Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc 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 peptidoglycan-hydrolyzing 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.

FIG. 1

Nucleotide sequence (nucleotides 1 to 869) and deduced amino acid sequences of the L. citreum DNA fragment encoding Mur (Lnmur) (accession number AF176553). The putative −10 and −35 sequences are double underlined, and the putative RBS is indicated with a dotted line. The stop codon is indicated with an asterisk, and a putative transcription terminator is underlined. The putative peptide signal cleavage site is indicated with an arrow.

FIG. 2

Alignment of the amino acid sequences of the Mur protein of L. citreum 22R (Lnmur) and the catalytic domains of AcmA of L. lactis MG1363, autolysin (EfAutol) of E. faecalis, muramidase-2 (Mur2) of E. hirae, and flagellar protein (FlgJ) of serovar Typhimurium. Alignment was made using the ClustalW program. Identical residues present in all the sequences are indicated with an asterisk, and similar residues are indicated with a point. Putative acidic residues present in the catalytic site are in bold letters.

FIG. 3

SDS-PAGE and renaturing SDS-PAGE analysis of the purified six-His-tagged L. citreum Mur protein. Coomassie blue staining was used for lanes 1 and 2. Lane 1, whole SDS cell extract of E. coli XL1-Blue harboring pTIL343 induced for 4 h with isopropyl-β-d-thiogalactopyranoside; lane 2, recombinant L. citreum Mur purified on Ni-nitrilotriacetic acid resin; lane 3, activity of the purified recombinant protein by renaturing SDS-PAGE containing 0.2% autoclaved M. lysodeikticus cells. The molecular masses (in kilodaltons) of standard proteins are indicated on the left.

FIG. 4

Renaturing SDS-PAGE analysis of the chimeric Mur-cA protein expressed in L. lactis MG1363acmAΔ1. Lane 1, L. lactis MG1363acmAΔ1 harboring plasmid pTIL344; lane 2, L. lactis MG1363acmAΔ1 harboring pTIL344 and pNZ9520 carrying nisRK genes. SDS cell extract of each strain was prepared from a noninduced culture and loaded onto polyacrylamide gel containing 0.2% autoclaved M. lysodeikticus cells. The molecular masses (in kilodaltons) of standard proteins are indicated on the left of the gel.

FIG. 5

Epifluorescent micrograph of L. lactis MG1363acmAΔ1 (A) and L. lactis MG1363acmAΔ1 harboring pTIL344 and pNZ9520 (B). Bacteria were grown in M17-glu medium and were not induced with nisin. Micrographs were taken after in situ hybridization of the lysozyme-treated bacteria with the universal probe EUB338.