Autolysis of Lactococcus lactis Is Increased upon d -Alanine Depletion of Peptidoglycan and Lipoteichoic Acids

Abstract

ABSTRACT Mutations in the genes encoding enzymes responsible for the incorporation of d -Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase ( alr ) mutant is strictly dependent on an external supply of d -Ala to be able to synthesize peptidoglycan and to incorporate d -Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when d -Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of d -Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the d -alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that d -alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis , LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking d -Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis , however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, d -Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis . Second, reduced amounts of d -Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.