Project description:Lysine acetylation is a major post-translational modification that plays an important regulatory role in almost every aspects in both eukaryotes and prokaryotes. Bacillus amyloliquefaciens, a Gram-positive bacterium, is very effective for the control of plant pathogens. Here, we conducted the first lysine acetylome in B. amyloliquefaciens through a combination of highly sensitive immune-affinity purification and high-resolution LC−MS/MS. Overall, we identified 3268 lysine acetylation sites in 1254 proteins. Acetylated proteins are associated with a variety of biological processes and a large fraction of these proteins are involved in metabolism. These data serves as an important resource for further elucidation of the physiological role of lysine acetylation in B. amyloliquefaciens.
Project description:Transcriptome analysis was used to investigate the global stress response of the Gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon that responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild-type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example citM, ylxF, yloA, ykoJ and several genes of the flgB operon. However, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction. Keywords: secretion stress response
Project description:Transcriptome analysis was used to investigate the global stress response of the Gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon that responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild-type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example citM, ylxF, yloA, ykoJ and several genes of the flgB operon. However, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction. Secretion stress was applied by overproducing the well-secreted AmyQ α-amylase (pKTH10 vector) from B. amyloliquefaciens. Besides examining secretion stress in wild-type cells, we compared transcriptome profiles of a cssS mutant strain under conditions of high-level AmyQ production. Samples for transcriptome analyses were collected at the late exponential growth stage (one hour before the transition point) and 3 hours upon entry in the stationary growth phase. Three independent cultures of each strain were used and cells were sampled for macroarray experiments. Duplicate spots were averaged in Array-Pro software (Media Cybernetics, Inc.) and the signal was normalized after background subtraction by calculation of the percentage of total signal per gene using Microsoft Excel.
Project description:Root exudates play an important role in plant-microbe interaction. The transcriptional profilings of plant growth-promoting rhizobacteria Bacillus amyloliquefaciens SQR9 in response to maize root exudates under static condition, were investigated by an Illumina RNA-seq for understanding the regulatory roles of the root exudates.
Project description:In this study we focus on two Saccharomyces cerevisiae (CEN. PK series) strains producing either insulin precursor or amylase and we compare the transcriptional regulation at different dilution rates, in particular with the objective to identify the relationship between cell metabolism and recombinant protein production. We found that anaerobic conditions showed high amount of amylase productions when comparing to aerobic conditions and the genome-scale transcriptional analysis suggested that genes related to the endoplasmic reticulum (ER), lipid synthesis and stress responses were generally up-regulated at anaerobic conditions. Moreover, we proposed a model for the electron transfer from ER to the final electron acceptor, fumarate under anaerobic conditions. Two Saccharomyces cerevisiae strains producing either insulin precursor or amylase were selected at different dilution rates in chemostat cultivation for RNA extraction and hybridization on Affymetrix microarrays. Biological triplicates were applied.
Project description:iTRAQ were used to analyze the difference in total protein expression levels of cultured strains after culturing with sucrose or mannitol
Project description:To establish the protein expression profile of Ba168, a high-resolution LC-MS/MS proteomic analysis was performed. A total of 1155 proteins were identified from 5233 unique peptides. GO and KEGG analysis revealed that a majority of the proteins were associated with biosynthesis and carbon metabolism pathways, such as biosynthesis of amino acids, peptidoglycan, and antibiotics. Then, we identified the antimicrobial proteins of Ba168. At least 16 potential antimicrobial-activity-related proteins were identified; 11 of these proteins have direct antimicrobial effects, while 5 of these proteins are associated with the formation of antimicrobial substances.
Project description:Our study showed that optimizing ncRNA expression can increase or lower the yield of alpha-amylase enzyme production in Bacillus subtilis while revealing a range of potentially novel ncRNAs.
Project description:The transcriptomic response of Bacillus amyloliquefaciens FZB42 to maize root exudates at OD600=3.0. This is a comprehensive analysis using the data of six microarray experiments (Exp1-2-3 and ExpABC respectively, 18 hybridization in total).