Table 1.
Strains used in this study.
Figure 1.
DNA appears in WT biofilms following attachment and is more pronounced under conditions that promote biofilm formation.
Biofilms of WT or ΔcprS 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.
Figure 2.
Increased extracellular DNA and lysis occur in biofilm-enhanced cultures.
A) Cell-free supernatants contain more DNA under biofilm-enhancing conditions. Supernatants were isolated from cultures (at a similar OD600) of WT, ΔcprS, ΔflhA, or ΔcprS ΔflhA grown in either MH alone or MH/DOC. Equal volumes were used as templates for qPCR. DNA amounts are normalized to WT in MH alone. Error bars represent the mean of three separate cultures. *p<0.0001; **p = 0.0015; NS p = 0.42, unpaired t-test. B) Lysis, independent of flagella, occurs under conditions that promote biofilms. Cell-free supernatants were isolated as in above. Both total cellular protein (‘Bacteria’) and supernatants (‘Media’) were analyzed by Western blotting with an antibody specific for the cytoplasmic response regulator CosR.
Figure 3.
Cell-free supernatants and exogenous DNA promote biofilms and DNA is necessary for biofilm formation.
A) Exogenous DNA enhances biofilms. Culture supernatants, concentrated for >3 kDa size components, or gDNA isolated from WT C. jejuni 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) Biofilm formation is inhibited by DNase I. Biofilms (WT/black bars or ΔcprS/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.
Figure 4.
DNase arrests biofilms following adherence. Biofilms of WT, ΔcprS, 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).
After the indicated times, biofilms were fixed, stained with DAPI, and visualized by confocal microscopy. Green: GFP-expressing bacteria; Blue: DAPI-stained DNA.
Figure 5.
The flagellum, but not motility, is absolutely required for biofilm formation.
A) Aflagellate mutants are defective for biofilm formation in WT and ΔcprS backgrounds. *p<0.0001; NS p>0.1 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.
Figure 6.
Aflagellate bacteria are defective for adherence; kinetics of biofilm formation is delayed in bacteria expressing paralyzed flagella.
Biofilms of WT, ΔflgR (aflagellate), and pflA (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.
Figure 7.
Biofilm formation confers stress tolerance in vitro. Standing cultures of the indicated strains (black bars, WT background; grey bars, ΔcprS background) were grown in MH broth with the indicated additions (labels below).
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 ΔcprS) in MH alone. Error bars represent the mean of three biological replicates. NS: not significant **p<0.0001 *p = 0.0018.
Figure 8.
Conditions that promote DNA release and biofilms also increase genetic exchange and UV tolerance.
Genetic exchange. WT bacteria, marked with StrR, were grown in mixed culture (1∶1) with either an isogenic WT strain marked with KanR or the ΔcprS mutant marked with KanR. 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).
Figure 9.
Model of C. jejuni biofilm formation. Evidence for the role of stress conditions, flagella and motility, eDNA release, and genetic exchange has been provided.
Biofilm formation also appears to confer tolerance of specific stresses, such as those that may be encountered during pathogenesis.