Ecori and xbai

The fliA-EcoRI-F/fliA-XbaI-R primers and flgM-EcoRI-F/flgM-XbaI-R primers (Table 2) were used to amplify the fliA and flgM genes, respectively, using Phusion® High-Fidelity DNA Polymerase (NEB). PCR was performed under standard conditions. The primer hybridisation temperatures were calculated with NEB Tm calculator. The amplified fragment and the pPSV35 shuttle vector [37 (link)] or pJN105 shuttle vector [39 (link)] were digested with EcoRI and XbaI (NEB) to generate cohesive ends. The coding region of the fliA gene was inserted into pPSV35 downstream of the PlacUV5 promoter and the coding region of the flgM gene into pJN105 downstream of the arabinose inducible promoter using T4-DNA-ligase (NEB). The resulting plasmids, pPSV35-fliA and pJN105-flgM, were used to transform E. coli Top10® cells by thermal shock. Plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific, Waltham, MA, USA) and verified by PCR with plasmid-specific primers and DNA sequencing.

The nucleotide sequence of the A2 lysin (LysA2) was obtained from GenBank (Nucleotide accession #AJ251789; Protein accession #NP_680500; NCBI; see supplementary materials). The sequence was codon optimized for E. coli, and synthesized and cloned into the pNW129 low copy expression plasmid (received from Deborah M. Hinton, NIDDK, NIH) by Genscript USA to make pSDL129. For recombinant protein expression, pSDL129 was transformed into E. coli BL21 DE3. Clone candidates were verified by both sequencing the insert (Macrogen, Rockville, MD, USA) and by digesting with EcoRI and XbaI (NEB). Several Lactobacillus species from our collection, including some species commonly found in OTC probiotic products, were used in our lytic activity screen. All strains used in our experiments are summarized in Table 1 below:

The hcp3 gene was amplified from P. fluorescens strain MFE01 with the hcp3-EcoRI-F and hcp3-XbaI-R primers (Table 2). The PCR conditions were as follows: 30 cycles with an annealing temperature of 58°C and an extension time of 40 s. The Phusion® High-Fidelity DNA polymerase (NEB) was used. The amplified fragment and the pPSV35 shuttle vector [39 (link)] were digested with EcoRI and XbaI (NEB) to generate cohesive ends, and the hcp3 gene was inserted into the vector by DNA ligase mediated ligation (NEB). The resulting plasmid, pPSV35-hcp3, was used to transform E. coli Top10® cells by thermal shock. Plasmid DNA was isolated with the GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific) and verified by PCR with primers binding to the plasmid. Fresh MFE01Δhcp3 colonies were obtained and washed twice with 1 mL cold sterile water before transformation with 5 μL pPSV35-hcp3 by electroporation in 1 mm electroporation cells at 1.8 KV for 5 milliseconds (Savant GTF100 Gene Transformer). LB was added and the mixture was incubated at 28°C for 1 h with shaking (180 rpm). Transformed bacteria were then selected by plating on LB agar supplemented with gentamycin and IPTG.