An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress

The novel insertion sequence ISS12 plays a key role in the tolerance of Pse udomonas putida S12, to sudden toluene stress. Under normal culturing condi tions the P, putida S12 genome contained seven copies of ISS12. However, a P. putida S12 population,growing to high cell density after sudden addition of a separate phase of toluene carried eight copies. The survival frequenc y of cells in this variant P. putida S12 population was 1000 times higher t han in "normal" P. putida S12 populations. Analysis of the nucleotide seque nce flanking the extra ISS12 insertion revealed integration into the sr-pg gene. srpS forms a gene cluster with srpR and both are putative regulators of the solvent resistance pump SrpABC. SrpABC makes a major contribution to solvent tolerance in P. putida S12 and is induced by toluene. The basal le vel of srp promoter activity in the P. putida S12 variant was seven times h igher than in wild-type P, putida S12. Introduction of the intact srpRS gen e cluster in the variant resulted in a dramatic decrease of survival freque ncy after a toluene shock. These findings strongly suggest that interruptio n of srpS by ISS12 upregulates expression of the solvent pump, enabling the bacterium to tolerate sudden exposure to lethal concentrations of toxic so lvents. We propose that P. putida S12 employs ISS12 as a mutator element to generate diverse mutations to swiftly adapt when confronted with severe ad verse conditions.