Project description:We adapted thermal proteome profiling (TPP) to study the thermostability of Escherichia coli proteins in vivo. We monitored the E. coli meltome and proteome at different growth phases, in a tolC knock-out mutant and after drug treatment.
Project description:Cyadox(CYA), as a new species of Quinoxaline 1, 4-dioxides and olaquindox(OLA) both showed higher antibacterial activity under anaerobic incubation. Microarray was used for global gene expression studies, which were further confirmed by real-time PCR. Cyadox and olaquindox mainly stimulated the expression of DNA repair genes as a response to the DNA damage. The induced gene sbmC was found to provide partial protection against the antibiotics of gyrase inhibitors like the quinolones and the ruvAB helped remove the topoisomerase IV-DNA cleavage complex caused by some type IIA topoisomerase poison antibiotics. It was inferred that the radical intermediate of cyadox reduction under anaerobic condition was responsible for the poison effect of IIA topoisomerases, which brought about DNA double stand breaks and other DNA damages in E. coli.
Project description:Purpose: This study aimed to identify the genes regulated by plasmid-encoded regulator C (PerC). Whole transcriptomes of WT typical enteropathogenic E. coli (tEPEC) strains E2348/69 and coisogenic null-perC mutant JPEP22 were analyzed to identify and quantify differentially expressed genes. Methods: RNA was isolated from the WT and null-perC mutant strains and RNA integrity (RIN) was determined to be 10 out of possible 10 for all samples by Aligent Bioanalyzer. rRNA was depleted and resulting mRNA was reverse-transcribed into cDNA. Libraries were multiplexed for discrimination between libraries, barcoded for sequencing, and amplified with random hexamers for 15 PCR cycles. Transcripts were sequenced for 50 bases in single-end fashion within one lane of an Illumina Hiseq 2000 flow cell. This yielded roughly 30 million reads per sample. Results: Differential gene expression (DGE) analysis showed that 157 genes were statistically significantly regulated using a false-discovery rate (FDR) of 10% (q ≤ 0.10). Of these genes, the perC-mutant strain had far greater transcripts for the fim operon genes, fewer transcripts of nitrate reductase genes and anaerobic metabolism genes, and fewer transcripts for Hfq-dependent ncRNAs compared to WT. Conclusions: Differential transcript abundance between the perC-mutant and WT strains indicate PerC's negative regulation of the fim genes and positive regulation of anaerobic metabolism and ncRNA genes.
Project description:In Escherichia coli, Lon is an ATP-dependent protease which degrades misfolded proteins and certain rapidly-degraded regulatory proteins. Given that oxidatively damaged proteins are generally degraded rather than repaired, we anticipated that Lon deficient cells would exhibit decreased viability during aerobic, but not anaerobic, carbon starvation. We found that the opposite actually occurs. Wild-type and Lon deficient cells survived equally well under aerobic conditions, but Lon deficient cells died more rapidly than the wild-type under anaerobiosis. Microarray analysis revealed that genes of the Clp family of ATP-dependent proteases were induced during aerobic growth but not during anaerobic growth. Thus, Clp may compensate for loss of Lon when cells are in an oxygen containing atmosphere. Under anaerobic carbon starvation conditions, Lon must be active to support survival. Keywords: Other
Project description:The purpose of this study is to investigate the changes of global gene expression in E. coli during an oxygen shift. All cultures were grown under aerobic or anaerobic conditions in M9 minimal media supplemented with glucose. Samples were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent, and total RNA was isolated from exponentially growing cells using a Qiagen RNeasy mini kit (protocols available at www1.qiagen.com). The RNA (10 µg) was then used as the template for cDNA synthesis, the product of which was fragmented, labelled, and hybridized to an Affymetrix E. coli Antisense Genome Array, which was washed and scanned to obtain an image. All of these steps were performed according to Affymetrix protocols (available at www.affymetrix.com). This SuperSeries is composed of the following subset Series:; GSE1106: aerobic knock-out; GSE1107: anaerobic knock-out Experiment Overall Design: Refer to individual Series
Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined anaerobic media. One sample was treated with 100 µM CORM-3, the other sample was a control. The volume (175 ml), temperature (37oC) and stirring (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats.
Project description:Mapping the occupancy of ArcA throughout the genome of Escherchia coli MG1655 K-12 using an affinity purified antibody under anaerobic and aerobic growth conditions. As a control, we also performed ChIP-chip onArcA in a ∆arcA mutant strain of Escherchia coli MG1655 K-12. Described in the manuscript The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli