Table 1.

Bacterial strains, plasmids, and primers used in this study

Strain, plasmid, or primerDescription or sequenceaReference or source
E. coli
 DH5αF′[F+φ80lacZΔM15] Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 [rK mK +] supE44 thi-1 gyrA relA1 27
 JP1111Hfr galE45 fabI392(Ts) relA1 spoT1 44
 BL21(DE3) E. coli B; F ompTrB mB (λDE3) 43
P. aeruginosa
 PAO1PAO1B. H. Holloway
 PAO200PAO1 with Δ(mexAB-oprM) 41
 PAO234PAO1 withfabI::Gmr-FRT This study
 PAO235PAO1 withfabI::FRT This study
Plasmids
 pET-15bApr; hexahistidine fusion and expression vectorNovagen
 pWSK29/30Apr; low-copy-number cloning and T7 expression vectors 47
 pFLP2Apr; source of Flp recombinase 21
 pPS856AprGmr; source of Gmr-FRTcassette 21
 pUCP21TApr; broad-host-range cloning vector 42
 pEX18TcTcr; sacB-based gene replacement vector 21
 pPS922Apr fabI + (ligation of a 880-bp BamHI-HindIII PCR fragment between the same sites of pWSK30; fabI-transcription driven by Plac)This study
 pPS925Apr fabI::Gmr-FRT (ligation of a 1,053-bp blunt-ended SacI fragment from pPS856 into theSmaI site of pPS922)This study
 pPS933Apr sacB+fabI::Gmr-FRT (ligation of a 1.95-kb BamHI-HindIII fragment between the same sites of pEX18Tc)This study
 pPS935Apr; H6-FabI expression vector (PCR-amplified 0.85-kbNdeI-BamHI fragment cloned between same sites of pET-15b)This study
 pPS967Apr fabI + (pUCP21T withBamHI-HindIII fragment from pPS922;fabI transcription driven by Plac)This study
 pPS1098Apr; H6-FabI expression vector (PCR-amplified 0.85-kb NdeI-BamHI fragment cloned between same sites of pET-15b); expresses H6-FabI mutant proteinThis study
Primers
 FabIU HindIII-GCAAGCttGCGGAAAACTGAGTAGCAGA
 FabID BamHI-ATGGatCCGCTTGAAGCGGCATTTTTC
 FabI-Nde NdeI-GGcatATGGGATTTCTCACAGGAAAAC
 FabI G>VCCACTCCGTCGtATTCGCTCCAG
  • a Plac, E. coli lac operon promoter. Primer sequences are printed 5′ to 3′; lowercase letters indicate nonmatching oligonucleotides used to either form the indicated motif as underlined or introduce other nonmotif changes indicated in lowercase letters.