Proteomic Analysis of Lactococcus lactis FM03 Under Prolonged Alkaline Stress

Lactococci are best known to thrive in acidic conditions. While their physiology in an acidic environment is well researched, their stress response towards an alkaline environment remains to be explored. In this study, we habituated Lactococcus lactis FM03 to pH 6, 7 and 8 in chemostats with a fixed dilution rate of 0.2 h^-1 and defined the alkaline-induced stress proteome. Based on an enrichment analysis of GO-terms, we identified key physiological adaptations, including cell wall reinforcement, an increase in the uptake and metabolism of peptides and amino acids, as well as elevated levels of translation-related proteins. Transmission electron microscopy revealed a doubling in cell wall thickness at pH 8 compared with pH 6, and we measured higher release of multiple amino acids in the supernatant at pH 8. Further, the compatible solute uptake system for glycine betaine (OpuA) was upregulated, indicating osmotic stress conceivably linked to low cyclic-di-AMP levels. Selected global stress proteins were upregulated at pH 8 (GroEL, HtrA, FtsH), and RelA increased, suggesting the activation of the stringent response, whereas acid resistance systems (F₀F₁-ATPases, ADI, GAD) decreased. Together, this work defined a distinct alkaline stress remodelling of the proteome in L. lactis FM03 under sustained alkaline growth.