Lactobacilli belong to the lactic acid bacteria, which play a key role in industrial and artisan food raw-material fermentation, including a large variety of fermented dairy products. Next to their role in fermentation processes, specific strains of Lactobacillus are currently marketed as health-promoting cultures or probiotics. The last decade has witnessed the completion of a large number of Lactobacillus genome sequences, including the genome sequences of some of the probiotic species and strains. This development opens avenues to unravel the Lactobacillus-associated health-promoting activity at the molecular level. It is generally considered likely that an important part of the Lactobacillus effector molecules that participate in the proposed health-promoting interactions with the host (intestinal) system resides in the bacterial cell envelope. For this reason, it is important to accurately predict the Lactobacillus exoproteomes. Extensive annotation of these exoproteomes, combined with comparative analysis of species- or strain-specific exoproteomes, may identify candidate effector molecules, which may support specific effects on host physiology associated with particular Lactobacillus strains. Candidate health-promoting effector molecules of lactobacilli can then be validated via mutant approaches, which will allow for improved strain selection procedures, improved product quality control criteria and molecular science-based health claims.

Lactobacilli belong to the lactic acid bacteria, which play a key role in industrial and artisan food raw-material fermentation, including a large variety of fermented dairy products. Next to their role in fermentation processes, specific strains of Lactobacillus are currently marketed as health-promoting cultures or probiotics. The last decade has witnessed the completion of a large number of Lactobacillus genome sequences, including the genome sequences of some of the probiotic species and strains. This development opens avenues to unravel the Lactobacillus-associated health-promoting activity at the molecular level. It is generally considered likely that an important part of the Lactobacillus effector molecules that participate in the proposed health-promoting interactions with the host (intestinal) system resides in the bacterial cell envelope. For this reason, it is important to accurately predict the Lactobacillus exoproteomes. Extensive annotation of these exoproteomes, combined with comparative analysis of species- or strain-specific exoproteomes, may identify candidate effector molecules, which may support specific effects on host physiology associated with particular Lactobacillus strains. Candidate health-promoting effector molecules of lactobacilli can then be validated via mutant approaches, which will allow for improved strain selection procedures, improved product quality control criteria and molecular science-based health claims.

Peptidoglycans provide bacterial cell walls with mechanical strength. The spatial organization of peptidoglycan has previously been difficult to study. Here, atomic force microscopy, together with cells carrying mutations in cell-wall polysaccharides, has allowed an in-depth study of these molecules.

Peptidoglycan (PG) N-acetyl muramic acid (MurNAc)O-acetylation is widely spread in Gram-positive bacteria and is generally associated with resistance against lysozyme and endogenous autolysins. We report here the presence of O-acetylation on N-acetylglucosamine (GlcNAc) in Lactobacillus plantarum PG. This modification of glycan strands was never described in bacteria. Fine structural characterization of acetylated muropeptides released from L. plantarum PG demonstrated that both MurNAc and GlcNAc are O-acetylated in this species. These two PG post-modifications rely on two dedicated O-acetyltransferase encoding genes, named oatA and oatB, respectively. By analyzing the resistance to cell wall hydrolysis of mutant strains, we showed that GlcNAc O-acetylation inhibits N-acetylglucosaminidase Acm2, the major L. plantarum autolysin. In this bacterial species, inactivation of oatA, encoding MurNAc O-acetyltransferase, resulted in marked sensitivity to lysozyme. Moreover, MurNAc over-O-acetylation was shown to activate autolysis through the putative N-acetylmuramoyl-L-alanine amidase LytH enzyme. Our data indicate that in L. plantarum, two different O-acetyltransferases play original and antagonistic roles in the modulation of the activity of endogenous autolysins.

BackgroundLactobacillus plantarum is commonly used in industrial fermentation processes. Selected strains are also marketed as probiotics for their health beneficial effects. Although the functional role of peptidoglycan-degrading enzymes is increasingly documented to be important for a range of bacterial processes and host-microbe interactions, little is known about their functional roles in lactobacilli. This knowledge holds important potential for developing more robust strains resistant to autolysis under stress conditions as well as peptidoglycan engineering for a better understanding of the contribution of released muramyl-peptides as probiotic immunomodulators.ResultsHere, we explored the functional role of the predicted peptidoglycan hydrolase (PGH) complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH-encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N-acetylglucosaminidase Acm2 and NplC/P60 D,L-endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell-wall integrity. We also showed by autolysis experiments that both PGHs are involved in the global autolytic process with a dominant role for Acm2 in all tested conditions, identifying Acm2 as the major autolysin of L. plantarum WCFS1. In addition, Acm2 and the putative N-acetylmuramidase Lys2 were shown to play redundant roles in both cell separation and autolysis under stress conditions. Finally, the analysis of the peptidoglycan composition of Acm2- and LytA-deficient derivatives revealed their potential hydrolytic activities by the disappearance of specific cleavage products.ConclusionIn this study, we showed that two PGHs of L. plantarum have a predominant physiological role in a range of growth conditions. We demonstrate that the N-acetylglucosaminidase Acm2 is the major autolysin whereas the D,L-endopeptidase LytA is a key morphogenic determinant. In addition, both PGHs have a direct impact on PG structure by generating a higher diversity of cleavage products that could be of importance for interaction with the innate immune system.

The peptidoglycan (PG) of Lactobacillus plantarum contains amidated meso-diaminopimelic acid (mDAP). The functional role of this PG modification has never been characterized in any bacterial species, except for its impact on PG recognition by receptors of the innate immune system. In silico analysis of loci carrying PG biosynthesis genes in the L. plantarum genome revealed the colocalization of the murE gene, which encodes the ligase catalyzing the addition of mDAP to UDP-N-muramoyl-D-glutamate PG precursors, with asnB1, which encodes a putative asparagine synthase with an N-terminal amidotransferase domain. By gene disruption and complementation experiments, we showed that asnB1 is the amidotransferase involved in mDAP amidation. PG structural analysis revealed that mDAP amidation plays a key role in the control of the L,D-carboxypeptidase DacB activity. In addition, a mutant strain with a defect in mDAP amidation is strongly affected in growth and cell morphology, with filamentation and cell chaining, while a DacB-negative strain displays a phenotype very similar to that of a wild-type strain. These results suggest that mDAP amidation may play a critical role in the control of the septation process.Peptidoglycan (PG) is a heteropolymer of glycan strands cross-linked by peptidic stems most often between the fourth and the third amino acids of the donor and the acceptor stem, respectively. The composition of this peptidic stem can vary from one bacterial species to another and, in Lactobacillus plantarum, is composed of L-Ala, D-Glu, meso-diaminopimelic acid (mDAP), and D-Ala. D-Lactic acid is found as the last moiety of the peptidic stem in PG precursors (5).Different amino acids found in PG have been reported as amidated in various species: D-Glu into D-iso-Gln (5, 9, 14, 21), mDAP into amidated mDAP (3,5,21), and D-Asp into D-Asn in species possessing a D-Asp as a cross bridge. Among these PG modifications, only the D-Asp amidation was characterized in Lactococcus lactis (23). D-Asp amidation is catalyzed by an asparagine synthase, AsnH, which is involved in autolysis control and resistance to cationic peptides (23).We have recently shown that both D-Glu and mDAP are highly amidated (100% and 94%, respectively) in L. plantarum (5). The functional role of these amidations remains poorly understood, except for their impact on PG recognition by the mammalian host innate immune system (2, 11, 14). As a typical prokaryotic structure, PG is sensed by pattern recognition receptors involved in bacterial detection (e.g., Nod1, Nod2, and Toll-like receptor 2[TLR2]), and PG modifications were shown to affect this process (2, 6, 11, 12). Unamidated mDAP is essential for Nod1 detection (2), whereas amidated mDAP was reported to modulate the recognition by TLR2 (11). Despite these important immunomodulatory properties, the genetic determinants of mDAP amidation have never been described, and the functional role of mDAP amidation in bacterial physiology remains completely unexplored.In this study, we identify the first mDAP...

Background: A range of peptidoglycan hydrolases (PGHs) contain low complexity domains of unknown function. Results: O-Glycosylation of the low complexity domain of the Lactobacillus plantarum autolysin Acm2 is a major negative modulator of enzymatic activity. Conclusion: O-Glycosylation represents an autoregulatory control mechanism of PGH activity.Significance: This is the first functional evidence that glycosylation controls the activity of a bacterial enzyme.

We show that in Lactococcus lactis, the gene asnH encodes the asparagine synthase involved in amidation of D-Asp present in peptidoglycan side chains and crossbridges. The level of D-Asp amidation in peptidoglycan has a strong effect on the sensitivity of bacteria to endogenous autolysins and to the cationic antimicrobials nisin and lysozyme.Peptidoglycan (PG), the main component of the cell wall in gram-positive bacteria, is a heteropolymer composed of glycan strands made of alternating N-acetylglucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) to which is attached a stem peptide (16). In the model lactic acid bacterium Lactococcus lactis, the stem peptide is composed of the pentapeptide chain L-Ala-␥-D-Glu-L-Lys-D-Ala-D-Ala. These peptidic chains can be cross-linked with a D-Asp residue (4, 11). D-Asp is transferred to the PG precursors by an aspartate ligase which ligates the ␤-carboxylate group of D-Asp to the ε-amino group of Lys (15). In the final mature L. lactis PG, about 75% of the side chain and crossbridge residues are amidated (4). However, neither D-iso-Asn nor D-Asn was shown to be a substrate for aspartate ligase (1, 15), indicating that amidation of the ␣-carboxyl group of D-Asp occurs after its incorporation into the PG precursor by aspartate ligase. Amidation could take place on the D-iso-Asp-containing lipid or on nascent PG (13). In this study, we identified for the first time the gene encoding the enzyme responsible for D-Asp amidation of the bacterial PG crossbridge.Analysis of the PG structure of an L. lactis asnH mutant. In the L. lactis MG1363 genome sequence, a gene, asnH (1,878 bp), encoding a putative asparagine synthase, is the first gene of the operon which also includes aslA, encoding the aspartate ligase involved in D-Asp incorporation into the PG interpeptide crossbridge. To investigate asnH function in L. lactis, we analyzed the PG structure of an asnH-negative mutant. This mutation was constructed in L. lactis MG1363 by single cross