Project description:We wished to identify Crl-regulated genes in stationary phase in E.coli, and whether those overlap with the previously identified regulon of RpoS. Therefore wildtype E.coli (MC4100) and its isogenic crl::cat mutant were grown at 30oC. Total RNA was extracted at on OD (578nm) of 4 (during entry into stationary phase) and then the analysis proceeded as described in detail in the protocoles. The experiment was repeated three times and the results from those experiments are presented here.
Project description:Lon protease plays vital roles in many biological processes in Pseudomonas syringae, including type III secretion systems (T3SS), transcription regulation, protein synthesis and energy metabolism. Lon also functions as a transcriptional regulator in other bacterial species (e.g., Escherichia coli and Brevibacillus thermoruber). Therefore, we hypothesise that Lon has dual functions in P. syringae. To reveal the molecular mechanisms of Lon as a transcriptional regulator and protease under different environmental conditions, we used a combination of transcriptome sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the genes or proteins regulated by Lon. As a transcriptional regulator, Lon bound to the promoter regions of PSPPH_4788, gacA, fur, gntR, clpS, lon and glyA and consequently regulated 1-dodecanol oxidation activity, motility, pyoverdine production, gluconokinase activity, N-end rule pathway, lon expression and serine hydroxymethyltransferase (SHMT) activity in King’s B medium (KB). In minimal medium (MM), Lon regulated SHMT activity and lon expression by binding to the promoter regions of glyA and lon, respectively. As a protease, Lon regulated the T3SS and metabolic pathways (e.g., amino acid metabolism). In MM, Lon regulated the polysaccharide metabolic process by controlling PSPPH_0514, AlgA, CysD and PSPPH_4991. Taken together, these data demonstrate that Lon acts as a transcriptional regulator or protease in different environments and tunes its virulence and metabolic functions accordingly.
Project description:Lon protease is known to regulate various transcriptional regulators in other bacterial organisms. To understand whether lon protease is involved in transcriptional changes in Vibrio cholerae, wholel-genome level transcriptional profiling was performed using custom microarrays. Transcriptomes of lonA mutant and wild-type strains were compared in this study.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) and transposon insertion mutagenesis (Tnseq) libraries of Lon deletions compared to wt Caulobacter crescentus. Methods: See Methods section of The Lon protease links nucleotide metabolism with proteotoxic stress for information regarding methods or contact lead correspondence. Briefly, Samples for RNAseq were extracted from wt and lon deletion strains grown to mid exponential phase. Methods: See Methods section of The Lon protease links nucleotide metabolism with proteotoxic stress for information regarding methods or contact lead correspondence. Briefly, Samples for Tnseq were generated by Eztn5 transposon mutagenesis. Conclusions: Our study represents the first detailed analysis of lon deletion comparison to wt caulobacter transcriptomes, with biologic replicates, generated by RNA-seq technology.
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:Microarray experiments were preformed comparing the effect of sub-inhibitory concentrations of ciprofloxacin on five independent cultures of PAO1 and the PAO1 Lon mutant
Project description:Growth curves wt and hns strains in rich dYT medium. Sample in Mid-exponential phase (ME), Transition to Stationary (TS) and Late Stationary phase (LS).
Project description:Effect of FIS and H-NS on gene expression at relexed and hypernagative supercoiling level using LZ41 and LZ54 strains. LZ41 and LZ54 strains contain drug-resistant alleles of different topoisomerase genes. LZ41 strain treatment norfloxacin strongly relaxes DNA, whereas in LZ54 strain the same treatment generates high negative supercoiling (Khodursky et al., 1995, PNAS 92:11801-5; Ziechedrich et al, 1997, Genes Dev. 11:2580-92).
Project description:Effect of supercoiling level on gene expression using LZ41 and LZ54 strains containing drug-resistant alleles of different topoisomerase genes. LZ41 strain treatment with norfloxacin strongly relaxes DNA, whereas in LZ54 strain the same treatment generates high negative supercoiling (Khodursky et al., 1995, PNAS 92:11801-5; Ziechedrich et al, 1997, Genes Dev. 11:2580-92).