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, ␤-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.

Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS) Promotes Biofilm Formation of Pseudomonas aeruginosa by Increasing Iron Availability

“…Depletion of rRNA was accomplished with the MICROB Express ™ bacterial mRNA enrichment kit (life technologies) according to the manufacturer's protocol. RNA-Seq was performed as described previously (Tettmann et al, 2014). Briefly, sequencing libraries were generated from 50 ng of rRNA depleted RNA samples following the Truseq RNA protocol (Illumina).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…A large conjugative plasmid encoding the OqxAB pump and type 3 fimbriae enabled E. coli to form a biofilm (892). In P. putida, inactivation of an extracytoplasmic function sigma factor, ECF-10, led to enhanced biofilm formation and the upregulation of the TtgABC pump with increased MDR (893). Upregulation of multiple proteins, including an uncharacterized RND pump and the OmpA and CarO OM proteins, also occurs in A. baumannii biofilms (894).…”

β-Glucanase Activity of the Oral Bacterium Tannerella forsythia Contributes to the Growth of a Partner Species, Fusobacterium nucleatum, in Cobiofilms

“…However, the transcription levels of BFO_0189 and BFO_0190 remained constant in the presence or absence of F. nucleatum (Table 1), suggesting that these regulators would have to be activated at the protein level following cell-to-cell contact to regulate the expression of ␤-glucanase operon. To support this notion, ECF sigma factors are involved in sensing environmental stress from antibiotics (16), heavy metals (17), oxidative stress (18), osmolytes (19), and temperature (20) in both Grampositive and -negative bacteria.…”