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

TEM Micrographs of wild-type (WT, NZ3900) and <i>pbp2b</i> mutant cells of <i>L</i>. <i>lactis</i>).

<p>(i and ii), <i>pbp2b</i> mutant cells with mis-oriented and asymmetrical septa; (iii), small chains of round cells; (iv), aggregate of unseparated cells; (v), cell with double septa. Arrows indicate PG outgrowths (piecrust) at the future septation site in WT. Scale bars, 500 nm.</p

Dynamics of FtsZ and Pbp2b during filament reversion.

<p>Methicillin-induced filaments expressing FtsZ-Ve (NZ3900 [pGIBLD031]) (A) or Ve-PBP2b (NZ3900 [pGIBLD031]) (B) were transferred to methicillin-free agar pads to study their localization dynamics by time-lapse microscopy (phase contrast (PC) and fluorescence) during filament reversion (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198014#pone.0198014.s019" target="_blank">S5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198014#pone.0198014.s024" target="_blank">S10</a> Movies). Localization of the protein at the constricting septum and future division sites is highlighted with red and orange arrows, respectively. Only the early steps of filament reversion are highlighted. Scale bars, 2 μm. (C) Schematic diagrams summarizing localization patterns of FtsZ-Ve (green) and Ve-PBP2b (yellow).</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

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

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

Localization of FtsZ and Pbp2b during filament formation.

<p><i>L</i>. <i>lactis</i> cells expressing FtsZ-Ve (NZ3900 [pGIBLD031]) (A) or Ve-PBP2b (NZ3900 [pGIBLD041]) (B) were grown in the presence of methicillin 1 μg ml^-1 to induce filament formation and visualized by phase contrast (PC) and fluorescence microscopy to examine the subcellular localization of the corresponding proteins at different stages of filament elongation. Schematic diagrams summarizing the data are shown below the microscopy pictures. The localization pattern of FtsZ-Ve and Ve-PBP2b are shown in green and yellow, respectively. Scale bars, 2 μm.</p

Complementation of <i>pbp2b</i> mutant with PBP2b wild type (PBP2b^WT) and the catalytic mutant of PBP2b (PBP2b*).

<p>(A) Images of wild-type (WT, NZ3900), <i>pbp2b</i> mutant, <i>pbp2b</i> + PBP2b^WT, and <i>pbp2b</i> + PBP2b* cells obtained by phase contrast (PC) and epifluorescence (membrane staining with FM4-64) microscopy. Cells were grown without (no nisin) or with nisin (0.05 ng ml^-1) as inducer of the expression of <i>pbp2b</i> gene variants. (B) Distribution of length to width ratios in the cell population (<i>n</i> = ~50) of WT, <i>pbp2b</i> mutant, <i>pbp2b</i> + PBP2b^WT and <i>pbp2b</i> + PBP2b*. For complementation, measurements were performed from cells grown with nisin 0.05 ng ml^-1 (N 0.05). (C) Relative septum position in dividing cells (% of deviation from the median position of cells stained with FM4-64) of the cell population of WT, <i>pbp2b</i> mutant, <i>pbp2b</i> + PBP2b^WT and <i>pbp2b</i> + PBP2b* (in presence of nisin 0.05 ng ml^-1, N 0.05).</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