Two LysR family transcriptional regulators, McbH and McbN, activate the operons responsible for the midstream and downstream pathways of carbaryl degradation in Pseudomonas sp. strain XWY-1, respectively

Abstract

Previously, a LysR family transcriptional regulator McbG that activates the mcbBCDEF gene cluster involved in the upstream pathway (from carbaryl to salicylate) of carbaryl degradation in Pseudomonas sp. strain XWY-1 has been identified by us ( Appl. Environ. Microbiol. 2021, 87(9): e02970-20.). In this study, we identified McbH and McbN, which activate mcbIJKLM cluster (responsible for the midstream pathway, from salicylate to gentisate) and mcbOPQ cluster (responsible for the downstream pathway, from gentisate to pyruvate and fumarate), respectively. They both belong to the LysR family of transcriptional regulators. Gene disruption and complementation study reveal that McbH is essential for transcription of the mcbIJKLM cluster in response to salicylate and McbN is indispensable for the transcription of the mcbOPQ cluster in response to gentisate. The results of electrophoretic mobility shift assay (EMSA) and DNase I footprinting showed that McbH binds to the 52-bp motif in the mcbIJKLM promoter area and McbN binds to the 58-bp motif in the mcbOPQ promoter area. The key sequence of McbH binding to mcbIJKLM promoter is a 13-bp motif that conforms to the typical characteristics of LysR family. However, the 12-bp motif that is different from the typical characteristics of the LysR family regulator binding site sequence is identified as the key sequence for McbN to bind to the mcbOPQ promoter. This study reveals the regulatory mechanism for the midstream and downstream pathway of carbaryl degradation in strain XWY-1 and further enriches the members of the LysR transcription regulator family. IMPORTANCE: The enzyme-encoding genes involved in the complete degradation pathway of carbaryl in Pseudomonas sp. strain XWY-1 include mcbABCDEF , mcbIJKLM and mcbOPQ . Previous studies demonstrated that the mcbA gene responsible for hydrolysis of carbaryl to 1-naphthol is constitutively expressed and the transcription of mcbBCDEF was regulated by McbG. However, the transcription regulation mechanisms of mcbIJKLM and mcbOPQ have not been investigated yet. In this study, we identified two LysR-type transcriptional regulators, McbH and McbN, which activate the mcbIJKLM cluster responsible for the degradation of salicylate to gentisate and mcbOPQ cluster responsible for the degradation of gentisate to pyruvate and fumarate, respectively. The 13-bp motif is critical for McbH to bind to the promoter of mcbIJKLM , and 12-bp motif different from the typical characteristics of the LTTR binding sequence affects the binding of McbN to promoter. These findings help to expand the understanding of the regulatory mechanism of microbial degradation of carbaryl.