Abstract
Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under standard laboratory conditions AcmD was mainly secreted into the culture supernatant. An L. lactis acmAacmD double mutant formed longer chains than the acmA single mutant, indicating that AcmD contributes to cell separation. This phenotype could be complemented by plasmid-encoded expression of AcmD in the double mutant. No clear difference in cellular lysis and protein secretion was observed between both mutants. Nevertheless, overexpression of AcmD resulted in increased autolysis when AcmA was present (as in the wild type strain) or when AcmA was added to the culture medium of an AcmA-minus strain. Possibly, AcmD is mainly active within the cell wall, at places where proper conditions are present for its binding and catalytic activity. Various fusion proteins carrying either the three LysM repeats of AcmA or AcmD were used to study and compare their cell wall binding characteristics. Whereas binding of the LysM domain of AcmA took place at pHs ranging from 4 to 8, LysM domain of AcmD seems to bind strongest at pH 4.
Highlights
Peptidoglycan, the major cell wall material of Gram-positive bacteria, is composed of chains of N-acetylmuramic acid and N-acetylglucosamine linked by means of β(1-4) glycosidic bonds
Impairment of cell separation activity of peptidoglycan hydrolases (PGH) leads to long chains of cells in the lactic acid bacteria Lactococcus lactis and Streptococcus thermophilus [25,37,40]
The lysis of the cells of lactic acid bacteria due to the action of its PGHs has been shown to be essential during cheese ripening for the release of intracellular proteins such as peptidases that contribute to flavor development [43]
Summary
Peptidoglycan, the major cell wall material of Gram-positive bacteria, is composed of chains of N-acetylmuramic acid and N-acetylglucosamine linked by means of β(1-4) glycosidic bonds. These bonds are cleaved by peptidoglycan hydrolases (PGH) during cell separation and cellular autolysis [8,16,25]. Impairment of cell separation activity of PGHs leads to long chains of cells in the lactic acid bacteria Lactococcus lactis and Streptococcus thermophilus [25,37,40]. The (auto) lysis of the cells of lactic acid bacteria due to the action of its PGHs has been shown to be essential during cheese ripening for the release of intracellular proteins such as peptidases that contribute to flavor development [43]
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