Insertion kit

For identification of the pColZ plasmid, transposon mutagenesis with a Tn5 transposon (EZ-Tn5™ < KAN-2 > Insertion Kit; Epicentre Biotechnologies, Madison, WI, USA) was used. Briefly, total plasmid DNA from the original producer of colicin Z was isolated using a QIAGEN Plasmid Midi Kit (Qiagen, Germany). This DNA was used in an in vitro transposon insertion reaction and transformed into E. coli DH10B. Plasmid DNA from 24 recombinant colonies (pColZ1-24) was isolated and the DNA in the vicinity of the inserted Tn5 transposon was sequenced (Eurofins Scientific, Brussels, Belgium) with Tn5 transposon sequencing primers (Supplementary Table S3). Next, sequencing primers were designed (Table S3) and plasmid DNA of recombinant strain ColZ1 (which was able to inhibit EIEC O164 indicator strain was used for sequencing of pColZ plasmid).
A non-enzymatic in vivo cloning strategy was used for recombinational cloning to E. cloni® 10 G (Lucigen, Middleton, WI, USA). For in vivo recombination, a digested pBAD/HisB vector (Thermo Fisher Scientific) was prepared and incubated with purified PCR product with 25 nt-long overlaps (Supplementary Table S3) that were complementary to the vector DNA.

Gene Splicing by Overlap Extension (gene SOEing) was used to create a fusion PCR product to replace fHbp in strain L91543 with the kanamycin resistance gene (kan) from EZ:Tn5 < KAN-2 > insertion kit (Epicentre) following the approach described by Horton (Horton, 1995 (link)). In the first round of PCRs, homology arms (HA1 and HA2) of approximately 600 bp flanking fHbp were amplified from genomic DNA of L91543 using primers, HA1_FHbp_Fwd and Rev_HA1_FHbp and HA2_FHbp_fwd and HA2_FHbp_rev, and the kan gene was amplified using primers Kan_fwd and Kan_Rev. As shown in Table 3, in bold are the regions that bind to kan, underlined are the regions that bind to the HA of interest and in italics are the regions that over-lap. The HA1 and kan PCR products obtained were gene-cleaned then used as template for the second round of PCR with primers HA1_FHbp_Fwd and Kan_Rev and the annealed product generated cleaned and used as template along with the HA2 PCR product for a third round of PCR with primers HA1_FHbp_Fwd and HA2_FHbp_rev. The final PCR product generated containing HA1-kan-HA2 was gene cleaned and sequenced for confirmation. The verified construct was used to transform strain L91543 with selection on kanamycin. Deletion mutants were confirmed by PCR and DNA sequencing and designated L91543ΔfHbp.

Acinetobacter baumannii ATCC 17978 transposon mutants were generated using the EZ-Tn5™ < KAN-2 > Insertion Kit (Epicentre Biotechnologies) as previously described [35 (link)]. Transformation of the transposome complex into ATCC 17978 was performed by electroporation [46 (link)]. A. baumannii 29D2 mutants were generated by making use of the strain’s ability for natural competence. The transforming DNA was isolated from the ATCC 17978 mutants described above. A suspension of DNA-accepting bacteria was generated by resuspending a few colonies (approx. 5 × 10⁶ CFU) in 100 µL of sterile phosphate buffered saline (PBS). The bacterial suspension was then mixed with equal volumes of the transforming DNA (~ 400 ng/µL). This mixture was stabbed into motility agar plates ten times, pipetting 2 µL of the mixture with each stabbing [16 (link)]. Controls were wildtype DNA and TE buffer without DNA that were mixed with bacterial suspensions and processed accordingly. The motility plates were incubated for 18 h at 37 °C. After incubation, the bacteria were flushed off the motility plates with 1 mL of sterile PBS and 100 µL (approx. 2 × 10⁸ CFU) was plated on selective agar plates (50 g/mL of kanamycin). After sub-culturing of selected colonies transformation was confirmed by PCR.