AtuR and MarR transcriptional repressors that sense fatty or naphthenic acids and regulate degradation or antimicrobial resistance operons in Pseudomonas sp. OST1909.

Abstract

Naphthenic acids (NA) are a complex family of acyclic and cyclic carboxylic acids that accumulate in tailings after bitumen extraction from oil sands in Alberta. In response to exposure to naphthenic acids, Pseudomonas sp. OST1909 induces the expression of operons regulated by TetR and MarR-type transcriptional repressors. This class of repressor releases from promoters after binding small molecules, leading to a de-repression of the target operons. We propose that these regulators sense fatty and naphthenic acids and control the expression of genes required to cope with their exposure. Plasmid-encoded transcriptional lux reporters to the atu degradation and mar antimicrobial resistance promoters were constructed that also included the corresponding repressor protein, controlled by various strength constitutive promoters. We provide evidence that the atuAR and marR promoters are directly repressed by AtuR and MarR, respectively. His-tagged MarR and AtuR proteins bound to their promoters containing palindromic binding sites in electrophoretic mobility shift assays (EMSA). Fatty and naphthenic acid compounds and mixtures prevented promoter DNA binding to the repressor proteins in EMSA assays, which suggested direct analyte binding to the protein. The marR-H biosensor construct was functional when introduced into E. coli, confirming the role of NA sensing and transcriptional repressor proteins. This study provides mechanistic insight into the use of NA-inducible promoters as bacterial biosensors for NA detection and identifies novel ligands for the MarR and TetR types of repressor proteins. ImportanceUnderstanding the gene expression responses to environmental pollutants is necessary to construct whole cell biosensors that can be employed as a monitoring technology. We have previously identified bacterial genes that were induced by exposure to naphthenic acids, a complex family of cyclic and polycyclic, alkyl substituted carboxylic acids that originate in oil sands but accumulate in the water used to extract bitumen from oil sands. In this study we focus on MarR and TetR-type regulatory proteins and their role in binding fatty acids and naphthenic acids, to further our understanding of how bacteria sense these compounds and mitigate a gene expression response.