Bacterial culture conditions and cultivation of eukaryotic cells
Bacteria were maintained at 42 °C on Columbia agar plates supplemented with sheep blood (COS) in anaerobe containers with a microaerophilic atmosphere, consisting of 5% O2, 10% CO2 and 85% N2 (CampyGen, ThermoScientific). Human colon carcinoma (Caco2) cells were maintained in 75 cm2 cell culture flasks in 30 ml of Dulbecco Minimal Essential Medium (DMEM) supplemented with 10% heat inactivated Fetal Calf Serum (FCS), 1× Non-Essential Amino acids (NEA) and 100 U/ml penicillin and 100 μg/ml streptomycin.
Bacterial cultures were prepared for growth experiments by firstly growing pre-cultures of the strains in 20 ml Mueller Hinton (MH) medium in 100 ml Erlenmeyer flasks for 24 h at 37 °C and 150 rotations per minute (rpm) under microaerophilic conditions. The ODs were adjusted to OD600 0.05 at the start of the growth experiments by inoculation of 20 ml MH medium with the prepared pre-cultures. Incubation conditions were as described for the pre-cultures.
Genome sequencing, assembly and annotation
Genome sequencing was carried out on the PacBio RSII (Pacific Biosciences, Menlo Park, CA) using P6 chemistry. Genome assembly was performed with the “RS_HGAP_Assembly.3” protocol included in SMRT Portal version 2.3.0. The chromosomal contig was trimmed, circularized, and adjusted to dnaA (A17_00001) as first gene. The extrachromosomal element was trimmed and circularized. In addition, genome sequencing of strain A17 was carried out on a NextSeq (Illumina, San Francisco, CA) and quality improvement of the long-read sequence was performed with Burrows-Wheeler Aligner (BWA) [31, 32] mapping the Illumina reads onto the contigs to obtain a final consensus sequence. A final quality of QV60 was confirmed. Automated genome annotation was carried out using Prokka . The complete genome has been deposited at GenBank under accession numbers CP028372 and CP028373.
Swarming motility assays
Soft agar swarming motility assays were performed as described in  with minor modifications. Liquid cultures were grown for 16 h at 37 °C under microaerophilic conditions and then adjusted to an OD600 of 0.025. These cultures were stabbed into 0.4% MH agar plates with a 1 μl inoculation loop. The plates were then incubated at 37 °C under microaerophilic conditions for 36 h, after which the diameters of the swarming motility zones were measured.
Eukaryotic cell invasion and adhesion
Invasion and adhesion assays were performed as described by Everest and coworkers  with minor modifications. CaCo2 cells were seeded in 24-well plates at a concentration of 2 × 105/well. After 16 h of incubation, CaCo2 cells were infected with ~ 2 × 106 CFU C. jejuni A17 derived mutants, which corresponds to a multiplicity of infection (MOI) of 10. Plates were centrifuged at 600 g for 5 min and incubated afterwards for two hours at 37 °C to allow the bacteria to invade the host cells. An aliquot of the bacterial suspension was plated on COS plates in serial dilutions, for a determination of the actual number of CFU added to each well. Following the 2 h incubation, the bacterial suspension was removed from the CaCo2 cells and the monolayer was washed three times with 1 ml DMEM before the addition of a 1 ml suspension of 100 μg/ml gentamycin in DMEM, followed by an incubation period of two hours at 37 °C. Afterwards, the cell monolayer was washed again three times with 1 ml of DMEM and lysed with 100 μl of 0.1% Triton X-100 in DMEM to release intracellular bacteria. After 10 min of incubation, 900 μl of DMEM was added and the number of viable bacteria in each well was determined by plating serial dilutions on COS plates. Percentage invasion was calculated by dividing the number of invaded bacteria by the number of viable bacteria that were added to the wells. Adhesion experiments were performed in the same way as described above with the only modification being an infection of the cell monolayer for 30 min rather than 2 h and no incubation step with gentamycin. Experiments were performed in technical triplicates and in at least biological duplicates.
Autoagglutination assays were carried out as described by Misawa and Blaser . Bacteria grown for 16 h on COS agar plates at 42 °C under microaerophilic conditions were resuspended in PBS (pH 7.4) and adjusted to an OD600 of 1. The bacterial suspensions were then added in a volume of 2 ml into glass tubes and incubated for 24 h at 37 °C under microaerophilic conditions without shaking. After incubation, 1 ml of the supernatant was carefully removed and the OD600 was measured. Relative autoagglutination was calculated by dividing the initial OD of the bacterial suspension by the final OD of the bacterial supernatant.
The biofilm assays were performed as described by Reeser and coworkers  with some modifications. Bacteria grown for 16 h on COS agar plates were resuspended in MH medium and adjusted to an OD600 of 0.05. 100 μl of the bacterial suspension was then added to each well of a 96-well plate and the plate was incubated for 48 h at 37 °C under microaerophilic conditions. Only MH medium was added to a separate row of wells to serve as the negative control. Following incubation, the bacterial cultures were removed from each well and the plate was dried for 30 min at 60 °C. 100 μl of 0.1% crystal violet resuspended in water was then added to each well and left to stain for 15 min at room temperature. Unbound crystal violet was removed from the wells, the wells were washed two times with 100 μl of water and the plate was dried for 15 min at 60 °C. The bound crystal violet was decolorized by adding 100 μl solutions of 80% ethanol and 20% acetone into each well for 15 min. After 15 min of decolorization, 90 μl of the bacterial solution was pipetted out of the wells and added to a fresh 96-well plate (as not all of the biofilm might have detached from the wells of the old plate after 15 min). The plate was then read at an absorbance of 570 nm with a microplate reader to quantify the amount of biofilm formed in each well. Results were normalized by subtracting absorbances obtained by the negative controls from these readings. The experiments were done for all strains in technical quadruplets and biological triplicates.
Δtlp12 knockout generation
The Δtlp12 knock-out mutant was generated by double homologous recombination that resulted in the insertion of a kanamycin resistance cassette, kanR, into the gene. For the construction of the knockout vector, a 546 bp 5′-fragment and a 493 bp 3′-fragment of the target gene in the C. jejuni strain A24 were amplified with the primers psk-5-TLP12-F/kan-5-TLP12-R and kan-3-TLP12-F/psk-3-TLP12-R, respectively. The psk-5-TLP12-F and psk-3-TLP12-R primers have 30 bp of oligonucleotides that are complementary to the pBluescript SKII vector attached to their 5′ and 3′ ends, respectively. The kan-3-TLP12-F and kan-5-TLP12-R primers have 30 bp of oligonucleotides that are complementary to the ends of the kanR gene attached to their 5′ and 3′ ends, respectively. The kanR gene was amplified using the Kan 1 and Kan 2 primers. The PCRs were performed as follows: 95 °C for 1 min, 35 cycles at 98 °C for 15 s, 58 °C for 15 s and 72 °C for 1 min and a final incubation at 72 °C for 5 min. The PCRBio HiFi Kit was used for all PCRs and PCR reactions were put together as per manufacturer’s recommendations with 1 U of PCRBIO HIFI polymerase, 400 nM forward and reverse primers and ~ 500 ng of genomic template DNA in 50 μl reactions. The pBluescript SKII vector was digested with BamHI and EcoRI to linearize the vector. The linearized vector, the 3′ and 5′ target gene fragments and the kanR gene were assembled using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB) and the assembled knockout vector was transformed into NEB 5-alpha competent E. coli. Plasmids were extracted from single colonies and the correct construction of the knockout vector was confirmed by Sanger sequencing using the psk-5-TLP12-F and psk-3-TLP12-R primers. The knockout vector was electroporated into C. jejuni A17 wild type competent cells using an ECM600 Electro Cell Manipulator (BTX) with the following settings: resistance – 2.5 kV, capacitance timing – 25 μF and resistance timing – 186 Ω. After electroporation, 100 μl SOC Outgrowth Medium (NEB) was added to the mixture which was then inoculated onto COS plates and incubated for 16 h at 37 °C under microaerophilic conditions to encourage the amplification of the generated mutants. Wild type strains in the background were then excluded by replating the bacteria onto COS plates containing kanamycin (10 μg/ml). Primer sequences are listed in Additional file 4.
The complementation of the Δtlp12 mutation was performed as described in  with some modifications. The complementation was performed by a plasmid-based, double-recombinational insertion of the tlp12-coding sequence linked to a chloramphenicol resistance cassette (camR) into one of the three rRNA loci of the A17 Δtlp12 mutant. For the construction of the tlp12 complementation vector, the tlp12 gene (A17_00255) from the wild type A17 genome was amplified using the TLP12_Complement Primer_Fw and TLP12_Complement Primer_Rev. At its 5′ end, the TLP12_Complement Primer_Fw has a Shine-Dalgarno sequence for improved binding of RNA polymerase after transcription of the gene and 36 bp of an oligonucleotides sequence that is complementary to the linearized pRRC vector at its 5′ end. The TLP12_Complement Primer_Rev primer has 36 bp of oligonucleotides that are complementary to the linearized pRRC vector at its 3′ end. The tlp12 gene was amplified as follows: 95 °C for 1 min, 35 cycles at 95 °C for 15 s, 65 °C for 15 s and 72 °C for 2 min and a final incubation at 72 °C for 5 min. The PCR reactions were put together as described previously with the PCRBio HiFi Kit. The pRRC vector was digested with XbaI and phosphatase treated with Antarctic Phosphatase (NEB), as per manufacturer’s recommendations. The linearized vector and the tlp12 gene were assembled and transformed into NEB 5-alpha competent E. coli as described previously. The correct construction of the knockout vector was confirmed by Sanger sequencing. Electroporation of the complementation vector into A17 Δtlp12 was performed as described before. The selective chloramphenicol concentration was 12.5 μg/ml. The complementation mutant was named Δtlp12::compl. The used primer sequences are listed in Additional file 4.
Chemotaxis syringe capillary assay
The chemotaxis assays were performed as described by Chandrashehkar et al. . All chemicals tested in the chemotaxis assays were suspended in PBS, adjusted to a pH of 7 and set to a concentration of 100 mM, a concentration that has previously been shown to result in the strongest chemotaxis response [7, 12]. Bacteria grown for 18 h on COS agar plates at 42 °C under microaerophilic conditions were resuspended in 1 ml of PBS (pH 7) and washed once by centrifuging at 4500 g for 5 min. The washed bacterial suspension was then adjusted to an OD600 of 0.5. 100 μl of the tested chemical solutions were drawn through a 27 G hypodermic needle (0.40 mm diameter × 20 mm long) into a 1 ml Luer syringe. Buffer alone served as a control. A 100 μl of the adjusted bacterial suspension was then drawn into a 200 μl disposable pipette and the tip was sealed with parafilm. The pipette tip was attached to the needle-syringe system such that most of the needle immersed into the bacterial suspension. The whole system was then incubated horizontally for 1 h at 42 °C under microaerophilic conditions, after which the needle-pipette tip system was detached from the syringe. The bacterial suspension in the syringe was then plated in 10-fold serial dilutions on COS plates for 48 h at 37 °C or 42 °C under microaerophilic conditions and the CFU were counted. The taxis toward a test compound was expressed by calculating the Relative Chemotaxis Ratio (RCR), which is the ratio of the number of bacteria in the syringe with a tested chemical to the number in the control (buffer only) after 1 h incubation. Capillary assays for glutamate and pyruvate were performed at least in biological triplicates, each with at least technical duplicates.
Statistical analysis were performed by two-sided, unpaired Student T-tests using Excel software.