The ttgGHI solvent efflux pump operon of Pseudomonas putida DOT‐T1E is located on a large self‐transmissible plasmid

Abstract

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of > 1% (v/v) toluene in the culture medium. A set of multidrug efflux pumps have been found to play a major role in the tolerance of this bacterium to organic solvents (Rojas et al., J Bacteriol 183: 3967-3973). In the course of studies of the mechanisms underlying solvent tolerance in DOT-T1E, we isolated a spontaneous solvent-sensitive mutant derivative which had lost the genes encoding the TtgGHI efflux pump, the most important extrusion element in quantitative terms. Genomic comparisons between the mutant and its parental strain by microarray analysis revealed that in addition to the ttgVW-ttgGHI gene cluster, another group of genes, highly similar to those found in the Tn4653A and ISPpu12 transposable elements of the TOL plasmid pWW0 from P. putida mt-2, were also absent from this strain. Further analysis demonstrated that strain DOT-T1E harboured a large plasmid (named pGRT1) that was lost from the solvent-sensitive mutant. Mapping analysis revealed that the ttgVW-ttgGHI genes and the Tn4653A-like transposon are borne by the pGRT1 plasmid. Plasmid pGRT1 is highly stable and its frequency of loss is below 10(-8) per cell per generation under a variety of growth conditions, including nutritional and physical stresses. The pGRT1 plasmid is self-transmissible, and its acquisition by the toluene-sensitive P. putida KT2440 and Pseudomonas aeruginosa PAO1 increased the recipient's tolerance to toluene up to levels similar to those exhibited by P. putida DOT-T1E. We discuss the importance and potential benefits of this plasmid for the development of bacteria with enhanced solvent tolerance, and its potential impact for bioremediation and whole-cell biotransformations.

An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a

“…Efflux pumps of the resistance-nodulation-division (RND) superfamily are common in Gram negative bacteria [7,28] and are well studied for their role in antibiotic resistance and solvent tolerance in many Pseudomonas species [29,30]. However, these may not be the native or dominant physiological functions of RND pumps in bacteria.…”

Bacterial chemotaxis towards aromatic hydrocarbons in Pseudomonas

“…From a quantitative point of view the most relevant pump is TtgGHI, encoded on the pGRT1 self-transmissible plasmid present in this strain (Rojas et al, 2003;Rodríguez-Herva et al, 2007). To test whether the extrusion of the chemoattractant was the reason for the strong chemotatic phenotype, a mutant strain, DOT-T1E-PS28 (Rojas et al, 2003), with a nonfunctional TtgGHI pump, was used.…”

Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

“…The genes which encode the TtgABC pump are located on the chromosome of HB3267. A secondary role in tetracycline resistance was assigned to the TtgGHI efflux pump, which is located on the pGRT1 plasmid in P. putida DOT-T1E [59], although the primary role of this pump in P. putida DOT-T1E appears to be solvent extrusion [58], [59], [60]. The ttgGHI genes are present in the chromosome of HB3267 and the operon is located in a 44 Kb genomic island (Figure 3) with no homology to the chromosomal sequences of other P. putida strains or with the rest of pGRT1 plasmid [60].…”