provided osmoprotectant glycine betaine helps the cell to rescue enzyme activities in the presence of high salt. A major effort of the cell during osmotic stress is the production of the compatible solute proline. This is achieved by the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways.
Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes.
Project description:Here, we investigated for the first time the systems-wide response of B. subtilis to different simultaneous stresses, i.e. nutrient limitation and high osmolarity. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses at the transcriptome, proteome, metabolome and fluxome levels. Our results indicate that the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. The accumulation of the exogenously provided osmoprotectant glycine betaine helps the cell to rescue enzyme activities in the presence of high salt. A major effort of the cell during osmotic stress is the production of the compatible solute proline. This is achieved by the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways. We applied transcriptomic, mass spectrometry-based protein, metabolite and 13C-metabolic flux analysis techniques to B. subtilis cells grown under well-controlled conditions in a glucose-limited chemostat at a growth rate of 0.1 h-1 under i) reference conditions, ii) in the presence of 1.2 M NaCl, and iii) in the presence of 1.2 M NaCl and 1 mM GB. Microarray hybridizations were performed with RNA from three biological replicates. The individual samples were labeled with Cy5; a reference pool containing equal amounts of RNA from all 9 samples was labeled with Cy3.
Project description:The gene expression of Bacillus subtilis 168 showed 3 major patterns including early expression, transition expression and late expression We monitored Bacillus subtilis gene expression by using microarray at differernt time points