UV-Vis absorbance changes upon photoexcitation and recovery for selected <i>As</i>LOV2 variants.

<p>Single exponential fits to the time dependent data are shown in red.</p

Effects on <i>As</i>LOV2 side chains during the photocycle.

<p>(A) Side chain positions in the chromophore binding pocket in the dark and light state crystal structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087074#pone.0087074-Halavaty1" target="_blank">[32]</a> (B) Chemical processes that occur to the FMN chromophore during the photocycle.</p

Chromophore and Photocycle parameters of <i>As</i>LOV2 variants.

†<p>Samples were air dried for 48 hours.</p

<i>As</i>LOV2 photocycle chemistry.

<p>Photon absorption results in the FMN being excited into a singlet then triplet state. The N5 of FMN becomes a strong nucleophile and removes the proton from the nearby C450 to form a covalent bond with the FMN C4a atom. Reduction of the FMNH state is inhibited by the inability of water to readily enter the chromophore binding pocket and catalyze the transfer of the proton back to the cysteine until a conformational change occurs involving residues N414 and Q513. The covalent bond is broken and the protein returns to the dark state.</p

Proposed environmental model.

<p>Environmental norspermidine may primarily derive from endogenously-produced norspermidine that is released during cell lysis or exported to the periplasm by an unknown transporter. It may also be provided by nearby eukaryotic organisms. Norspermidine may also act as a quorum sensing molecule, allowing <i>V</i>. <i>cholerae</i> to detect this signal, recognize that it is in the presence of other <i>Vibrios</i>, and respond appropriately by forming the <i>Vibrio</i> polysaccharide. In this way, norspermidine may allow <i>V</i>. <i>cholerae</i> to persist in its biofilm form in its natural environment.</p

Effects of <i>ΔpotD1</i>, <i>nspC</i>::<i>kan</i>^<i>R</i>, <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔpotD1</i>, and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔnspSΔpotD1</i> mutations, with and without exogenous norspermidine, on biofilm formation in <i>V</i>. <i>cholerae</i>.

<p>Biofilms were formed in borosilicate tubes in LB broth for 18 h at 27°C and quantified as described in Materials and Methods. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type. The values for WT and <i>nspC</i>::<i>kan</i>^<i>R</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186291#pone.0186291.g004" target="_blank">Fig 4</a> are the same as these experiments were performed simultaneously.</p

Effects of vibriobactin synthesis and utilization on biofilm formation in <i>V</i>. <i>cholerae</i>.

<p><b>(A)</b> Biofilm formation of <i>ΔvibF</i> and <i>viuA</i>::<i>tet</i>^<i>R</i> mutants. <b>(B)</b> Biofilm formation of <i>ΔvibF</i>, <i>nspC</i>::<i>kan</i>^<i>R</i>, and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔvibF</i> mutants. Biofilms were formed in borosilicate tubes in LB broth for 24 h at 27°C and quantified as described in Materials and Methods. EDDA was added to chelate iron to generate iron-deplete conditions. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type.</p

Role of NspS, MbaA, and NspC on biofilm formation in <i>V</i>. <i>cholerae</i>.

<p><b>(A)</b> Biofilm assay of <i>ΔnspS</i>, <i>nspC</i>::<i>kan</i>^<i>R</i>, and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔnspS</i> mutations, with and without exogenous norspermidine. <b>(B)</b> Biofilm assay of <i>nspC</i>::<i>kan</i>^<i>R</i>, <i>ΔmbaA</i>, and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔmbaA</i> mutations, with and without exogenous norspermidine. Biofilms were formed in borosilicate tubes in LB broth for 18 h at 27°C and quantified as described in Materials and Methods. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type.</p

Role of NspS and NspC on cellular polyamine content in <i>V</i>. <i>cholerae</i>. Polyamine composition of <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔnspS</i> cells with and without exogenous norspermidine.

<p>Polyamines were extracted from cells, derivatized by benzoylation and analyzed by HPLC as described in Materials and Methods. Labeled peaks on the chromatogram correspond to putrescine (put), diaminopropane (dap), cadaverine (cad), norspermidine (nspd), and spermidine (spd). AU₂₅₄, absorbance units at 254 nm. Only 4–14 minutes of a 40-minute run are plotted for clarity.</p

Primers.

<p>Primers.</p