Impact of Salt and Nutrient Content on Biofilm Formation by <i>Vibrio fischeri</i>

<div><p><i>Vibrio fischeri</i>, a marine bacterium and symbiont of the Hawaiian bobtail squid <i>Euprymna scolopes</i>, depends on biofilm formation for successful colonization of the squid’s symbiotic light organ. Here, we investigated if culture conditions, such as nutrient and salt availability, affect biofilm formation by <i>V</i>. <i>fischeri</i> by testing the formation of wrinkled colonies on solid media. We found that <i>V</i>. <i>fischeri</i> forms colonies with more substantial wrinkling when grown on the nutrient-dense LBS medium containing NaCl relative to those formed on the more nutrient-poor, seawater-salt containing SWT medium. The presence of both tryptone and yeast extract was necessary for the production of “normal” wrinkled colonies; when grown on tryptone alone, the colonies displayed a divoting phenotype and were attached to the agar surface. We also found that the type and concentration of specific seawater salts influenced the timing of biofilm formation. Of the conditions assayed, wrinkled colony formation occurred earliest in LBS(-Tris) media containing 425 mM NaCl, 35 mM MgSO₄, and 5 mM CaCl₂. Pellicle formation, another measure of biofilm development, was also enhanced in these growth conditions. Therefore, both nutrient and salt availability contribute to <i>V</i>. <i>fischeri</i> biofilm formation. While growth was unaffected, these optimized conditions resulted in increased <i>syp</i> locus expression as measured by a P_<i>sypA</i><i>-lacZ</i> transcriptional reporter. We anticipate these studies will help us understand how the natural environment of <i>V</i>. <i>fischeri</i> affects its ability to form biofilms and, ultimately, colonize <i>E</i>. <i>scolopes</i>.</p></div

Impact of salts on <i>V</i>. <i>fischeri</i> pellicle formation.

<p>KV4366 was grown in LBS and diluted to an OD₆₀₀ of 0.3 in LBS(-Tris) and LBS(-Tris) with 425 mM NaCl, 35 mM MgSO4, or 5 mM CaCl2, and LBS-Opt. Triplicate cultures were grown statically in the center wells of a 24-well plate at 24°C and imaged after 20, 24, and 48 h. One sample of the triple cultures was disrupted with a toothpick at each of the three time points; for each time point, images of the undisrupted and disrupted pellicles are presented from left to right.</p

Composition of media used in this work.

<p>Composition of media used in this work.</p

<i>V</i>. <i>fischeri</i> strains and plasmids used in this study.

<p><i>V</i>. <i>fischeri</i> strains and plasmids used in this study.</p

Magnesium sulfate promotes wrinkled colony morphology.

<p>KV4366 was grown in LBS and spotted onto medium containing 1.0% tryptone, 0.5% yeast extract, 342 mM NaCl, and 0–100 mM MgSO₄. Colonies were imaged at the same magnification at the indicated times and disturbed with a toothpick at the last time point. Asterisks indicate the first time point at which wrinkling became visible.</p

Optimal media promotes wrinkled colony morphology and <i>syp</i> locus transcription.

<p>KV4366 was grown in LBS and spotted onto LBS(-Tris) (A) or LBS(-Tris) with 425 mM NaCl (B), 35 mM MgSO₄ (C), 5 mM CaCl₂ (D), or LBS-Opt (E). Images were captured at the same magnification at the indicated times, and asterisks indicate the time at which wrinkling became visible. At the last time point colonies were disturbed with a toothpick. Images in parts A-D are selections from Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169521#pone.0169521.g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169521#pone.0169521.g004" target="_blank">4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169521#pone.0169521.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169521#pone.0169521.g006" target="_blank">6</a>. To measure <i>syp</i> locus transcription, Δ<i>sypA</i> mutant strains carrying a P_{<i>sypA-lacZ</i>} transcriptional reporter and a plasmid overproducing RscS (pKG11/Δ<i>sypA</i>) or vector control (pKV69/Δ<i>sypA</i>) were grown in LBS(-Tris) or LBS-Opt at 24°C for 24 h and assayed for β-galactosidase activity (F). Error bars represent the standard deviation of at least three biological replicates. *, <i>P</i><0.05. To assess the effect of LBS-OPT on growth, KV4366 was grown with shaking at 24°C (blue circles) and 28°C (red triangles) in LBS(-Tris) (solid line) or LBS-Opt (dotted line), and the absorbance at 600 nm was measured each hour (G).</p

Impact of tryptone and yeast extract on wrinkled colony morphology.

<p>KV4366 was grown in LBS and spotted onto medium containing only 342 mM NaCl and tryptone (A) or yeast extract (B) at amounts ranging from 0.25–1.0%, as indicated. Images were captured at the same magnification after 24 or 48 h, and at the last time point colonies were disturbed with a toothpick.</p

Sodium chloride promotes wrinkled colony morphology.

<p>KV4366 was grown in LBS and spotted onto medium containing 1.0% tryptone, 0.5% yeast extract, and 210–500 mM NaCl as indicated. Colonies were imaged at the same magnification at the indicated time points and disturbed with a toothpick at the last time point. Asterisks indicate the first time point at which wrinkling became visible.</p

Wrinkled colony formation on two complex media, LBS and SWT.

<p>Cultures of ES114 (wild-type, non-biofilm induced) and KV4366 (RscS-overproducing, biofilm-induced) were grown in LBS and spotted onto LBS or SWT. Images were captured at the same magnification following growth for 48 h.</p

Impact of nutrients and seawater salts on wrinkled colony morphology.

<p>KV4366 was grown in LBS and spotted onto LBS (A), LBS(-Tris) (B), SWT (C), HTY-SW (D), or LTY-NaCl (E). Images were captured at the same magnification at the indicated time points. At the last time point, colonies were disturbed with a toothpick. The 48 h images for LBS and SWT are the same as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169521#pone.0169521.g001" target="_blank">Fig 1</a>. Asterisks indicate the first time point at which wrinkling became visible.</p