Analysis of the peptidoglycan hydrolase complement of Lactococcus lactis: identification of a third N-acetylglucosaminidase, AcmC

Abstract

The peptidoglycan hydrolase (PGH) complement of Lactococcus lactis was identified by amino acid sequence similarity searching of the L. lactis IL-1403 complete genome sequence. Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins. Four of these PGHs, AcmA to AcmD, contain a catalytic domain homologous to that of enterococcal muramidase, but they have different domain structures. The fifth one (YjgB) has sequence similarity with the active-site domain of peptidoglycan-specific endopeptidases. The three new PGH-encoding genes identified in this study are all actively transcribed in L. lactis subsp. cremoris MG1363. The relative abundance of their transcripts varied during growth and was maximal during the early exponential growth phase. The three encoded proteins have peptidoglycan-hydrolyzing activities which are detected only at acidic pHs by zymography. Like AcmA and AcmB, AcmC has N-acetylglucosaminidase activity rather than the N-acetylmuramidase activity predicted by sequence similarity.

FIG. 1.

Schematic representation of the domain structure of the five PGHs of L. lactis IL-1403. The total number of amino acids (aa), the calculated molecular mass (M.M.), and the calculated isoelectric point (pI) of each protein, with or without a putative signal peptide (SP), are indicated.

FIG. 2.

Amino acid sequence alignment of the putative catalytic domains of YjgB of L. lactis IL-1403, dl-endopeptidase II of B. sphaericus (EPII), and the LytE and LytF endopeptidases of B. subtilis. The alignment was performed with the ClustalW 1.74 program. *, identical amino acids in all four sequences. The motif Asp-Cys-Ser-Gly, characteristic of the dl-endopeptidase II family and containing the putative Cys residue present in the catalytic site, is shaded. The numbers refer to amino acid sequence positions.

FIG. 3.

Growth phase-dependent expression of PGH genes. (A) Northern blot analysis of PGH gene expression. Total RNAs were prepared from L. lactis MG1363 cells grown in CDM to different OD₆₅₀ values and analyzed by Northern blotting with probes specific for each gene. The hybridization signals were imaged and quantified with a Storm system. The transcript amount was standardized by the amount of 16S rRNA in each sample. (B) Growth was monitored by OD₆₅₀ measurements.

FIG. 4.

PAGE and zymogram analysis of purified AcmC-His, AcmD-His, and YjgB-His. Electrophoresis was done in the presence (AcmC-His and AcmD-His) or absence (YjgB-His) of SDS. Lanes 1, 4, and 6, Coomassie blue staining; lanes 2, 5, and 7, gel containing 0.2% (wt/vol) M. lysodeikticus cells; lane 3, gel containing 0.4% (wt/vol) L. lactis cells. Renaturation and activity detection were performed at pH 4.0 (citrate phosphate buffer) or at pH 6.0 (50 mM MES buffer) with 1% (vol/vol) Triton X-100. The apparent molecular masses are indicated in kilodaltons.

FIG. 5.

RP-HPLC analysis of soluble muropeptides released by AcmC from B. subtilis vegetative peptidoglycan. The structures determined for the two main muropeptides (1 and 2) are indicated. MurNAc, N-acetylmuramic acid; GlcNAc, N-acetylglucosamine; mDAP, meso-diaminopimelic acid.

FIG. 6.

PGH complement of L. lactis. The schematic structure of the L. lactis peptidoglycan and the sites of cleavage by the different PGHs. MurNAc, N-acetylmuramic acid; GlcNAc, N-acetylglucosamine.