Project description:Transcription profiles in BL21, BL21/pOri1 and BL21/pOri2 were analysed using DNA microarray technology. BL21, BL21/pOri1 or BL21/pOri2 strains were cultured at chemostat status and harvested after the cultivation arrived steady status. Keywords: Effects of plasmid DNA on Escherichia coli metabolism
Project description:PhoP is considered a regulator of virulence despite being conserved in both pathogenic and non-pathogenic Enterobacteriaceae. While Escherichia coli strains represent both non-pathogenic commensal isolates and numerous virulent pathotypes, the PhoP virulence regulator has only been studied in commensal E. coli. To better understand how conserved transcription factors contribute to virulence, we characterized PhoP in pathogenic E. coli. Loss of phoP significantly attenuated E. coli during extraintestinal infection. This was not surprising since we demonstrated that PhoP differentially regulated the transcription of >600 genes. In addition to survival at acidic pH and resistance to polymyxin B, PhoP was required for repression of motility and oxygen-independent changes in the expression of primary dehydrogenase and terminal reductase respiratory chain components. All phenotypes have in common a reliance on an energized membrane. Thus, we hypothesized that PhoP mediated these effects by regulating genes that generate a proton motive force. Indeed, bacteria lacking PhoP exhibited a hyper-polarized membrane, and dissipation of the transmembrane electrochemical gradient increased the susceptibility of the phoP mutant to acidic pH, while inhibiting respiratory generation of the proton gradient restored resistance to antimicrobial peptides independent of lipopolysaccharide modification. These findings demonstrate a connection between PhoP, virulence, and the energized state of the membrane. Comparison of gene expression between wild-type CFT073 and a CFT073 phoP deletion mutant during logarithmic phase growth in LB medium. Three biological replicates were compared from each strain.
Project description:Transcriptional analysis of the effects of the deletion of the sRNAs glmY and glmZ in EHEC Wt, ΔglmY, and ΔglmZ strains were grown for 6 hours in low glucose DMEM under static conditions and then processed according to the manufacters instructions.
Project description:The only target locus of transcription factor BglJ known to date is the bgl operon, and activation of bgl by BglJ requires RcsB. Transcription factor LeuO is involved in stress responses and known as antagonist of H-NS. To identifiy novel targets of BglJ, we overexpressed BglJ in Escherichia coli K12 and measured differential gene expression by performing DNA microarray analysis. Moreover, to analyze whether all targets of BglJ require RcsB, we overexpressed BglJ in an rcsB deletion background. In addition, we overexpressed LeuO to identifiy targets of LeuO.
Project description:Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its co-factor NusG is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global co-localization of the nucleoid-associated protein H-NS with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.
Project description:The impact of 2',3'-cyclic nucleotide phosphodiesterase (CNPase) on global gene expression in E. coli was investigated by heterologous expression of mammalian CNPase in a strain containing a deletion of the 'rna' gene. 'rna' encodes an endoribonuclease capable of hydrolyzing RNA into 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) such that deletion of 'rna' drastically reduces 2',3'-cNMPs levels to undetectable levels. Expression of CNPase in this strain allowed any effects of CNPase unrelated to 2',3'-cNMPs to be observed.
Project description:We replaced the natural pnp locus with the human cDNA and studied the transcriptomes of 3 strains, namely the wt pnp+ (C-1a), the mutant with pnp ORF deletion (C-5691) and the strain with the substitution of the bacterial ORF with the human one (C-6001), before and 4 minutes after the addition of rifampicin to inhibit transcription.