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

Contrôle du cycle cellulaire par les muropeptidases de type NlpC/P60 chez Lactobacillus plantarum

The cell wall is an essential structure for the life of most bacterial species. Peptidoglycan (PG) is one of the major compounds of the cell wall. It is composed of glycan strands linked together by peptide side chains. Two types of enzymes are required for PG biosynthesis. Penicillin binding proteins (PBPs) insert the new material in the pre-existing structure. Concomitantly, peptidoglycan hydrolases (PGHs) cleave the PG meshwork to allow the insertion of this new material. Growth of rod-shaped bacteria requires that these two key processes occurred properly. First, bacteria elongate and second, they divide to produce two daughter cells of equal size and composition. Dedicated PBPs and PGHs are involved in these two cell-cycle pathways. In L. plantarum that is used as model in this work, 12 PGHs were previously identified. Members belonging to the four families of enzymatic activities are found. In this work, we studied the role of NlpC/P60 endopeptidases in the cell cycle. This family contains four enzymes: LytA, LytB, LytC and LytD. LytA and LytB are modular enzymes composed of two accessory domains (LysM and AST) in addition of their catalytic domain. LytC and LytD have only one accessory domain of unknown function associated to their catalytic domain. First, we assigned a potential function to each enzyme by simple and double gene inactivation. LytA is implicated in cell elongation and septum positioning. LytB is involved in the spatio-temporal regulation of cell division. LytC and LytD are potential PG recycling enzymes. Second, we studied LytA and LytB in more details. Indeed, these two enzymes have similar modular organizations with LysM and AST domains. However, these accessory domains could not be exchange without affecting the efficiency of the enzyme. This indicates that accessory domains followed a co-evolution to give specific functions to LytA and LytB. Third, we inactivate key proteins of the cell cycle. By phenotypic comparison, we conclude that LytB could be controlled by a FtsEX-like complex. Moreover, the comparison of the phenotype of LytA inactivation with those of MreB1 and PBP2b confirmed the implication of LytA in the elongation process. Further work will consist in the identification of interactions between cell cycle proteins and LytA or LytB using co-localization and co-purifications experiments.La paroi est indispensable à la vie de la majorité des bactéries. Celle-ci est composée principalement de peptidoglycane (PG) qui forme une structure tridimensionnelle contenant de brins de glycanes pontées entre eux par des chaines peptidiques latérales. Deux types d’enzymes agissent sur cette structure afin de lui permettre de grandir lors de la croissance de la cellule bactérienne tout au long de deux processus essentiels : la division et l’élongation. Les synthases du PG ou penicillin binding proteins (PBPs) intègrent le nouveau matériel dans le réseau de PG tandis que les hydrolases du PG (PGHs) coupent dans la structure préexistante afin de faciliter l’insertion du nouveau matériel. Chez L. plantarum, utilisé comme modèle dans ce travail, 12 PGHs ont été identifiées parmi lesquelles, quatre endopeptidases de type NlpC/P60, LytA, LytB, LytC et LytD qui font l’objet de ce travail. Par une approche d’inactivation génique, une fonction potentielle pour chaque endopeptidase a été déterminée. LytA est impliquée dans le processus d’élongation et de positionnement du septum. LytB participe à la régulation spatio-temporelle de la division. LytC et LytD sont quant à elles des enzymes potentiellement impliquées dans le recyclage du PG. LytA et LytB sont deux enzymes modulaires comprenant en plus de leur domaine catalytique de type NlpC/P60 un domaine AST non organisé et un domaine LysM de liaison au PG. La complémentarité et la co-évolution de ces domaines pour assurer la spécificité de fonction de chaque enzyme a été mise en évidence grâce à l’utilisation de protéines hybrides. L’inactivation de différents acteurs-clef du cycle cellulaire a confirmé la participation de LytA dans l’élongation. De plus, LytB pourrait être contrôlé par un homologue de FtsEX. L’étude des interactions de ces protéines sera nécessaire pour confirmer les rôles de LytA et LytB.(SC - Sciences) -- UCL, 201

Binding mechanism of the peptidoglycan hydrolase Acm2: low affinity, broad specificity

Peptidoglycan hydrolases are bacterial secreted enzymes that cleave covalent bonds in the cell-wall peptidoglycan,thereby fulfilling major physiological functions during cell growth and division. Although the molecular structure and functional roles of these enzymes have been widely studied, the molecular details underlying their interaction with peptidoglycans remain largely unknown, mainly owing to the paucity of appropriate probing techniques. Here, we use atomic force microscopy to explore the binding mechanism of the major autolysin Acm2 from the probiotic bacterium Lactobacillus plantarum. Atomic force microscopy imaging shows that incubation of bacterial cells with Acm2 leads to major alterations of the cell-surface nanostructure, leading eventually to cell lysis. Single-molecule force spectroscopy demonstrates that the enzyme binds with low affinity to structurally different peptidoglycans and to chitin, and that glucosamine in the glycan chains is the minimal binding motif. We also find that Acm2 recognizes mucin, the main extracellular component of the intestinal mucosal layer, thereby suggesting that this enzyme may also function as a cell adhesion molecule. The binding mechanism (low affinity and broad specificity) of Acm2 may represent a generic mechanism among cell-wall hydrolases for guiding cell division and cell adhesion

Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation.

Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca(2+) exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca(2+) uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca(2+)/H(+) antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn(2+) to the cells

PBP2b plays a key role in both peripheral growth and septum positioning in Lactococcus lactis

Lactococcus lactis is an ovoid bacterium that forms filaments during planktonic and biofilm lifestyles by uncoupling cell division from cell elongation. In this work, we investigate the role of the leading peptidoglycan synthase PBP2b that is dedicated to cell elongation in ovococci. We show that the localization of a fluorescent derivative of PBP2b remains associated to the septal region and superimposed with structural changes of FtsZ during both vegetative growth and filamentation indicating that PBP2b remains intimately associated to the division machinery during the whole cell cycle. In addition, we show that PBP2b-negative cells of L. lactis are not only defective in peripheral growth; they are also affected in septum positioning. This septation defect does not simply result from the absence of the protein in the cell growth machinery since it is also observed when PBP2b-deficient cells are complemented by a catalytically inactive variant of PBP2b. Finally, we show that round cells resulting from β-lactam treatment are not altered in septation, suggesting that shape elongation as such is not a major determinant for selection of the division site. Altogether, we propose that the specific PBP2b transpeptidase activity at the septum plays an important role for tagging future division sites during L. lactis cell cycle

Filamentation of <i>L</i>. <i>lactis</i> induced by methicillin treatment.

<p>(A) Micrographs of wild-type (NZ3900) cells treated by methicillin (1 μg ml^-1) obtained by transmission electron microscopy (TEM). Arrows indicate incomplete septa. Scale bars, 500 nm. (B) Identification of methicillin-targeted PBPs by Bocillin-FL staining competition assay. Membrane extracts from wild-type and <i>pbp2a</i> mutant cells were incubated with 0, 1, 2, 4 or 8 μg ml^-1 of methicillin prior to add Bocillin-FL. Note the sharp decrease in PBP2x band intensity in the profile of the <i>pbp2a</i> mutant. In wild-type extracts, selective blocking of PBP2x by methicillin is masked by the co-migrating PBP2a band. The two bottom panels show the relative fluorescence intensity of each band (in arbitrary units, A.U.) normalized to the fluorescence intensity measured in absence of methicillin (first lane of each gel).</p

Model for FtsZ-directed dynamics of PBP2b during vegetative and filamentation cycles of <i>L</i>. <i>lactis</i>.

<p>FtsZ structures are shown in green. PBP2b is depicted as a yellow oval. Peripheral localization of the proteins is shown as a ring of the corresponding color. Direction of peripheral and septal growth is shown by grey arrows. The vegetative cell cycle (left) is divided in two separate phases as determined by FtsZ rings structural changes and spatio-temporal localization of PBPs, including PBP2b. During the elongation-only phase (two top cells), FtsZ equatorial ring exhibits a dynamic structure and the PBP2b-dependent peripheral growth mediates cell elongation at mid-cell. At the time of cell constriction, the Z ring segregates into 3 discrete rings; a central constricting ring directing cell division and two lateral rings that move apart as peripheral growth continues from PBP2b located at the septum. After completion of cell division, the elongation-specific PBP2b relocates to the new equatorial FtsZ rings of the newborn cells to reinitiate the cell cycle. The methicillin-induced filamentation cycle (right) results from PBP2x inhibition and reactivation following methicillin removal. During filamentation, PBP2b-dependent peripheral growth mediates cell elongation from a pre-septal position. During filament reversion, FtsZ-directed dynamics of PBP2b takes place as observed during the vegetative cycle but in a highly hierarchical manner starting from the center of the filament.</p

Cell elongation and dynamics of FtsZ during the vegetative cell cycle of <i>L</i>. <i>lactis</i>.

<p>(A) Cell length (in μm) was measured for 11 individual cells analyzed by time-lapse microscopy (representative example shown on the top) and the resulting length curves were aligned on the start of cell constriction (T₀ = 0 min). The cell cycle is separated in two phases without obvious transition between: i. cell elongation only and ii. combined elongation and division during and after constriction. (B) Time-lapse imaging of FtsZ-Ve during cell elongation and division. <i>L</i>. <i>lactis</i> cells expressing the FtsZ-Ve fluorescent protein (NZ3900 [pGIBLD031]) were grown on agar pads and visualized by phase contrast (PC, top row) and epifluorescence (FtsZ-Ve, lower row) microscopy (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198014#pone.0198014.s003" target="_blank">S3 Fig</a> [Cell #1] and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198014#pone.0198014.s015" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198014#pone.0198014.s016" target="_blank">S2</a> Movies). Pictures were taken every 10 min. Observed structural changes of the FtsZ ring (green line) are schematically represented for different steps of the cell cycle. The shaded green band depicts the fuzzy aspect of FtsZ structures during early elongation phase. Scale bar, 2μm.</p

Inhibition of cell elongation in <i>L</i>. <i>lactis</i> by amoxicillin treatment.

<p>(A) Length to width ratios of wild-type (WT, NZ3900), WT treated with amoxicillin 0.1 μg ml^-1 (WT + Amo 0.1), and <i>pbp2b</i> mutant. Mean values (<i>n</i> = ~ 50 cells) ± standard deviations. Statistical analysis of the difference between length to width ratios was performed by a <i>t</i> test using WT as reference. **, <i>P</i> < 0.01. (B) Micrographs of WT cells treated by amoxicillin (0.1 μg ml^-1) obtained by transmission electron microscopy (TEM). Arrows indicate PG outgrowths (piecrust) at the future septation site. Scale bars, 500 nm.</p

Localization of PBPs and PBP2b during the vegetative cell cycle of <i>L</i>. <i>lactis</i>.

<p>(A) Labelling of <i>L</i>. <i>lactis</i> PBPs by Bocillin-FL staining. Membranes of wild type (WT), <i>pbp1a</i>, <i>pbp1b</i>, <i>pbp2a</i>, <i>pbp2</i> and <i>dacA</i> mutant cells were purified, incubated with Bocillin-FL (+ Bocillin-FL), and separated on SDS polyacrylamide gel. Bocillin-FL-labeled PBP bands were revealed by fluorescence scanning. Dotted lines indicate auto-fluorescent bands detected in wild-type extracts prior to Bocillin-FL staining. Colored arrowheads mark the absence of PBP1b (1b, black), PBP2a (2a, light blue), PBP2b (2b, red), PBP1a (1a, yellow) and DacA (purple) in the mutant profiles, except for PBP2x whose deleted mutant is not viable. (B) Localization of PBPs with respect to FtsZ during the cell cycle. Cells expressing FtsZ-Ve (NZ3900 [pGIBLD031]) were stained with Bocillin™650/665 and visualized by phase contrast (PC) and epifluorescence (FtsZ-Ve and Bocillin) microscopy. Merge shows the superimposition of both fluorescent patterns. Scale bar, 2μm. <i>L</i>. <i>lactis</i> cell cycle was reconstituted from individual cells taking the beginning of cell constriction (as visualized by phase contrast) as the demarcation between elongation-only and combined elongation + division. PBP staining by Bocillin™650/665 is depicted in red on the cell cycle diagram shown below the pictures. Scale bar, 2μm. (C) Cells expressing the Venus-PBP2b fusion (Ve-PBP2b) (NZ3900 [pGIBLD041]) were visualized by phase contrast (PC) and epifluorescence microscopy. Scale bar, 2μm. A complete cell cycle was reconstituted from representative cells as reported in panel B. Ve-PBP2b fluorescence pattern is depicted in yellow on the cell cycle diagram shown below the pictures. The two bottom panels show fluorescence intensity maps (in arbitrary units, A.U.) from low (blue) to high (red) intensity). Cells (<i>n</i> = 20) were chosen before and after cell constriction based on phase contrast imaging and their normalized Ve-PBP2b fluorescent profiles were superimposed.</p