Bacterial strains and cultivation
All strains used in this work were stored at -80°C in 20% glycerol stocks. During genetic manipulations, C. jejuni 81–176 (ATCC® BAA-2151™) was routinely cultivated at 42°C under microaerobic conditions (5% O2, 10% CO2, 85% N2) in a multi-gas incubator (MCO-18M, Schoeller, Germany) on the Mueller-Hinton (MH) agar or broth (Hi-Media, India), eventually supplemented either with chloramphenicol (20 µg/mL, Merck, USA) for the cultivation of luxS deletion, substitution and insertion inactivated mutant strains, or kanamycin (50 µg/mL, Merck, USA) for the cultivation of luxS complemented strains. For biofilm formation, C. jejuni was pre-cultivated on Karmali agar (Oxoid, UK). Escherichia coli DH5α (Q5® Site-Direct Mutagenesis Kit, NEB, USA) was cultivated aerobically at 37°C on the Lysogeny agar (LB-A) or broth (LB; Hi-Media, India), eventually supplemented either with kanamycin (50 µg/mL; bacteria carrying a plasmid with the complementation cassette), chloramphenicol (20 µg/mL; bacteria carrying plasmids containing the cassettes for deletion, substitution or insertion inactivation) or ampicillin (100 µg/mL; bacteria carrying plasmids with partially assembled cassettes before the insertion of chloramphenicol or kanamycin selection marker. For the bioluminescence assay, Vibrio campbellii ATCC® BAA-1117™, designated as Vibrio harveyi BB170 (AI-2 sensitive reporter strain), and V. campbellii ATCC® BAA-1119™ designated as Vibrio harveyi BB152 (AI-2 positive control) were cultivated on the Autoinducer bioassay (AB) agar or broth (ATCC Medium: 2746) aerobically at 30°C.
Genetic engineering of Escherichia coli DHα and Campylobacter jejuni 81–176
For all cloning experiments, amplification parameters of the specific regions of C. jejuni 81–176 were designed using the FastPCR software (Kalendar et al. 2017). Primers for site-specific mutagenesis with substitution and insertion inactivation cassettes were designed in the NEBaseChanger® (NEB, USA). Genomic DNA of C. jejuni 81–176 was isolated according to the protocol described by He (2011). Competent E. coli DH5α cells were prepared and transformed by routine heat-shock transformation according to the protocol described by Sambrook and Russell (2006). The electro-competent cells of C. jejuni were prepared according to the protocol described by Wassenaar et al. (1993) with the modification of replacing the saponin agar plates with MH agar without supplement. All plasmids used in this study were isolated by the GenElute HP Plasmid Miniprep Kit (Merck, USA). The restriction enzymes used in this study (XbaI, BamHI, NcoI, EcoRI and SacI) were purchased from NEB, USA. Ligase T4 DNA was used for ligations, and Q5® High-Fidelity DNA Polymerase was used for all PCRs (both NEB, USA). The accuracy of assembled cassettes in both E. coli and transformed C. jejuni strains was confirmed by PCR and Sanger sequencing. All PCR programs used in this study are available in Online Resource 1.
Construction of the luxS deletion cassette
To prepare the deletion cassette (Fig. 1; Fig. S1a, Online Resource 1), border sequences of approx. 600 bp in front of and behind the luxS gene were amplified by two primer sets containing specific restriction sites (Set 1 and Set 2; Table 1). The fragments from PCR reactions were purified by Wizard® SV Gel and PCR Clean-Up System (Promega, USA). Subsequently, approximately 1 µg of each product was digested with 10 units (U) of appropriate restriction enzymes, together with 1 µg of plasmid pGEM®-T easy vector (Promega, USA). Digested fragments and plasmids were electrophoretically separated in 1% agarose gel, cut out, and purified using the Wizard kit. The concentration of all purified fragments was measured using Nanophotometer™ (Implen, Germany) and the appropriate ligation ratio (F1:F2:plasmid = 3:3:1) was calculated in silico using NEBioCalculator® (NEB, USA). Ligation was performed according to a protocol provided by the manufacturer of T4 ligase. After an overnight ligation at 16°C, 5 µl of the ligation mixture was introduced into competent E. coli DH5α by heat shock as described by Sambrook and Russell (2006). Transformed cells were cultivated 24h at 37°C on LB agar containing 100 µg/mL of ampicillin. Resulting plasmids were isolated from 10 randomly selected colonies. The assembly of plasmids was verified by restriction analysis with BamHI, as a unique BamHI site was created between the F1 and F2 fragments (Fig. S1a, Online Resource 1). Finally, chloramphenicol acetyltransferase (cat, GenBank: M35190.1) was amplified from plasmid pRY111 (Yao et al. 1993) by primer Set 3 (Table 1). After its purification and BamHI treatment, the cassette was ligated into the created BamHI site, replacing the luxS gene. The final plasmid was transformed into E. coli DH5α as mentioned above. Colonies containing the deletion cassette were selected on the LB-A plate with chloramphenicol (20 µg/mL). The sequence of the deletion cassette was verified by Sanger sequencing using primer Set 4 (Table 1). Data from sequencing are available in Online Resource 1 (Fig. S1b and Fig. S1c).
Construction of the luxS substitution cassette
Substitution cassette (Fig. 1; Fig. S2a, Online Resource 1) was created as follows. A fragment of 1.2 kbp containing the luxS gene with approx. 600 bp in front of and 100 bp behind the gene was amplified by primer Set 5 containing modified regions with restriction sites (Table 1). The PCR product was purified and 1 µg was digested with 10 U of SacI and BamHI, together with plasmid pUC19 (NEB, USA). Products of digestion were then treated as described in the previous chapter, and they were subsequently ligated and transformed into competent E. coli DH5α by heat shock. Transformed cells were cultivated 24h at 37°C on LB agar containing 100 µg/mL of ampicillin, 50 µg/mL of IPTG, and 40 µg/mL of X-Gal, which allows the blue-white screening as described by Juers et al. (2012). After cultivation, 10 random white colonies were selected to confirm the presence of the fragment of interest by colony PCR using primer Set 5 (Table 1). A point substitution 256A previously identified by Plummer et al. (2011) was incorporated into the luxS gene using specifically designed primer Set 6 (Table 1) and Site-Direct Mutagenesis Kit (NEB, USA). To confirm the successful substitution, a small region containing the luxS gene was amplified with modified primer Set 7 (Shagieva et al. 2020) and subsequently cloned between the NcoI and EcoRI restriction sites of the pGEM-T easy vector. The substitution was confirmed by Sanger sequencing using the commercially available M13/forward primer (Fig. S2b, Online Resource 1). Finally, the complementary border sequence (approx. 500 bp) located behind the luxS gene was amplified (primer Set 8, Table 1), purified, and digested by BamHI and XbaI. The products of digestion were ligated at a standard ratio of 3:1, incorporating the border sequence behind the first fragment containing the point mutation 256A (Fig. S2a, Online Resource 1). After transformation into E. coli, positive colonies were confirmed by restriction analysis with BamHI and XbaI enzymes. Finally, the cat cassette was cloned between both fragments, into the BamHI site, and positive colonies were selected on the LB-A plates with chloramphenicol (20 µg/mL).
Construction of the cassette for insertion inactivation of luxS
The modified primer Set 9 containing restriction sites for NcoI and EcoRI (Table 1) was used for amplification of the fragment of approx. 1.5 kbp containing the luxS gene with border sequences on both sides. This fragment was purified from the PCR reaction and subsequently digested with NcoI and EcoRI, together with the plasmid pGEMT easy vector. The products of digestion were then electrophoretically separated, purified, ligated, and transformed into the E. coli DH5α as described in the sections above. Transformed cells were cultivated for 24h at 37°C on LB agar containing 100 µg/mL of ampicillin, 50 µg/mL of IPTG, and 40 µg/mL of X-Gal, which allows the blue-white screening. Positive (white) colonies were confirmed by the colony PCR using primer set 9 (Table 1). In silico analysis of the luxS gene revealed a nucleotide sequence similar to the BamHI restriction site, suitably situated close to the middle of the luxS, requiring substitution of three nucleotides. Using the primer Set 10 (Table 1), where the forward primer carried the required substitution, and Q5® Site-Direct Mutagenesis Kit (NEB, USA), the substitution was introduced and the BamHI site was created and confirmed by the restriction analysis. Into the BamHI unique restriction site in the middle of luxS, the amplified cat cassette was cloned as described above, disrupting the luxS gene (Fig. S3a, Online Resource 1). Positive colonies were selected on the LB-A plates with chloramphenicol (20 µg/mL). All modifications were confirmed using Sanger sequencing (Fig. S3b and Fig. S3c, Online Resource 1).
Construction of the luxS complementation cassette
A fragment containing a functional copy of the luxS gene (approx. 1.2 kbp) was amplified (primer Set 11, Table 1), purified, and digested with NcoI and BamHI. Plasmid pGEMT easy vector containing the complete deletion cassette was digested in the same way as the fragment. After electrophoretic separation in 1% agarose gel, the characteristic band of the pGEM-T easy vector containing the fragment F2 was cut out and purified (Fig. S4b, Online Resource 1), and was subsequently ligated with the functional copy of the luxS gene. Positive colonies were confirmed by colony PCR using primer Set 11 (Table 1). For complementation, the kanamycin resistance cassette from plasmid pRY107 was amplified using the modified primer Set 12 (Table 1; Shang et al. 2016) and was cloned between both fragments into the BamHI site (Fig. S4a, Online Resource 1). Positive colonies were selected on the LB-A plate with kanamycin (50 µg/mL). The correct assembly of the complementation cassette was confirmed through the Sanger sequencing (Fig. S4c, Fig. S4d, and Fig. S4e, Online Resource 1) using commercially available primers M13 and primer SubF (Table 1) to assure a precise sequencing of the long construct.
Transformation of Campylobacter jejuni 81–176 by cassette mutagenesis
The transformation was performed as described by Wassenaar et al. (1993). Briefly, the PCR with primer set 13 was performed for amplification of deletion, substitution, insertion inactivation, and complementation cassettes (1 µg of DNA), which were subsequently electroporated into freshly prepared electro-competent cells of C. jejuni 81–176 using MicroPulser™ (Bio-Rad, Hercules, California, USA) and pre-cooled 0.2 cm cuvettes (Bio-Rad, Hercules, California, USA). For each electroporation experiment, exactly one pulse of 2500 V was used. After electroporation, the cuvette was rinsed two times with 100 µL of pre-warmed MH broth, and each suspension was transferred (without spreading) on a surface of pre-warmed MH agar (without antibiotics) and incubated microaerobically at 42°C overnight. Subsequently, the culture was harvested with 1 mL of the pre-warmed MH broth and 100 µL aliquots were spread on MH agar plates containing 20 µg/mL of chloramphenicol and incubated for 3–7 days. After the incubation, colonies grown on the selective agar plate were individually inoculated on the surface of fresh antibiotic-supplemented plates and were incubated microaerobically at 42°C overnight. To confirm the recombination, a part of each grown overnight culture was harvested with a sterile inoculation loop, and its DNA was isolated by thermal lysis. The presence of mutagenic cassettes within the genome was confirmed by PCR (primer Set 13, Table 1). After visualization of products in 1% agarose gel, the remaining cells of positive cultures were collected with pre-warmed MHB containing 20% glycerol and stored at -80°C.
To complement the mutant strains, the complementation cassettes were electroporated into electro-competent cells of the respective luxS mutant strains. Positive colonies were selected on the MH agar plates with kanamycin (50 µg/mL).
The presence/absence of the luxS gene was confirmed by PCR (primer Set 14, Table 1). For final sequencing of the incorporated cassettes (mutagenic/complementation), these were amplified from DNA of positive colonies (primer Set 9, Table 1) and cloned into the pGEM®-T easy vector as described above. All sequences were deposited to the GenBank under the following numbers: deletion MN640707; substitution MN738698; insertional inactivation MN738697; and complementation MN711445.