Single point mutation in the transcriptional regulator PnpR renders Pseudomonas sp. strain WBC-3 capable of utilizing 2-chloro-4-nitrophenol

Abstract Pseudomonas sp. strain WBC-3 mineralizes para -nitrophenol (PNP) as a growth substrate. The genes responsible for PNP degradation constitute 3 operons: pnpA , pnpB , and pnpCDEFG . As a chlorinated PNP, 2-chloro-4-nitrophenol (2C4NP) serves as a substrate for the PNP 4-monooxygenase PnpA; however, strain WBC-3 is unable to utilize 2C4NP. The LysR-type transcriptional regulator (LTTR) PnpR activates the expression of the 3 catabolic operons in response to PNP. Furthermore, an additional LTTR PnpM responds to 2C4NP other than PNP to initiate the expression of pnpCDEFG operon. Using a long-term directed evolution selection, spontaneous mutants of strain WBC-3 that grew on 2C4NP were obtained. The 2C4NP + strains had single mutation in codon 6 of the gene encoding PnpR, with substitution of cysteine for serine. The variant PnpR S6C drove the PNP-independent expression of pnpA and pnpB operons in an almost constitutive manner but allowed higher levels of induction upon addition of inducers. However, expression of pnpCDEFG operon was still PNP-dependent together with active PnpR S6C . When the pnpR S6C allele was introduced into a pnpR -deleted WBC-3 mutant, the corresponding strain acquired the ability to grow on 2C4NP but was still able to utilize PNP for growth.