Decreased growth rate and lack of tunicamycin sensitivity of <i>ΔgbcO</i> mutant.

<p>(<b>A</b>) Growth curves of NEM316 WT (solid squares), Δ<i>gbcO</i> mutant (circles) and Δ<i>gbcO</i>pTCVΩ<i>gbcO</i> (empty squares) strains. Cultures were performed in TH medium without antibiotics at 37°C in 96 wells plates in triplicate. Optical densities were recorded at 600 nm in a Tecan M200 apparatus with 5 sec agitation before measure. Average values of a typical experiment are presented. (<b>B</b>) Effect of various concentrations of tunicamycin on the growth rate of WT (solid squares), <i>ΔgbcO</i> (black circles) and Δ<i>gbcO</i>pTCVΩ<i>gbcO</i> (empty squares) strains. Tunicamycin, a general inhibitor of UDP-GlcNAc:lipid phosphate carrier transferase activities, inhibits the growth of WT and complemented strains but not that of <i>ΔgbcO</i> mutant suggesting that GbcO carries this activity. Experiments were performed in triplicate and results are reported as a percentage of the growth rate in absence of tunicamycin. Error bars represent ± S.E. of triplicate experiments.</p

Fluorescent immunolocalization of the putative peptidoglycan hydrolase PcsB.

<p>Exponentially growing NEM316 WT, <i>ΔgbcO</i> mutant and Δ<i>gbcO</i>pTCVΩ<i>gbcO</i> complemented strains were harvested, transferred to glass slide, and fixed. IFM with anti-PcsB serum and DAPI staining were performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002756#s3" target="_blank">Materials and Methods</a>.</p

Structure of GBC and proposed scheme of GBC synthesis.

<p>(<b>A</b>) The multiantennary GBC is shown linked to an N-acetyl muramic (NAM) moiety, a component of PG. (<b>B</b>) The figure depicts the first steps of GBC synthesis where GbcO is proposed to catalyze the transfer of UDP-GlcNAc to a lipid phosphate carrier.</p

Electron microscopy imaging of NEM316 WT, <i>ΔgbcO</i> mutant, and complemented strains.

<p>Bacteria were harvested in mid-log phase (OD_{600 nm} = 0.5), fixed, and prepared as described in Supporting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002756#s3" target="_blank">Materials and Methods</a> (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002756#ppat.1002756.s005" target="_blank">Text S1</a></b>) (<b>A</b>) Representative views of scanning electron microcopy analysis illustrating the morphological alterations (size, form, and cell division abnormalities) due to <i>gbcO</i> inactivation. (<b>B, C</b>) Transmission electron microscopy views of uranyl acetate stained thin cryosections at two magnifications (see scale bars). The presence of the pellicle (electron dense outer layer) at the surface of WT and complemented strains observed at the higher magnification is highlighted with black arrows. An open triangle depicts the equatorial ring (EqR), a zone of active peptidoglycan synthesis seen in almost all WT and complemented cells but absent in the <i>ΔgbcO</i> mutant cells.</p

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

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

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

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</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