Strains and plasmids.

<p>Strains and plasmids.</p

GDP-mannose 4,6 dehydratase (Gmd) activity is required for EPS synthesis in <i>C</i>. <i>crescentus</i>.

<p>(A) EPS phenotypes on PYE-sucrose (left) and PYE media (right). NA1000 has a fully mucoid (EPS⁺) phenotype and NA1000ΔMGE has a dry, non-mucoid (EPS^-) phenotype when cultured on PYE-sucrose (left). The NA1000Δ<i>gmd1</i> and NA1000Δ<i>gmd2</i> single mutants show slightly reduced or markedly reduced EPS expression, respectively. The NA1000Δ<i>gmd2</i>Δ<i>gmd1</i> double mutant has a dry, non-mucoid (EPS^-) phenotype, grows slowly, and shows slightly reduced colony size. The NA1000Δ<i>rsaA</i> mutant has a wildtype, fully mucoid (EPS⁺) phenotype. The different EPS phenotypes are subtle and do not photograph well when they are grown on plain PYE media (right). (B) Merged Differential Interference Contrast (DIC) and fluorescence images of mixed EPS⁺ (NA1000, non-fluorescent) and EPS^- (NA1000ΔMGE, YFP-expressing, yellow) cells stained with a Texas Red conjugated lectin that specifically binds fucose residues (<i>Lotus tetragonolobus</i>, red). The lectin binds to EPS⁺ cells, but not EPS^- cells. (C) Representative DIC and fluorescence images of mixed EPS⁺ (NA1000, GFP-expressing, green) and EPS^- (NA1000ΔMGE, YFP-expressing, yellow) showing TRITC-Dextran exclusion. The black regions surrounding the cells correspond with the capsule (EPS) and EPS⁺ (GFP⁺ cells, indicated with arrowheads) cells show larger zones of exclusion than EPS^- (YFP⁺ cells, indicated with arrows) cells in this negative stain assay. Only isolated cells unambiguously expressing a fluorescent protein (arrows, arrowheads) were included in quantitative analyses while non-fluorescent and adjacent cells (asterisk) were excluded. (D) Quantitative analysis of the zones of exclusion from the experiment described in panel (C) show that NA1000 (black squares) and NA1000Δ<i>gmd1</i> (green triangles) make more EPS than NA1000ΔMGE (blue circles) and NA1000Δ<i>gmd2</i> (yellow triangles) (ANOVA F(3, 1657) = 63.19, P < 0.0001, NA1000 vs. NA1000ΔMGE t(1675) = 10.85 p < 0.0001, NA1000 vs. NA1000Δ<i>gmd2</i> t(1657) = 7.241 p < 0.0001, NA1000 vs. NA1000Δ<i>gmd1</i> t(1657) = 0.8581 p > 0.9999) when grown under the same conditions.</p

EPS production may be disadvantageous in phage-free environments.

<p>Strains labeled with two different fluorophores (GFP, mCherry) were mixed and relative fluorescence measured over the course of 8 days. (A–C) In control experiments where strains of the same genetic background expressing different fluorescent proteins were mixed, no significant change in the ratio of GFP:mCherry fluorescence was detected. Conversely, when strains of different genotypes were mixed, those producing more EPS appear to be at a competitive disadvantage (D) NA1000 and NA1000ΔMGE, (E) NA1000 and NA1000Δ<i>gmd2</i>, or (F) NA1000ΔMGE and NA1000Δ<i>gmd2</i>. The difference in fluorescence ratio between GFP and mCherry was assessed at the beginning (Day 1) and end (Day 8) of the experiment using an ANOVA with Bonferroni correction for multiple comparisons. Comparisons that resulted in significant differences are indicated with an asterisk (*) in the figure. (Day 1: ANOVA F(17, 558) = 0.0009178, P > 0.9999; Day 8: ANOVA F(17, 554) = 16.40, P < 0.0001, NA1000-GFP vs. NA1000-mCherry, t(554) = 1.051, p > 0.9999, NA1000ΔMGE-GFP vs. NA1000ΔMGE-mCherry t(554) = 0.5479, p > 0.9999, or NA1000Δ<i>gmd2</i>-GFP vs. NA1000Δ<i>gmd2</i>-mCherry t(554) = 2.479, p = 0.1213, NA1000-mCherry vs. NA1000ΔMGE-GFP t(554) = 8.892, p = < 0.0001, NA1000-GFP vs. NA1000ΔMGE-mCherry t(554) = 10.11, p = < 0.0001, NA1000-mCherry vs. NA1000Δ<i>gmd2</i>-GFP t(554) = 5.038, p = < 0.0001, NA1000-GFP vs. NA1000Δ<i>gmd2</i>-mCherry t(554) = 4.655, p = < 0.0001, NA1000ΔMGE-mCherry vs. NA1000Δ<i>gmd2</i>-GFP t(554) = 3.099, p = < 0.0184, NA1000ΔMGE-GFP vs. NA1000Δ<i>gmd2</i>-mCherry t(554) = 2.201, p = 0.2532).</p

Gmd activity is required for maintenance of S-layer dependent phage CR30 susceptibility.

<p>Both NA1000 (EPS⁺, top) and NA1000ΔMGE (EPS^-, middle) support plaque formation when challenged with phage CR30 lysates (10²–10⁴ pfu/mL). Note that NA1000ΔMGE is about 10-fold more sensitive to CR30 infection under these conditions, consistent with previous findings [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190371#pone.0190371.ref016" target="_blank">16</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190371#pone.0190371.ref024" target="_blank">24</a>]. In contrast, NA1000Δ<i>gmd</i>1Δ<i>gmd2</i> (EPS^-, bottom) does not support phage CR30 plaque formation; a result that is consistent with the hypothesis that this strain sheds the S-layer because it is unable to synthesize N-acetylperosamine and assemble normal LPS O-antigen (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190371#pone.0190371.g001" target="_blank">Fig 1</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190371#pone.0190371.ref030" target="_blank">30</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190371#pone.0190371.ref031" target="_blank">31</a>].</p