Three distinct glycosylation pathways are involved in the decoration of Lactococcus lactis cell wall glycopolymers

Extra-cytoplasmic sugar decoration of glycopolymer components of the bacterial cell wall contributes to their structural diversity. Typically, the molecular mechanism that underpins such a decoration process involves a three-component glycosylation system (TGS) represented by an undecaprenyl-phosphate (Und-P) sugar-activating glycosyltransferase (Und-P GT), a flippase, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer. Here, using bioinformatic analyses, CRISPR-assisted recombineering, structural analysis of cell wall-associated polysaccharides (CWPS) through Maldi-Tof MS and methylation analysis, we report on three such systems in the bacterium Lactococcus lactis. On the basis of sequence similarities, we first identified three gene pairs, csdAB, csdCD, and csdEF, each encoding an Und-P GT and a PolM GT, as potential TGS component candidates. Our experimental results show that csdAB and csdCD are involved in Glc side chain addition on the CWPS components rhamnan and polysaccharide pellicle (PSP), respectively, whereas csdEF plays a role in galactosylation of lipoteichoic acid (LTA). We also identified a potential flippase encoded in the L. lactis genome (llnz_02975, cflA) and confirmed that it participates in the glycosylation of the three cell wall glycopolymers rhamnan, PSP, and LTA, thus indicating that its function is shared by the three TGSs. Finally, we observed that glucosylation of both rhamnan and PSP can increase resistance to bacteriophage predation and that LTA galactosylation alters L. lactis resistance to bacteriocin

Distinct and Specific Role of NlpC/P60 Endopeptidases LytA and LytB in Cell Elongation and Division of Lactobacillus plantarum

Peptidoglycan (PG) is an essential lattice of the bacterial cell wall that needs to be continuously remodeled to allow growth. This task is ensured by the concerted action of PG synthases that insert new material in the pre-existing structure and PG hydrolases (PGHs) that cleave the PG meshwork at critical sites for its processing. Contrasting with Bacillus subtilis that contains more than 35 PGHs, Lactobacillus plantarum is a non-sporulating rod-shaped bacterium that is predicted to possess a minimal set of 12 PGHs. Their role in morphogenesis and cell cycle remains mostly unexplored, except for the involvement of the glucosaminidase Acm2 in cell separation and the NlpC/P60 D, L-endopeptidase LytA in cell shape maintenance. Besides LytA, L. plantarum encodes three additional NlpC/P60 endopeptidases (i.e., LytB, LytC and LytD). The in silico analysis of these four endopeptidases suggests that they could have redundant functions based on their modular organization, forming two pairs of paralogous enzymes. In this work, we investigate the role of each Lyt endopeptidase in cell morphogenesis in order to evaluate their distinct or redundant functions, and eventually their synthetic lethality. We show that the paralogous LytC and LytD enzymes are not required for cell shape maintenance, which may indicate an accessory role such as in PG recycling. In contrast, LytA and LytB appear to be key players of the cell cycle. We show here that LytA is required for cell elongation while LytB is involved in the spatio-temporal regulation of cell division. In addition, both PGHs are involved in the proper positioning of the division site. The absence of LytA activity is responsible for the asymmetrical positioning of septa in round cells while the lack of LytB results in a lateral misplacement of division planes in rod-shaped cells. Finally, we show that the co-inactivation of LytA and LytB is synthetically affecting cell growth, which confirms the key roles played by both enzymes in PG remodeling during the cell cycle of L. plantarum. Based on the large distribution of NlpC/P60 endopeptidases in low-GC Gram-positive bacteria, these enzymes are attractive targets for the discovery of novel antimicrobial compounds

The CWPS Rubik's cube: Linking diversity of cell wall polysaccharide structures with the encoded biosynthetic machinery of selected Lactococcus lactis strains

The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designatedcwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A-C). In the present study, thecwpsloci of 107 lactococcal strains were compared, revealing the presence of a fourthcwpsgenotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link betweencwpsgenotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in thecwpscluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galfand phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain

Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG

Peer reviewe

Analysis of the Peptidoglycan Hydrolase Complement of Lactobacillus casei and Characterization of the Major γ-D-Glutamyl-L-Lysyl-Endopeptidase

Peptidoglycan (PG) is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs) which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties

PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis

Background: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H2O2) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H2O2. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displaye

Role mechanisms and control of lactic acid bacteria lysis in cheese

Lysis of dairy starters is a prerequisite for optimum cheese maturation, since intracellular starter enzymes, particularly peptidases, can then play their role. Here we describe the different methods used to detect starter lysis in situ and current knowledge concerning the impact of lysis on cheese ripening, particularly the increase of free amino acids due to early lysis and the reduction of bitterness by hydrolysis of large hydrophobic peptides. Recent results obtained on the impact of lysis on lipolysis and amino acid catabolism are also described. Then, we present current knowledge regarding the mechanisms involved, focussing mainly on the model most investigated: Lactococcus lactis. Recent advances concerning the molecular characterization of peptidoglycan hydrolases are summarized (sequence, structure, regulation) together with current knowledge of the relationship between lysogeny and lysis. Lastly, we review the different approaches proposed to control or induce lysis in situ. In conclusion, we point out unaddressed questions

Editorial : Post-translational modifications in bacteria – The dynamics of bacterial physiology

International audienc

Etude du système autolytique de Lactococcus lactis (caractérisation biochimique et génétique des hydrolases du peptidoglycane)

L'autolyse bactérienne désigne l'éclatement de la cellule résultant de la dégradation du peptidoglycane, par des enzymes appelées hydrolases du peptidoglycane (PGH) ou autolysines. L'autolyse des bactéries lactiques joue un rôle important dans le développement de la flaveur et des qualités organoleptiques des fromages en cours de l'affinage. La maîtrise de ce phénomène apparaît comme un moyen de mieux contrôler et d'accélérer l'affinage, processus lent et coûteux. A cette fin, nous avons entrepris une étude exhaustive du contenu en PGHs de Lactococcus lactis, bactérie lactique largement utilisée en industrie laitière. Jusqu'à présent, AcmA était l'unique PGH caractérisée chez L.lactis. A partir de la séquence complète du génome de L.lactis subsp lactis IL1403, nous avons dressé l'inventaire des gènes codant des PGHs. Quatre nouvelles PGHs appartenant à deux familles sur la base des similarités de séquence, ont été identifiées: AcmB, AcmC et AcmD, comme AcmA, appartiennent à la famille de la muramidase d'E.hirae et YjgB à celle de l'endopeptidase de B.sphaericus. Nous avons montré que l'ensemble des PGHs constitue un système autolytique fonctionnel et établi que, malgré leurs homologies de séquence avec la muramidase d'entérocoque, AcmB et AcmC sont des N-acétyl-glucosaminidases. Les nouvelles PGHs ont la particularité d'être actives à pH acide et de présenter des spécificités différentes sur le peptidoglycane de différentes espèces bactériennes. Dans notre étude, nous avons caractérisé, plus particulièrement, AcmB et établi qu'elle contribue à l'autolyse cellulaire au côté d'AcmA. En revanche, YjgB n'y contribue pas. Les travaux réalisés au cours de cette thèse ont permis de progresser sur la caractérisation moléculaire des PGHs et d'acquérir une vue d'ensemble du système autolytique de L.lactis.Bacterial autolysis is defined as the cell disintegration resulting from peptidoglycan degradation by so called peptidoglycan hydrolases (PGH) or autolysins. In the case of lactic acid bacteria, which are widely used in dairy industry, cell autolysis is known to have a positive impact on flavour development and organoleptic properties during cheese ripening. In order to control this phenomenon, we investigated in details the PGH content of Lactococcus lactis. Up to now, only the major PGH named AcmA, was characterized in L.lactis. In the complete genome sequence of L.lactis subsp lactis IL1403 we identified four new PGHs, by sequence homology search. AcmB, AcmC and AcmD, like AcmA, belong to the E.hirae muramidase family and YjgB to the B.sphaericus endopeptidase family. We established that the five PGHs constituted a functional autolytic system and that though presenting sequence similarity with enterococcal muramidase, AcmB and AcmC have N-acetyl-glucosaminidase specificity. All the new PGHs are active only at acidic pH and exhibit different specificities towards the peptidoglycan originating from different bacterial species. We have focused our work on AcmB and shown that AcmB contributes to cellular autolysis, its hydrolytic action being potentiated by the major autolysin AcmA action. YjgB doesn't contribute to cellular autolysis. This work provides PGH molecular characterization and an overview on L lactis autolytic system.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF