Project description:Abstract of associated manuscript: Daptomycin is the first of a new class of cyclic lipopeptide antibiotics used against multidrug-resistant Gram-positive pathogens. The proposed mechanism of action involves disruption of the functional integrity of the bacterial membrane in a Ca2+-dependent manner. We have used transcriptional profiling to demonstrate that treatment of Bacillus subtilis with daptomycin strongly induces the lia operon including the autoregulatory LiaRS two-component system (homologous to Staphylococcus aureus VraSR). The lia operon protects against daptomycin and deletion of liaH, encoding a phage shock protein A (PspA)-like protein, leads to 3-fold increased susceptibility. Since daptomycin interacts with the membrane, we tested mutants with altered membrane composition for effects on susceptibility. Deletion mutations of mprF (lacking lysyl-phosphatidylglycerol) or des (lipid desaturase) increased daptomycin susceptibility, whereas overexpression of MprF decreased susceptibility. Conversely, depletion of the cell for the anionic lipid phosphatidylglycerol led to increased resistance. Fluorescently-labeled daptomycin localized to the septa and in a helical pattern around the cell envelope and was delocalized upon depletion of phosphatidylglycerol. Together, these results indicate that the daptomycin-Ca2+ complex interacts preferentially with regions enriched in anionic phospholipids and leads to membrane stresses that can be ameliorated by PspA family proteins. Bacillus subtilis W168, WT (+DAP) vs. WT (-DAP). The experiment was conducted in triplicate using three independent total RNA preparations. For WT-rep1 and WT-rep2, daptomycin treated samples were labeled with Alexa Fluor 647 and untreated samples with Alexa Fluor 555. For WT-rep3, the daptomycin treated sample was labeled with Alexa Fluor 555 and the untreated sample with Alexa Fluor 647.
Project description:Abstract of associated manuscript: Daptomycin is the first of a new class of cyclic lipopeptide antibiotics used against multidrug-resistant Gram-positive pathogens. The proposed mechanism of action involves disruption of the functional integrity of the bacterial membrane in a Ca2+-dependent manner. We have used transcriptional profiling to demonstrate that treatment of Bacillus subtilis with daptomycin strongly induces the lia operon including the autoregulatory LiaRS two-component system (homologous to Staphylococcus aureus VraSR). The lia operon protects against daptomycin and deletion of liaH, encoding a phage shock protein A (PspA)-like protein, leads to 3-fold increased susceptibility. Since daptomycin interacts with the membrane, we tested mutants with altered membrane composition for effects on susceptibility. Deletion mutations of mprF (lacking lysyl-phosphatidylglycerol) or des (lipid desaturase) increased daptomycin susceptibility, whereas overexpression of MprF decreased susceptibility. Conversely, depletion of the cell for the anionic lipid phosphatidylglycerol led to increased resistance. Fluorescently-labeled daptomycin localized to the septa and in a helical pattern around the cell envelope and was delocalized upon depletion of phosphatidylglycerol. Together, these results indicate that the daptomycin-Ca2+ complex interacts preferentially with regions enriched in anionic phospholipids and leads to membrane stresses that can be ameliorated by PspA family proteins.
Project description:Transcriptional response of Bacillus subtilis to daptomycin in wild-type and in a daptomycin resistant mutant. Bacillus subtilis 168, WT (-DAP) vs. DapR1 (-DAP), WT (+DAP) vs. DapR1 (+DAP), DapR1 (+DAP) vs. DapR1 (-DAP). Each experiment was conducted at least twice using two independent total RNA preparations. For daptomycin untreated comparison between 168 WT and DapR1 mutant, DapR1 was labeled with Alexa Fluor 647 and WT was labeled with Alexa Fluor 555. For daptomycin treated experiments between WT and DapR1, DapR1 was labeled with Alexa Fluor 647 and WT with Alexa Fluor 555. For treated vs. untreated DapR1, the DAP treated samples were labeled with Alexa Fluor 647 and the untreated with Alexa Fluor 555. For dye swap, untreated DapR1 was labeled with Alexa Fluor 647 and DAP treated with Alexa Fluor 555.
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:Comparison of the transcriptome of Bacillus subtilis under going membrane protein overproduction in the wild type, sigW and cssRS deletion strains. We demonstrate that the dynamics of the stress systems involved in membrane overproduction are far more complicated than was first hypothesised and that many more systems than SigW and CssRS are involved in membrane protein overexpression stress. Interestingly the cssRS genes are repressed in the sigW deletion strain.