Peptidoglycan Structure Analysis of Lactococcus lactis Reveals the Presence of an l , d -Carboxypeptidase Involved in Peptidoglycan Maturation

Abstract

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has L,D-carboxypeptidase activity and is involved in peptidoglycan maturation.Peptidoglycan is the major component of the gram-positive bacterial cell wall and ensures its rigidity and stability. Although its basic structure is characteristic of a given bacterial species, peptidoglycan is in a dynamic state throughout the bacterial life span, and its structure is the result of complex biosynthetic, maturation, and degradation reactions (11). Structural analysis of the peptidoglycan constituent muropeptide is a powerful method that allowed elucidation of the roles of biosynthesis enzymes involved in the design of cell wall architecture (3) and to characterize changes in peptidoglycan structure leading to antibiotic resistance (1,8,16). Also, the technique allowed revelation of peptidoglycan covalent modifications, such as O-acetylation or de-N-acetylation, which could play essential roles in the control of the activities of exogenous (25) and endogenous (17) cell wall-degrading enzymes.Lactococcus lactis is the model gram-positive lactic acid bacterium. Its peptidoglycan hydrolase complement was previously characterized (7,12,13,22). Bacterial peptidoglycan hydrolases are involved in different cellular functions during growth, such as cell separation, cell wall turnover, and cell wall expansion (21). Their activities can also lead to bacterial autolysis by hydrolysis of the protective cell wall peptidoglycan. Since these potentially lethal enzymes are synthesized during bacterial growth, their activities should be controlled. As mentioned above, covalent structural modification of peptidoglycan is one of the proposed mechanisms that could control peptidoglycan hydrolase activity (17, 21). Thus, the analysis of the L. lactis peptidoglycan structure constitutes the basis for further studies of the mechanisms that regulate synthesis and degradation of the L. lactis cell wall. Earlier studies revealed that L. lactis (formerly Streptococccus lactis) has A4␣-type peptidoglycan, with a monomer primary structure (GlcNAcMurNAc-L-Ala-␣-D-Glu-L-Lys-D-Ala) and a D-Asp in the interpeptide bridge, attached to the ε-amino group of Lys (19). In this study, we achieved detailed analysis of the muropeptide composition of Lactococcus lactis. Also, using the method developed, we identified an L,D-carboxypeptidase in L. lactis involved in peptidoglycan maturation.Muropeptide composition of L. lactis MG1363. Lactococcus lactis subsp. cremoris MG1363 was grown on M17 medium conta...

Structural basis for the evolution of vancomycin resistance D , D -peptidases

“…Structure-based multiple sequence alignment ( (25,26), and/or self-resistance of a glycopeptide producer, such as VanY n (27). This evolutionary model is consistent with sequence analysis and extensive phylogenetic reconstruction (Fig.…”

Hydrolysis of peptidoglycan is modulated by amidation of meso‐diaminopimelic acid and Mg2+ in Bacillus subtilis

“…The methanol gradient (from 0% in buffer A to 20% in buffer B) was developed at a flow rate of 0.5 ml min −1 . Muropeptides were analyzed directly by matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) mass spectrometry, as previously described (Courtin et al ., 2006). The peaks identified by MALDI‐TOF were integrated in the Agilent software to estimate the amount of each muropeptide.…”

Peptidoglycan N-acetylglucosamine deacetylation decreases autolysis in Lactococcus lactis

“…It was then cloned into the NcoI and SacI restriction sites of the pJIM2242 Cell wall peptidoglycan preparation. Peptidoglycan from L. lactis strains was prepared as described previously (Courtin et al, 2006). Cells from a 500 ml exponentially growing culture (OD 600 0.3) were chilled on ice and harvested by centrifugation (4200 g, 10 min, 4 uC).…”

“…The enzyme was inactivated by boiling the sample for 3 min and insoluble material was removed by centrifugation. The soluble muropeptides were reduced with sodium borohydride and then separated by reversedphase HPLC using a Hypersil ODS column (C18; 25064.6 mm internal diameter; 5 mm particle size; Thermo Hypersil-Keystone) at 50 uC using ammonium phosphate buffer and a methanol linear gradient, as described previously (Courtin et al, 2006). Peaks were analysed without desalting by MALDI-TOF MS with a Voyager DE STR mass spectrometer (Applied Biosystems), as described previously (Courtin et al, 2006).…”

“…Its structure can be analysed by identification of the constituent muropeptides obtained after muramidase digestion. Recently, we performed a detailed analysis of L. lactis peptidoglycan structure and observed several deacetylated muropeptides (Courtin et al, 2006). A gene (xynD) encoding a putative peptidoglycan deacetylase was identified in the L. lactis IL1403 genome on the basis of sequence similarity with the peptidoglycan GlcNAc deacetylase PgdA from Streptococcus pneumoniae (Vollmer & Tomasz, 2000).…”