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: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:The bacterial Lon protease participates in a variety of biological processes. In Pseudomonas syringae, mutation of lon is known to activate hrpL and a few hrpL-regulated genes in the rich medium. The elevated expression of hrpL and hrpL-regulated genes results from of increased stability of HrpR, the transcriptional activator of hrpL, in the lon mutant. Here we conducted a microarray analysis to determine genes that are differently expressed in the lon- mutant of P. s. pv. tomato DC3000 grown in the rich medium KB. Most genes induced in the lon- mutant belong to the HrpL-regulon or are related to the transcription, protein synthesis, and energy metabolism. The major group of genes reduced in the lon- mutant is related to cell wall biogenesis. The HrpL-regulated genes exhibit different induction patterns in the lon- mutant, suggesting the involvement of regulators additional to HrpL in regulating these genes. Compared with the wild type bacteria, the lon- mutants exhibit elevated hrpL expression in KB medium but reduced hrpL expression in the minimal medium (MM). The reduced hrpL RNA is correlated with the reduced hrpR and hrpS RNAs, suggesting that the Lon-mediated regulation of hrpL involves different mechanisms in KB and MM. Consistent with the reduced expression of the hrpL in MM, the lon- mutants are less pathogenic on host plants. Keywords: Expression profiles of lonB mutant in KB three samples ( biological replicates) of mutation were analyzed
Project description:The bacterial Lon protease participates in a variety of biological processes. In Pseudomonas syringae, mutation of lon is known to activate hrpL and a few hrpL-regulated genes in the rich medium. The elevated expression of hrpL and hrpL-regulated genes results from of increased stability of HrpR, the transcriptional activator of hrpL, in the lon mutant. Here we conducted a microarray analysis to determine genes that are differently expressed in the lon- mutant of P. s. pv. tomato DC3000 grown in the rich medium KB. Most genes induced in the lon- mutant belong to the HrpL-regulon or are related to the transcription, protein synthesis, and energy metabolism. The major group of genes reduced in the lon- mutant is related to cell wall biogenesis. The HrpL-regulated genes exhibit different induction patterns in the lon- mutant, suggesting the involvement of regulators additional to HrpL in regulating these genes. Compared with the wild type bacteria, the lon- mutants exhibit elevated hrpL expression in KB medium but reduced hrpL expression in the minimal medium (MM). The reduced hrpL RNA is correlated with the reduced hrpR and hrpS RNAs, suggesting that the Lon-mediated regulation of hrpL involves different mechanisms in KB and MM. Consistent with the reduced expression of the hrpL in MM, the lon- mutants are less pathogenic on host plants. Keywords: Expression profiles of lonB mutant in KB
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. Three biological replicates of wild-type and lonA mutant strains were used for this study. Reference RNA sample was harvested from wild-type strain.
Project description:Using Mycoplasma pneumoniae as a model organism, we conditionally depleted the two essential ATP-dependent proteases (Lon and FtsH) of this bacterium, by engineering three strains carrying a Lon and/or FtsH inducible expression locus. An integrative comparative study combining label-free shotgun proteomics and RNA-seq allowed us to decipher the global cellular response to Lon and FtsH depletion and to define protease substrates in this genome-reduced organism.
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 Lon protein is a protease implicated in virulence of many pathogenic bacteria, including some plant pathogens. However, little is known about the role of Lon in bacteria from genus Dickeya. This group of bacteria include important potato pathogens, with the most aggressive species, D. solani. To determine the importance of Lon for pathogenicity and response to stress conditions of bacteria, we constructed a D. solani Δlon strain. The mutant bacteria showed increased sensitivity to certain stress conditions, in particular osmotic and high-temperature stresses. Furthermore, qPCR analysis showed an increased expression of the lon gene in D. solani under these conditions. The deletion of the lon gene resulted in decreased motility, lower activity of secreted pectinolytic enzymes and finally delayed onset of blackleg symptoms in the potato plants. In the Δlon cells, the altered levels of several proteins, including virulence factors and proteins associated with virulence, were detected by means of MS-SWATCH analysis. These included components of the type III secretion system and proteins involved in bacterial motility. Our results indicate that Lon protease is important for D. solani to withstand stressful conditions and effectively invade the potato plant.