Construction of allelic exchange mutants

To construct the bfmS-DK2 allelic exchange mutant, a ~3.3-kb PCR product covering ~1-kb upstream of bfmS, the bfmS gene, and ~0.95-kb downstream of bfmS was amplified from P. aeruginosa DK2 genomic DNA using primers bfmS-allelic-DK2-F (with Eco RI site) and bfmS-allelic-DK2-R (with Hind III site). The fragments were subsequently digested and cloned into Eco RI/Hind III–digested gene replacement vector pEX18Tc (table S1). The resultant plasmid, pEX18Tc-For-bfmS^DK2, was electroporated into the ΔbfmS mutant (with gentamicin resistance cassette) (table S1) with selection for both tetracycline and gentamicin resistance, which typically indicates a single-crossover event. Colonies were further screened for tetracycline and gentamicin sensitivity and loss of sucrose (5%) sensitivity, which typically indicates a double-crossover event and thus marks the occurrence of gene replacement. The bfmS-DK2 mutant was further confirmed by PCR and DNA sequencing. For generating bfmS-DK2^H238, plasmid pEX18Tc-For-bfmS^{L181P/E376Q/ H238A} was constructed using the primer pair BfmS(H238A)-F/BfmS(H238A)-R, a QuikChange II site-directed mutagenesis kit (StrataGene, catalog no. 200518), and pEX18Tc-For-bfmS^DK2 plasmid DNA.

To construct the bfmS^R393H (MPAO1-bfmS^R393H) allelic exchange mutant, PCR product was amplified from P. aeruginosa MPAO1 genomic DNA using primers bfmS-allelic-DK2-F (with Eco RI site) and bfmS-allelic-DK2-R (with Hind III site) and then cloned into pEX18Tc, yielding pEX18Tc-For-bfmS^PAO1 plasmid. Subsequently, the resultant plasmid, pEX18Tc-For-bfmS^R393H (table S1), was constructed using the primer pair R393H-F/R393H-R and a QuikChange II site-directed mutagenesis kit (StrataGene, catalog no. 200518). The pEX18Tc-For-bfmS^R393H was further electroporated into the ΔbfmS mutant (with gentamicin resistance cassette) (table S1) to generate the bfmS^R393H allelic exchange mutant as described above.

To construct bfmS-DK2²ΔbfmR, pEX18Ap::bfmRUGD plasmid (table S1) was electroporated into bfmS^DK2 allelic exchange mutant with selection for gentamicin resistance. To construct bfmS-DK2ΔgtrS and bfmS^R393H∆gtrS mutants, pEX18Ap::gtrSUGD plasmid (table S1) was electroporated into bfmS-DK2 and bfmS^R393H allelic exchange mutants, respectively, with selection for gentamicin resistance. Colonies were screened for gentamicin sensitivity and loss of sucrose (5%) sensitivity, which typically indicates a double-crossover event and thus marks the occurrence of gene replacement. PCR and DNA sequencing further confirmed the replacement of the gene allele.

A similar strategy was used to construct the DK2-bfmS^PAO1 and AU15431-bfmS^PAO1 mutants. Briefly, pEX18Tc-For-bfmS^PAO1 plasmid was electroporated into DK2-ΔbfmS and AU15431-ΔbfmS (table S1), respectively. Colonies were screened for tetracycline and gentamicin sensitivity and loss of sucrose (5%) sensitivity.