Bacterial strains or plasmids used in this study.

<p>Bacterial strains or plasmids used in this study.</p

Mutagenesis of the arylsulfatase gene <i>astA</i> with <i>aph</i>(7″) or <i>aac</i>(3)IV non-polar markers and complementation of Δ<i>astA</i> via genomic insertion with pRRH or pRRA.

<p>(A) Loci arrangement of <i>astA</i> single-gene operon in <i>C. jejuni</i> 81–176. (B) Deletion of <i>astA</i> with either <i>aph</i>(7″) or <i>aac</i>(3)IV from pAC1H or pAC1H. (C) Introduction of promoterless <i>astA</i> into pRRH or pRRA in the same orientation as the <i>cat</i> promoter created pRRH+<i>astA</i> or pRRA+<i>astA</i> and resulted in polycistronic expression of <i>astA</i> with <i>aph</i>(7″) or <i>aac</i>(3)IV. (D) Promoterless <i>astA</i> inserted in the opposite orientation to the <i>cat</i> promoter (designated pRRH+<i>astA</i> (reverse) or pRRA+<i>astA</i> (reverse) (E) Insertion of the endogenous <i>astA</i> promoter and <i>astA</i> in the opposite orientation to the <i>cat</i> promoter in pRRH and pRRA created pRRH+(p)<i>astA</i> (reverse) and pRRA+(p)<i>astA</i> (reverse). Only <i>Hyg^R</i> plasmids/strains are depicted in B–E, but both <i>Hyg^R</i> and <i>Apr^R</i> plasmids represented with <i>Hyg^R</i> in C, D and E were integrated into the genome of the Δ<i>astA</i> strain, DRH461. (F) Arylsulfatase activity of the deletion and complementation strains was assessed by spotting 10 µL of OD-standardized cultures onto MH agar plates supplemented with the chromogen XS cleaved by arylsulfatase. A blue-green color indicates activity, and the spots correspond to labels on the bar graph below. (G) Quantification of arylsulfatase activity from broth cultures to assess transcription of <i>astA</i>.</p

Synthesis of plasmids containing <i>aph</i>(7″) or <i>aac</i>(3)IV as non-polar hygromycin B and apramycin resistance markers.

<p>(A) Schematic of ultramers designed to amplify <i>aph</i>(7″) or <i>aac</i>(3)IV. The 5′ ultramers 5631 and 5633, for <i>aph</i>(7″) or <i>aac</i>(3)IV respectively, include <i>Mfe</i>I, <i>Kpn</i>I and <i>Sma</i>I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for <i>Sma</i>I and <i>Bam</i>HI, and restriction sites for <i>Xba</i>I, <i>Nde</i>I, <i>Pst</i>I and <i>Sph</i>I. (B) The amplified <i>aac</i>(3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker (<i>Apr^R</i>). MCS sites that cut <i>aac</i>(3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The <i>Mfe</i>I- and <i>Sph</i>I-digested <i>aph</i>(7″) amplification product was cloned into pBAD24 digested with <i>Eco</i>RI (<i>Mfe</i>I-compatible) and <i>Sph</i>I. The <i>Mfe</i>I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.</p

Adaptation of the pRRC gene delivery and expression system to harbor hygromycin B or apramycin resistance, and testing of genome-integrated markers for detrimental effects of resistance genes.

<p>(A) Schematic of pRRC, which inserts into any of 3 rRNA clusters in the genome by homologous recombination. (B) Inverse PCR amplification of pRRC with primers 5705 (<i>Kpn</i>I) and 5706 deleted the chloramphenicol resistance gene but conserved the <i>Campylobacter</i>-optimized <i>cat</i> promoter. (C) The inverse PCR product was digested with <i>Kpn</i>I and <i>Xba</i>I, and ligated to similarly digested <i>aph</i>(7″) or <i>aac</i>(3)IV from pAC1H or pAC1A to create pRRH and pRRA respectively (only pRRH is shown). (D) Restriction digest analysis confirmed the function of all introduced sites. (E) The resistance markers from pRRK, pRRC, pRRH and pRRA were inserted into the <i>C. jejuni</i> 81–176 genome, and each resulting strain was analyzed for microaerobic growth and survival in shaken Mueller-Hinton (MH) broth by counting CFU over 48 hours at both 42°C (left panel) and 37°C (right panel). (F) To determine if the introduction of either marker contributed any fitness cost that could affect competitiveness against wild-type or the other marked strains, a competition assay was performed. Equal numbers of wild-type marked with hygromycin, apramycin, chloramphenicol and kanamycin resistance markers were co-cultured with unmarked wild-type in shaking MH broth at 37°C under microaerobic conditions. CFU were assessed by plating a dilution series on MH agar. (G) CFU were further assessed from the co-culture by plating on MH only (the total CFU, same data as in F) or MH supplemented with each antibiotic, representing the number of bacteria resistant to each antibiotic.</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

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

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

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