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Plasticity in AAA+ proteases reveals substrate specificity niches

ABSTRACT: Purpose: The goal of this study is to compare RNA-seq libraries of wildtype Caulobacter crescentus with two lon deletion strains (delta lon and delta lon clpX*). Methods: See Methods section of "Plasticity in AAA+ proteases reveals substrate specificity niches" for information regarding methods or contact lead correspondence. Briefly, Samples for RNAseq were extracted from wt and lon deletion strains grown to stationary phase. Conclusions: Our study represents the first detailed analysis of lon deletions (delta lon and delta lon clpX*) comparison to wt caulobacter transcriptomes, with biologic replicates, generated by RNA-seq technology in stationary phas

ORGANISM(S): Caulobacter vibrioides NA1000

PROVIDER: GSE166765 | GEO | 2022/02/12

REPOSITORIES: GEO

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The Lon protease links nucleotide metabolism with proteotoxic stress

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.

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Project description:With this analysis we aim to identify proteins that are substrates of LarA-activated Lon protease in Caulobacter crescentus. We will compare samples of wild type cells either harboring an empty vector (vector control, VC) or overexpressing 3xFLAG-tagged LarA (F-LarA). Based on our previous data we hypothesize that proteins affected by LarA-overexpression which results in Lon protease activation will decrease in levels compared to the vector control sample. The aim of this analysis is the identification of the interactome of the Lon protease in Caulobacter crescentus. It is a follow-up analysis of a project in which we were looking for novel substrates of the Lon protease in Caulobacter crescentus. To narrow down the list of potential substrates we obtained through the first analysis, we have purified the protease (Lon WT) as well an inactive TRAP mutant and interacting proteins using a Twin-Strep-tag (Iba lifesciences). As a control, a cell lysate without Strep-tag containing protein was included in the purification set-up. The aim is to identify the proteins that specifically interact with the Lon protease by mass spectrometry.

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Expression analysis of Caulobacter crescentus NA1000 delta-CCNA_00382 (ccrM) [DccrM] vs Caulobacter crescentus NA1000 wild type [WT]

Project description:Investigation of whole genome gene expression level changes in a Caulobacter crescentus NA1000 delta-CCNA_00382 (ccrM) mutant, compared to the wild-type strain. The mutations engineered into this strain render it incapable of methylating its genome on the adenine at GANTC motifs. References for strains : WT: Marks, M.E., Castro-Rojas, C.M., Teiling, C., Du, L., Kapatral, V., Walunas, T.L. and Crosson, S. (2010) The genetic basis of laboratory adaptation in Caulobacter crescentus. J Bacteriol, 192, 3678-3688; Collier, J. and Shapiro, L. (2009) Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter. J Bacteriol, 191, 5706-5716. DccrM: Gonzalez, D. and Collier, J. (2013) DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus. Mol Microbiol, 88, 203-218. A six chip study using total RNA recovered from three separate wild-type cultures of Caulobacter crescentus NA1000 and three separate cultures of a triple mutant strain, Caulobacter crescentus NA1000 delta-CCNA_00382 (ccrM), in which the ccrM gene coding for a DNA methyltransferase methylating the adenine in GANTC motifs is truncated and its product inactive. Each chip measures the expression level of 3933 genes from Caulobacter crescentus NA1000 with 3 probes per gene and with three-fold technical redundancy.

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Expression analysis of Caulobacter crescentus NA1000 delta-CCNA_00382 (ccrM) [DccrM] vs Caulobacter crescentus NA1000 wild type [WT]

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