Endogenous peptidoglycan (PG)-hydrolyzing enzymes, the autolysins, are needed to relax the rigid PG sacculus to allow bacterial cell growth and separation. PGs of pathogens and commensal bacteria may also be degraded by hydrolases of animal origin (lysozymes), which act as antimicrobials. The genetic mechanisms regulating PG resistance to hydrolytic degradation were dissected in the Gram-positive bacterium Lactococcus lactis. We found that the ability of L. lactis to counteract PG hydrolysis depends on the degree of acetylation. Overexpression of PG O-acetylase (encoded by oatA) led to bacterial growth arrest, indicating the potential lethality of oatA and a need for its tight regulation. A novel regulatory factor, SpxB (previously denoted as YneH), exerted a positive effect on oatA expression. Our results indicate that SpxB binding to RNA polymerase constitutes a previously missing link in the multistep response to cell envelope stress, provoked by PG hydrolysis with lysozyme. We suggest that the two-component system CesSR responds to this stress by inducing SpxB, thus favoring its interactions with RNA polymerase. Induction of PG O-acetylation by this cascade renders it resistant to hydrolysis.