Biofilm formation confers stress tolerance <i>in vitro</i>. Standing cultures of the indicated strains (black bars, WT background; grey bars, Δ<i>cprS</i> background) were grown in MH broth with the indicated additions (labels below).

<p>Biofilm formation was impaired by addition of DNase (90 U mL⁻¹). Sub-MIC levels of DOC were included where indicated. Total OD₆₀₀ of three independent cultures, following 2 days growth and resuspension by vortexing was measured. Cultures were normalized to the strain background (WT or Δ<i>cprS</i>) in MH alone. Error bars represent the mean of three biological replicates. NS: not significant **p<0.0001 *p = 0.0018.</p

DNA appears in WT biofilms following attachment and is more pronounced under conditions that promote biofilm formation.

<p>Biofilms of WT or Δ<i>cprS</i> were grown on glass coverslips in MH broth alone or MH/DOC (0.05%). At indicated times post-inoculation, coverslips were fixed, stained with DAPI, and visualized by confocal microscopy. Green: GFP-expressing bacteria; Blue: DAPI-stained DNA.</p

Conditions that promote DNA release and biofilms also increase genetic exchange and UV tolerance.

<p>Genetic exchange. WT bacteria, marked with Str^R, were grown in mixed culture (1∶1) with either an isogenic WT strain marked with Kan^R or the Δ<i>cprS</i> mutant marked with Kan^R. Cultures were grown in either MH broth alone or MH/DOC. Cells were removed at indicated time points and CFUs were determined on the appropriate antibiotics. Error bars represent the mean of three biological replicates. *p<0.1 vs. WT+WT (MH).</p

Model of <i>C. jejuni</i> biofilm formation. Evidence for the role of stress conditions, flagella and motility, eDNA release, and genetic exchange has been provided.

<p>Biofilm formation also appears to confer tolerance of specific stresses, such as those that may be encountered during pathogenesis.</p

Strains used in this study.

<p>Strains used in this study.</p

Aflagellate bacteria are defective for adherence; kinetics of biofilm formation is delayed in bacteria expressing paralyzed flagella.

<p>Biofilms of WT, Δ<i>flgR</i> (aflagellate), and <i>pflA</i> (paralyzed flagella) were grown on coverslips for 36 h, fixed, stained with DAPI, and visualized by confocal microscopy. Green: GFP-expressing bacteria; Blue: DAPI-stained DNA.</p

Cell-free supernatants and exogenous DNA promote biofilms and DNA is necessary for biofilm formation.

<p><b>A</b>) Exogenous DNA enhances biofilms. Culture supernatants, concentrated for >3 kDa size components, or gDNA isolated from WT <i>C. jejuni</i> grown for 24 h on MH plates (500 ng) were included in fresh MH broth. Tubes were then inoculated with WT, and following 2 days growth, biofilms were quantified with crystal violet. *p = 0.08; **p = 0.003 (vs. MH alone). <b>B</b>) Biofilm formation is inhibited by DNase I. Biofilms (WT/black bars or Δ<i>cprS</i>/grey bars) were grown in either MH alone, MH/DOC, (0.05%) MH/DNase (90 U mL⁻¹), or MH/DOC/DNase, followed by CV staining after 2 days growth. Error bars represent the mean of three biological replicates. *p<0.005 vs. counterpart without DNase.</p

DNase arrests biofilms following adherence. Biofilms of WT, Δ<i>cprS</i>, or WT in MH/DOC (0.05%) were grown on coverslips in the presence (top panels) or absence (bottom panels) of DNase (90 U mL⁻¹).

<p>After the indicated times, biofilms were fixed, stained with DAPI, and visualized by confocal microscopy. Green: GFP-expressing bacteria; Blue: DAPI-stained DNA.</p

The flagellum, but not motility, is absolutely required for biofilm formation.

<p><b>A</b>) Aflagellate mutants are defective for biofilm formation in WT and Δ<i>cprS</i> backgrounds. *p<0.0001; NS p>0.1 <b>B</b>) Only non-flagellate bacteria remain completely defective in biofilm-promoting media. *p<0.0001; NS, p = 1. Indicated strains were grown in static culture for 2 days in either MH broth alone or MH/DOC, followed by staining and quantification with crystal violet.</p