R-pyocin mutant generation.

We generated R-pyocin mutants by deleting the genes coding for the R-pyocin tail fiber and its chaperone assembly gene (PA0620 and PA0621 in the PAO1 reference strain). We achieved this using a double homologous recombination method with the help of our deletion gene construct. Our 1,076-bp gene construct was generated from a two-stage PCR. In the first-stage PCR, we amplified the upstream and downstream genes PA0619 and PA0622 (flanking our genes of interest, PA0620 and PA0621) separately using primer pairs L0619H3/R0619 and L0622/R0622BHI. This first stage generated 454- and 637-bp amplicons, respectively. In the second stage, we used the two amplicons from the first stage as the templates, bringing the engineered 15-bp complementary ends together by splicing of overlapping extensions using the end primers L0619H3 and R0622BHI. Next, we cloned the gene construct into HindIII/BamHI-digested pME3087 (28) to produce plasmid pOO1, which was used to transform E. coli S17-1 strains. We then integrated pOO1 into P. aeruginosa chromosomes by conjugating P. aeruginosa strains with transformed S17-1 cells. We selected our P. aeruginosa transconjugants using agar plates containing 150 µg/ml Tc plus 15 μg/ml nalidixic acid. We picked individual colonies from our selective plates and cultured them in overnight broths without antibiotics in order to enrich for tetracycline-sensitive cells. We then made fresh cultures (1:100 dilution) from the overnight broth and incubated until they reached an OD₆₀₀ of 0.1 ± 0.05. We added tetracycline and carbenicillin in turn at 1-h intervals to achieve final concentrations of 20 µg/ml and 2,000 µg/ml, respectively. Then we washed our cells using sterile LB and repeated the enrichment process (in plain and antibiotic broths). After washing the cells, we plated out their dilutions (10⁻⁵ to 10⁻⁸) on LB agar to harvest individual colonies. We performed a replica plating of individual colonies using LB and 25-µg/ml Tc agar plates. This step selected for unmarked mutants with double crossovers, and since they were sensitive to tetracycline, we picked them from the LB plates. We further confirmed the deletion mutation by PCR using primer pair L0619H3 and R0622BHI, thus, recovering our 1,076-bp deletion gene construct. We tested for the successful attenuation of pyocin activity by performing spot assays using cell extracts from these null-R-pyocin mutants and comparing the results with cell extracts from wild-type bacteria.