An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress

Area of Science:

• Microbiology
• Genetics
• Biotechnology

Background:

• Pseudomonas putida S12 exhibits tolerance to toxic solvents like toluene.
• Sudden exposure to solvents can be lethal to bacterial populations.
• Insertion sequences are mobile genetic elements that can influence gene expression and bacterial adaptation.

Purpose of the Study:

• To investigate the role of the insertion sequence ISS12 in Pseudomonas putida S12's tolerance to sudden toluene stress.
• To elucidate the genetic mechanism underlying enhanced solvent tolerance in P. putida S12.
• To explore the potential of ISS12 as a mutator element for bacterial adaptation.

Main Methods:

• Comparative genomic analysis of P. putida S12 populations under normal and toluene-stressed conditions.
• Nucleotide sequence analysis to identify insertion sites of ISS12.
• Gene expression analysis of the srpABC operon and its regulators.
• Experimental validation of ISS12's effect on solvent tolerance.

Main Results:

• A variant P. putida S12 population exposed to toluene showed an increase in ISS12 copy number and a 1000-fold higher survival rate.
• The extra ISS12 copy was found inserted into the srpS gene, part of the srpRS gene cluster regulating the SrpABC solvent resistance pump.
• The variant exhibited a seven-fold higher basal srp promoter activity, and disruption of srpS by ISS12 up-regulated SrpABC expression, enhancing toluene tolerance.

Conclusions:

• ISS12 acts as a mutator element in P. putida S12, facilitating rapid adaptation to sudden toxic solvent stress.
• Interruption of the srpS gene by ISS12 enhances the expression of the SrpABC solvent efflux pump, crucial for solvent tolerance.
• This mechanism allows P. putida S12 to survive lethal concentrations of toxic solvents by up-regulating protective systems.