Subcellular localization of OatA^TM10-YFP and OatB^TM10-YFP fusions.

<p>A) Schematic representation of fusion proteins (YFP, yellow star) and corresponding micrographs in phase contrast (PC) microscopy and fluorescence microscopy (YFP). Upper panels, <i>oatA</i> mutant producing cytoplasmic YFP (OatA^−/YFP) used as control; middle panels, <i>oatA</i> mutant producing OatA^TM1–10-YFP (OatA^−/OatA^TM1–10-YFP); lower panels, <i>oatB</i> mutant producing OatB^TM1–10-YFP (OatB^−/OatB^TM1–10-YFP). Induction of expression was performed with 10 ng/ml of nisin. Bar scale, 2.0 µm. B) Fluorescence ratio (FR; AU, arbitrary unit) between the fluorescence measured at mid-cell position and pole. A^−/A™, <i>oatA</i> mutant producing OatA^TM1–10-YFP and B^−/B™, <i>oatB</i> mutant producing OatB^TM1–10-YFP. Lines represent the mean value (n = 20, 3 independent replicates).</p

Effect of expression of <i>min::yfp</i> fusions on cell morphology and subcellular localization.

<p>Effect of expression of <i>minC::yfp</i> (A, MinC-YFP) and <i>minD::yfp</i> (B, MinD-YFP) in <i>L. plantarum</i> wild-type (WT) and <i>oatA</i> mutant (OatA⁻) without nisin induction (0 ng/ml) and with 2.5 ng/ml of nisin. Micrographs were obtained in bright field (BF) microscopy and fluorescence microscopy (YFP). For MinD-YFP (nisin 2.5 ng/ml), minicells are indicated by arrows in WT and three selected branched cells (insets) are added for OatA⁻. Bar scale, 2.0 µm.</p

Morphological aberrations induced by the overproduction of different variants of OatA in the <i>oatA</i> mutant.

<p>Induction was performed with 20 ng/ml of nisin. A) Selection of cells showing curvature (labeled C), asymmetrical septation (labeled A), dual septation (labeled D) observed in bright field (BF) or phase contrast (PC) microscopy and fluorescent microscopy (FM4–64, membrane staining). A^−/A⁺⁺⁺, <i>oatA</i> mutant overexpressing <i>oatA</i>^WT; A^−/A^{* +++}, <i>oatA</i> mutant overexpressing <i>oatA</i>^D510A/S511A; A^−/A™⁺⁺⁺, <i>oatA</i> mutant overexpressing <i>oatA</i>^TM1–10<i>::yfp</i>. Bar scale, 2.0 µm. B) Selection of two tripartite cells (I and II) showing a central mini-cell (labeled MC) resulting from a dual septation event observed in bright field (BF) microscopy and fluorescent microscopy (YFP, OatA^TM1–10-YFP fluorescence; DAPI, DNA staining).</p

Temporal localization of OatA^TM1–10-YFP during the cell cycle.

<p>Upper panels, selection of cells at different division stages observed in bright field (BF) microscopy and fluorescent microscopy (FM4–64, membrane staining; YFP, OatA^TM1–10-YFP fluorescence). Induction of <i>oatA^TM1–10::yfp</i> expression was performed with 10 ng/ml of nisin. I, septal localization of the YFP fusion prior to membrane invagination; II, co-localization of the YFP fusion and the septal membrane; III, polar localization at the end of septation; IV, reinitiation of septal localization in daughter cells. Bar scale, 1.0 µm. Lower panel, schematic representation of the cell cycle. Colors and numbers refer to above micrographs. Yellow represent a merge between FM4–64 and YFP fluorescences.</p

Study of the uncoupling between elongation and septation phases by time-lapse experiments.

<p>A) Cell length (µm) of the mother cell during the division process for the wild type (solid line) and the <i>oatA</i> mutant (dashed line). Each line corresponds to one individual cell. Time (min) was arbitrarily fixed to zero at the last view before any detectable cell invagination in bright field. Correspondences between time and division state are drawn upside of the graph for the wild type. One representative time-lapse experiment of three independent experiments for each strain (n ≥3 for each). All examined cells of the wild type and the <i>oatA</i> mutant (n = 15 for each) display their respective phenotype. B) Cell length increase (µm) measured during the division process after 5 min (T10–T5; T5, initial invagination), 10 min (T15–T5), and 15 min (T20–T5; T20, final septation). Time intervals are reported in panel A. Symbols: WT, wild-type; A⁻, <i>oatA</i> mutant; A^−/A⁺<i>oatA</i> mutant complemented with <i>oatA</i>^WT; A^−/A^*<i>oatA</i> mutant complemented with <i>oatA</i>^D510A/S511A; A^−/A™, <i>oatA</i> mutant complemented with <i>oatA</i>^TM1–10<i>::yfp</i>. All the variants of <i>oatA</i> are expressed at low basal level in absence of the nisin inducer. Mean values of 5 cells in each time-lapse experiment. Significance based on <i>t</i>-test; **, p-value <0.01.</p

Bacterial strains and plasmids.

#<p>Cm^r and Amp^r indicate resistance to chloramphenicol and ampicillin, respectively.</p

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

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