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...

SummaryBacterial attachment to solid matrices depends on adhesive molecules present on the cell surface. Here we establish a positive correlation between peptidoglycan (PG) breaks, rather than particular molecules, and biofilm-forming capacity in the Grampositive bacterium Lactococcus lactis . The L. lactis acmA strain, which is defective in PG hydrolase, adhered less efficiently than the wild-type (wt) strain to different solid surfaces and was unable to form biofilms. These phenotypes were abolished by addition of lysozyme, a PG hydrolytic enzyme. Thus, the presence of PG breaks introduced by PG hydrolase, and not the AcmA protein itself, appears to be responsible for biofilm formation. Two different genetic screens confirmed the importance of PG breaks in L. lactis biofilm formation. Using the chainforming ability of the acmA strain as a phenotypic indicator of PG integrity, we selected for insertional mutants generating short chains. Five independent mutants were all mapped to ponA , which encodes the PG synthesis enzyme PBP1A. Double acmA ponA mutants displayed increased adhesion and biofilmforming capacity. Direct selection for strains with increased biofilm-forming capacity resulted in the isolation of another five mutations in ponA. Based on these results, we conclude that PG breaks are important for both adhesion and biofilm formation in L. lactis.

The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P nisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.Lactococcus lactis is a gram-positive lactic acid bacterium that is widely used in the production of fermented food products, and as such, it is considered a food-grade microorganism. Experimental data and genomic analyses indicate that only a few proteins are naturally secreted in L. lactis (4,32,38), and a plasmid-free strain does not produce the extracytoplasmic protease PrtP (13). These features have drawn the attention of researchers to the potential use of L. lactis for secretion of proteins of biotechnological interest. Thus, L. lactis has been extensively engineered for production and export of heterologous proteins with high added value, such as antigens or enzymes (2, 6, 8-12, 20, 22, 23, 31, 35). For this purpose, several genetic tools have been developed for L. lactis, and the potential of this organism as a prokaryotic host for heterologous protein production has been confirmed (7,9,23,40).Systems that allow controlled levels of expression of foreign proteins in L. lactis may offer certain advantages over constitutive systems (8). The nisin-controlled expression (NICE) system (7, 18), based on a combination of the P nisA promoter and the nisRK regulatory genes, has proven to be highly versatile (8,17,18) and has already been used to express different heterologous proteins (2,6,10,11,35).Protein export to the cell surface or into the medium is a preferred means of protein expression for several biotechnological applications (9, 23). However, poor expression and proteolytic degradation of heterologous proteins are limiting factors for stable protein production in bacteria. In Escherichia coli and Bacillus subtilis, several exported proteases that are associated with turnover of both natural and foreign proteins have been described (15,25,26,30,37). In contrast to E. coli and B. subtilis, L. lactis has a unique extracellular housekeeping protease, HtrA (high-temperature requirement), as demonstrated by construction of an L. lactis htrA-IL1403 mutant strain (previously designated htrA [33...

In bacterial communities one bacterium can influence the growth of other members of the population. These interactions may be based on nutritional factors or may occur via bacterial signaling molecules that are released in the medium. We present an example, showing that in addition to the above means of interactions, muramidases, enzymes that specifically cleave peptidoglycan chains, can also mediate interactions between bacteria. Using fluorescent in situ hybridization we demonstrate that Lactococcus lactis muramidase AcmA can hydrolyze the cell wall of Streptococcus thermophilus, without affecting viability. This intercellular activity of the lactococcal muramidase results in chain disruption of streptococci in vivo. Our data lead us to propose that chains can give growth advantages to streptococci in aerobic conditions.

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