title: Knockout of extracytoplasmic function sigma factor ECF-10 affects stress resistance and biofilm formation in Pseudomonas putida KT2440.
abstract: Pseudomonas putida is a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome of P. putida reveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, β-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes were ttgA, ttgB, and ttgC, which code for the major multidrug efflux pump TtgABC in P. putida KT2440. Investigation of an ECF-10 and ttgA double-knockout strain and a ttgABC-overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance in P. putida KT2440.
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces, Karlsruhe, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Tettmann
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces, Karlsruhe, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Dötsch
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics, Eggenstein-Leopoldshafen, Germany.
authors.forename: Olivier
authors.initials: O
authors.lastname: Armant
authors.affiliation: James Hogg Research Centre, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Fjell
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces, Karlsruhe, Germany joerg.overhage@kit.edu.
authors.forename: Joerg
authors.initials: J
authors.lastname: Overhage
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018441:Biofilms; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D016958:Pseudomonas putida / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism; D013312:Stress, Physiological
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D012808:Sigma Factor