Project description:The entire set of flagellar structural components and flagellar-specific transcriptional regulators, as well as much of the core chemotaxis machinery, is encoded into a >70 kbp cluster in Pseudomonas putida KT2440 genome. We have performed RNA-seq of the wild-type strain in order to identify operon boundaries and promoters location in this cluster.
Project description:The sigma factor FliA (σ28) has been described to activate the expression of several chemoreceptor-encoding genes and the late flagellar genes in Pseudomonas putida, enabling synthesis of the filament, an stator complex and completion of the flagella-associated chemotaxis machinery. The activity of FliA is repressed in the cytoplasm by the anti-sigma factor FlgM upon completion of the flagellar hook. In this study we aim to identify genome-wide targets of regulation by FliA in P. putida KT2442 (a spontaneous rifampicin-resistant mutant of the reference strain KT2440) by performing RNA-seq experiments using a fliA deletion mutant and a constitutively active strain that combines the deletion of flgM with ectopic production of FliA.
Project description:Here we demonstrate by experimental evolution and genetics the rapid and repeatable evolutionary re-wiring of regulatory pathways, where the cellular nitrogen regulatory system acquired the ability to “cross-talk” with the flagellar regulon. This rewiring restored flagella to aflagellate strains of Pseudomonas fluorescens devoid of the master regulator fleQ via a repeatable two-step evolutionary pathway. Step 1 initiates cross-talk by increasing intracellular levels of phosphorylated NtrC , a distant homologue of FleQ, which begins to commandeer control of the fleQ regulon whilst constitutively activating nitrogen uptake and assimilation genes. Step 2 is a switch-of-function mutation (NtrC’) that further redirects NtrC towards activation of motility and away from nitrogen uptake. Evolved NtrC’ emerges with a novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps to adopt new functionality.
Project description:To get insights in the electrogenic anaerobic lifestyle of P. putida KT2440 cultivated in a bioelectrochemical system (BES), we employed whole genome microarray expression profile.
Project description:Vibrio campbellii is a gram-negative bacterial pathogen that is both free-living and a pathogen of marine organisms and exhibits swimming motility via a single, polar flagellum. Swimming motility is a critical virulence factor in V. campbellii pathogenesis, and disruption of the flagellar motor significantly decreases host mortality. However, while V. campbelli encodes homologs of flagellar and chemotaxis genes conserved by other members of the Vibrionaceae, the regulatory network governing these genes have not been explored. We systematically deleted all 63 known flagellar and chemotaxis genes in V. campbellii and examined their effects on motility compared to their homologs in other Vibrios. We specifically focused on assessing the roles of the core flagellar regulators of the flagellar regulatory hierarchy established in other Vibrios: rpoN, flrA, flrC, and fliA. Although V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other Vibrios, we observed two critical differences: the σ54-dependent regulator FlrA is dispensable for motility, and Class II gene expression is independent of σ54 regulation. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae.
Project description:P. putida KT2440 was mixed in a conjugation filter with either E. coli BW27783-NxR bearing no plasmid (NP), E. coli BW27783-NxR bearing pSU2007 (ardC+) or E. coli BW27783-NxR bearing pLGM25 (ardC-). RNA-seq results showed that: (a) R388 genes involved in conjugation are highly upregulated in the ardC+ condition with respect to the ardC- condition. (b) a number of donor E. coli genes and pathways involved in flagellar motility, SOS and stress responses and different metabolic pathways are downregulated in the ardC- condition with respect to NP or ardC+ conditions. (c) SOS genes are upregulated in recipient P. putida cells when receiving ardC+ containing plasmid with respect to NP or ardC- conditions.
Project description:Bacterial transcription factors (TFs) regulate gene expression to adapt to changing environments; when combined, the TF’s regulatory actions comprise transcriptional regulatory networks (TRNs). The chromatin immunoprecipitation (ChIP) assay is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It enables the genome-wide study of transcription factor binding sites (TFBSs) and gene regulation. Although rapidly accumulating publicly-available ChIP data are a valuable resource for the study of gene regulation, there are no full datasets of key regulators in Pseudomonas putida. Here, we present the genome-wide binding for major TFs in P. putida.