Project description:Long-term use of antibiotics has engendered a large number of resistant pathogens, which pose a serious threat to human health. Here, we investigated the mechanism of fusaricidin antibacterial activity toward Bacillus subtilis and characterized the pathways responsible for drug resistance. We found that σ(w), an extracytoplasmic function sigma factor, plays an important role in the resistance to fusaricidins during the initial 5 minutes of drug addition. Approximately 18 genes were induced more than 3-fold, of which 66.7% are known to be regulated by σ(w). Over the following 3 h, fusaricidins induced 194 genes more than three-fold, and most were associated with classes of antibiotic-responsive stimulons. Moreover, the fusaricidin treatment increased the catabolism of fatty and amino acids but strongly repressed glucose decomposition and gluconeogenesis. In summary, our data provide insight into the mechanism of fusaricidin activity, on which we based our suggested strategies for the development of novel antibiotic agents.
Project description:Transcriptional profiling analysis was used to reveral the genetic changes of B.subtilis after addition of high levels of iron. This study is to reveal the effect of high levels of iron on B.subtilis' metabolism and identify the potential resistance mechanism to high levels of iron.
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: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:Bacillus subtilis strain AG174 was grown in the presence and absence of benzoate (30 mM). Benzoate was used in order to equalize the external and internal pH. The cultures were grown at external pH 7.0, and the addition of benzoate did not change the external pH. Microarray analysis was performed on cDNA synthesized from bacterial RNA. Real-time PCR of highly up-regulated genes confirmed the results of the microarray. These data were compared to previous B.subtilis microarray results, where genes regulated by external pH were identified.
Project description:Characterization of the putative genetic determinants of the VBNC state in a known spore-forming Gram-positive organism Bacillus subtilis 168. The VBNC state was induced under osmotic stress and aminoglycoside treatment. The transcriptome landscape of VBNC cells was compared to the viable, antibiotic sensitive B. subtilis cells and to the viable cells with no antibiotic treatment.
Project description:The sigma(B)-dependent general stress response in the common soil bacterium Bacillus subtilis can be elicited by a range of stress factors, such as starvation or an ethanol-, salt-, or heat-shock, via a complex upstream signaling cascade. Additionally, sigma(B) can be activated by blue light, via the phototropin homologue YtvA, a component of the environmental branch of the signaling cascade. The genome-wide transcriptomes of B. subtilis reported here show that sigma(B) can activated by blue as well as red light via RsbP/RspQ, the energy branch of sigma(B) upstream signaling cascade. A 16 chip genome-wide expression study using RNA recovered from B.subtilis strain PB565/pYtvA under light and dark conditions before and after induction with IPTG, and from B.subtilis strain PB565 under light and dark conditions. Each B.subtilis subsp. subtilis strain 168 NC_000964 chip measures the expression level of 4,104 genes in two-fold from with eight 60-mer probe pairs (PM/MM) per gene.
Project description:Transcriptional profiling analysis was used to reveral the genetic changes of B.subtilis after addition of high levels of iron. This study is to reveal the effect of high levels of iron on B.subtilis' metabolism and identify the potential resistance mechanism to high levels of iron. 1_1 is the independent biological replicate of sample 2_1, while sample 1_2 is the independent biological replicate of sample 2_2. Bacteria collected from each B.subtilis culture at 0min were mixed and used as ch2 source for each sample. The ch1 sources of sample 1_1 and 2_1 are bacteria collected from cultures without addition of Fe3+ at 20min, while the ch1 sources of sample 2_1 and 2_2 are bacteria collected from cultures with addition of 4mM Fe3+ at 20min.
Project description:With the improper application of fungicides, Phytophthora sojae begins to develop resistance to fungicides, and biological control is one of the potential ways to control it. We screened two strains of Bacillus; Bacillus amyloliquefaciens JDF3 and Bacillus subtilis RSS-1, which had an efficient inhibitory effect on P. sojae. They could inhibit mycelial growth, the germination of the cysts, and the swimming of the motile zoospores. To elucidate the response of P. sojae under the stress of B. amyloliquefaciens and B. subtilis, and the molecular mechanism of biological control, comparative transcriptome analysis was applied. Transcriptome analysis revealed that the expression gene of P. sojae showed significant changes, and a total of 1616 differentially expressed genes (DEGs) were detected. They participated in two major types of regulation, namely "specificity" regulation and "common" regulation. They might inhibit the growth of P. sojae mainly by inhibiting the activity of ribosome. A pot experiment indicated that B. amyloliquefaciens and B. subtilis enhanced the resistance of soybean to P. sojae, and their control effects of them were 70.7% and 65.5%, respectively. In addition, B. amyloliquefaciens fermentation broth could induce an active oxygen burst, NO production, callose deposition, and lignification. B. subtilis could also stimulate the systemic to develop the resistance of soybean by lignification, and phytoalexin.
Project description:The aim of our study is to elucidate the gene expression changes in rice in response to colonization by a plant growth promoting rhizobacteria such as the Bacillus subtilis through microarray high throughput technology. In particular, the effect of B.subtilis on root exudation (secretion of phytochemicals through roots) will be analysed. For this rice plantlets were grown in hydroponics and treated with B.subtilis RR4 for 48 hrs. The root samples of the control and treated plants were then used for the microarray experiment. The data obtained through microarray revealed genes related to cell wall modification, phytohormone synthesis, defense response, root exudation, etc. to be differentially regulated in response to B.subtilis RR4. Real time PCR analysis of few chosen genes (OsMS, OsALMT, OsABC, OsSDH, etc) also confirmed the validity of the microarray data. The initial responses of a plant in response to colonization by the microbe will be changes in cell wall of the plant tissues and the secretion of phytochemicals to attract/repel the colonizing beneficial/pathogenic organism. From analysis of microarray data we found the cell wall related genes which aid in root colonization and the root exudate related genes (biosynthesis and transport) which play a role in providing nutrition for the bacterial growth to be differentially regulated significantly. Analysis of specific genes and their biosynthesis pathways indicated that rice plants responded positively to root colonization by B.subtilis RR4. Notable among the exudation related genes such as Malate synthase and ALMT were found to be upregulated which indicates the significant role played by organic acids particularly malate in recruiting the PGPR towards the plant roots. This recruitment will thereby facilitate plant growth. Subsequently, these genes can be engineered in crop plants to recruit beneficial bacteria which might further open new avenues for improved crop production.