Identification of Mur, an Atypical Peptidoglycan Hydrolase Derived fromLeuconostoc citreum

Abstract

A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycanhydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.Bacteria produce one or several peptidoglycan hydrolases (PGHs), which are capable of hydrolyzing covalent bonds in the peptidoglycan of their own cell envelope (for reviews, see references 46 and 49). Some of them, named autolysins, are able to trigger cell autolysis. PGHs are located in the cell wall and are involved in various cellular functions, including cell wall expansion, cell wall turnover, or cell separation. On the basis of their cleavage site in the peptidoglycan, four types of PGHs are defined: (i) N-acetyl-muramidases, (ii) N-acetyl-glucosaminidases, (iii) N-acetyl-muramoyl-L-alanine amidases, and (iv) peptidases. Most of the PGHs characterized so far have a modular structural organization with two domains: a catalytic domain containing the enzyme active site and a cell wall binding domain composed of several amino acid repeats (22,30).Autolysis of lactic acid bacteria (LAB) used as starters for cheese manufacturing plays an important role in flavor development during ripening (for reviews, see references 13 and 18). It has been shown that lysis of Lactococcus lactis starter strains leads to the release of intracellular peptidases in the cheese curd, and as a result more free amino acids (which are aroma precursors) are produced and hydrophobic bitter peptides are degraded (12,35,52).The PGH activities present in L. lactis were studied by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which allowed their detection after renaturation in a substrate-containing gel. Several activity bands were evidenced by this technique (9, 36, 40, 44). The major autolysin, AcmA, was characterized at the genetic level. It is an N-acetyl-muramidase that is required for proper cell separation after cell division (9) and is involved in autolysis observed during stationary phase after growth in liquid medium (10).Leuconostocs are heterofermentative LAB used as cheese starters in association with lactococci. They contribute to the development of cheese organoleptic properties by metabolizing citrate to diacetyl, an important flavor compound, an...

Functional characterization of domains found within a lytic enzyme produced by Streptococcus equi subsp. zooepidemicus

“…They are clearly related to cellular peptidoglycan hydrolases which are essential for various cellular functions, including cell wall expansion, cell wall turnover, and cell separation [20], but unlike these enzymes they do not remain cell associated. Their release into the extracellular medium, combined with their high target speci¢city, supports the concept that these enzymes are produced for the speci¢c purpose of killing closely related bacterial species.…”

Analysis of the Peptidoglycan Hydrolase Complement of Lactococcus lactis : Identification of a Third N- Acetylglucosaminidase, AcmC

“…Like Mur1 from Streptococcus thermophilus (25) and Mur from Leuconostoc citreum (14), AcmC contains a catalytic domain but lacks a specific cell wall binding domain. In contrast, like AcmA, AcmD has a modular structural organization with two domains, namely an N-terminal catalytic domain (residues 26 to 169) fused to a C-terminal cell wall binding domain.…”

Cell Wall Attachment of a Widely Distributed Peptidoglycan Binding Domain Is Hindered by Cell Wall Constituents

“…Nearly all cell wall hydrolases seem to consist of a catalytic domain and usually, but not always, a domain containing a number of specific amino acid repeats (5,6). Mur1 of Streptococcus thermophilus and Mur, the N-acetylmuramidase of Leuconostoc citreum, do not contain such repeats (7,8). Peptidoglycan hydrolyzing activity of Mur could be detected in vitro, but Mur on its own was not able to complement an acmA mutation in L. lactis.…”

“…A clear function of the cell wall binding domain of a peptidoglycan hydrolase is to direct the enzyme to its site of action, to the poles of the cell in the case of AcmA. Mur and Mur1 probably bind less specifically to the cell wall, probably even to the whole cell surface, which was already suggested for Mur (8).…”

“…Peptidoglycan hydrolyzing activity of Mur could be detected in vitro, but Mur on its own was not able to complement an acmA mutation in L. lactis. However, expression of a Mur-cA fusion protein was able to play the role of AcmA in cell separation after cell division in L. lactis acmA (8).…”