A transcription Factor-sRNA cascade enhances acid tolerance in Lactococcus lactis by rewiring arginine metabolism

Lactococcus lactis is a vital starter culture in dairy and food industries. However, rapid lactic acid accumulation during fermentation leads to self-induced acid stress that limits cell growth, metabolic performance, and the production of high-value products such as nisin. Understanding and enhancing its acid tolerance in L. lactis is therefore crucial for improving the efficiency and productivity of industrial food fermentation processes. Here, we elucidated a novel multi-layered regulatory cascade comprising a transcription factor and a small non-coding RNA (sRNA) that governs acid stress adaptation in L. lactis F44, a nisin producer. We identified the TetR-family transcription factor AcrR1 as an upstream repressor of sRNA s042 through DNA pulldown and EMSA assays. DNase I footprinting precisely mapped its binding site to a 16-bp sequence within the s042 promoter. Under acid stress, acrR1 was downregulated, derepressing s042 transcription. The induced s042 post-transcriptionally activated the arginine regulators ArgR and AhrC by directly interacting with their mRNAs. This shifted the regulatory balance towards ArgR-AhrC complex formation, which derepressed the arginine deiminase (ADI) pathway while repressing biosynthesis, thereby enhancing acid tolerance. Our findings uncovered a sophisticated cascade-like regulation of AcrR1-s042-ArgR/AhrC that, in response to acid stress, fine-tuned the ADI pathway to confer acid resistance in L. lactis.