Control of a programmed cell death pathway in Pseudomonas aeruginosa by a ppGpp-responsive antiterminator [RNA-Seq]
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ABSTRACT: In the opportunistic pathogen Pseudomonas aeruginosa the alp system encodes a programmed cell death (PCD) pathway that is switched on in a subset of cells in response to DNA damage. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified operon, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. We further show that the activity of AlpA is potentiated by the alarmone ppGpp, which is produced by the cell in response to stress. Through its responsiveness to ppGpp, AlpA can integrate environmental cues into the decision to execute a PCD pathway that is linked to the virulence of the organism.
Project description:In the opportunistic pathogen Pseudomonas aeruginosa the alp system encodes a programmed cell death (PCD) pathway that is switched on in a subset of cells in response to DNA damage. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified operon, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. We further show that the activity of AlpA is potentiated by the alarmone ppGpp, which is produced by the cell in response to stress. Through its responsiveness to ppGpp, AlpA can integrate environmental cues into the decision to execute a PCD pathway that is linked to the virulence of the organism.
Project description:In the opportunistic pathogen Pseudomonas aeruginosa the alp system encodes a programmed cell death (PCD) pathway that is switched on in a subset of cells in response to DNA damage. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified operon, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. We further show that the activity of AlpA is potentiated by the alarmone ppGpp, which is produced by the cell in response to stress. Through its responsiveness to ppGpp, AlpA can integrate environmental cues into the decision to execute a PCD pathway that is linked to the virulence of the organism.
Project description:Escherichia coli RelA is a ribosomal factor with strong (p)ppGpp synthesis activity that is dramatically activated in the presence of deacylated tRNA in the ribosomal A-site. RelA is a unique enzyme in that it is directly positively regulated by its product, alarmone nucleotide (p)ppGpp. Using HDX-MS, mulecular docking, biochemsitry, and microbiology approaches, we localise the (p)ppGpp binding site of E. coli RelA and uncover the molecular mechanism of RelA regulation by the alarmone.
Project description:The stringent response is an adaptive physiological response triggered by different conditions of nutritional or environmental stress and aimed at increasing survival under harsh conditions. This response is mediated the signalling nucleotides guanosine tetraphosphate (ppGpp) and pentaphospate (pppGpp), collectively known as (p)ppGpp. In this study we aim to identify genome-wide targets of regulation by the stringent-response associated alarmone (p)ppGpp in Pseudomonas putida by performing RNA-seq experiments using the wild-type KT2440tel strain and a KT2440tel-derivative bearing deletions of the (p)ppGpp synthetase-encoding genes relA and spoT (ppGpp0 mutant).
Project description:The impact on gene expression by the alarmone (p)ppGpp during growth and non-growth was determined by comparing the transcriptome of R. etli CFN42 wild type and rel mutant. This allowed us to better understand the pleiotropic stress phenotype of the rel mutant as well as identifying specific (p)ppGpp-dependent stress regulators.
Project description:This study elucidates the whole proteome changes in the wild type and gnetically modified strains of Vibrio cholerae. Label-free SWATH-MS is used for protein quantitation following genetic modifications in alarmone (p)ppGpp metabolizing genes and transcription factor DksA.