Regulation of acetate tolerance by small ORF-encoded polypeptides modulating efflux pump specificity in Methylomonas...

Methanotrophs have emerged as promising hosts for the biological conversion of methane into value-added chemicals, including various organic acids. Understanding the mechanisms of acid tolerance is essential for improving organic acid production. WatR, a LysR-type transcriptional regulator, was initially identified as involved in lactate tolerance in a methanotrophic bacterium Methylomonas sp. DH-1. In this study, we investigated the role of WatR as a regulator of cellular defense against weak organic acids and identified novel target genes of WatR.

By conducting an investigation into the genome-wide binding targets of WatR and its role in transcriptional regulation, we identified genes encoding an RND-type efflux pump (WatABO pump) and previously unannotated small open reading frames (smORFs), watS1 to watS5, as WatR target genes activated in response to acetate. The watS1 to watS5 genes encode polypeptides of approximately 50 amino acids, and WatS1 to WatS4 are highly homologous with one predicted transmembrane domain. Deletion of the WatABO pump genes resulted in decreased tolerance against formate, acetate, lactate, and propionate, suggesting its role as an efflux pump for a wide range of weak organic acids. WatR repressed the basal expression of watS genes but activated watS and WatABO pump genes in response to acetate stress. Overexpression of watS1 increased tolerance to acetate but not to other acids, only in the presence of the WatABO pump. Therefore, WatS1 may increase WatABO pump specificity toward acetate, switching the general weak acid efflux pump to an acetate-specific efflux pump for efficient cellular defense against acetate stress.

Our study has elucidated the role of WatR as a key transcription factor in the cellular defense against weak organic acids, particularly acetate, in Methylomonas sp. DH-1. We identified the genes encoding WatABO efflux pump and small polypeptides (WatS1 to WatS5), as the target genes regulated by WatR for this specific function. These findings offer valuable insights into the mechanisms underlying weak acid tolerance in methanotrophic bacteria, thereby contributing to the development of bioprocesses aimed at converting methane into value-added chemicals.