Table 1.

Strains and plasmids used in this work

Strain or plasmidRelevant characteristicsSource or reference
Strains
E. coliJM109[F′ traD36 proA+B+ lacI qΔ(lacZ)M15] recA1 endA1 gyr96 hsdR17(rK mK +)supE44 relA1 thiΔ(lac-proAB)Promega, Madison, Wis.
E. coli MC1061F araD139 Δ(ara leu)7697 galE15 galK16Δ(lac)X74 rpsL hsdR2(rK mK +) mcrA mcrB1 Bio-Rad, Hercules, Calif.
E. coliBL21(DE3)(pLysS)F ompT hsdSB (rB mB ) dcm gal λ(DE3) (pLysS)Novagen, Madison, Wis.
E. coli VECO2065MC1061 ΔPdef::PBAD; PBAD-regulated def geneThis study
E. coli VECO2068VECO2065 ΔtolC This study
S. aureus ATCC 25923American Type Culture Collection, Manassas, Va.
S. aureus 1-63Clinical isolateH. F. Chambers III (12)
S. aureus NCTC 8325-4 28
S. aureusRN4220 14
S. aureusVSAU6011ATCC 25923 fmt (A108E); actinonin-resistant mutantThis study
S. aureus VSAU6012ATCC 25923fmt (G117V); actinonin-resistant mutantThis study
S. aureus VSAU6013ATCC 25923 fmt(E157stop); actinonin-resistant mutantThis study
S. aureus VSAU60141-63 fmt (frameshift, codon 180); actinonin-resistant mutantThis study
S. aureusVSAU7108RN4220 ΔdefA::aac-aph(pPV158-1)This study
S. aureus VSAU7136RN4220 Δfmt::aac-aph This study
Plasmids
 pAW8 tet oriE. coli oriS. aureus ; shuttle vector 33
 pAW9 tet oriE. coli ; shuttle vector 33
 pBAD/Myc-HisB bla araCPBAD Invitrogen, Carlsbad, Calif.
 pBS-SK+ bla lacZαStratagene, La Jolla, Calif.
 pBS-SK bla lacZαStratagene, La Jolla, Calif.
 pBS-KS+ bla lacZαStratagene, La Jolla, Calif.
 pBSII-SK+ bla lacZαStratagene, La Jolla, Calif.
 pBSL99 kan American Type Culture Collection, Manassas, Va.
 pBT2 bla cat repF(Ts); shuttle vector, temperature sensitive for replication in S. aureus 7
 pET20b+ blaPT7 Promega, Madison, Wis.
 pGO1 aac-aph 32
 pGO514 aac-aph 27
 pKO3 sacB cat repA(Ts) 16
 pRDC19 erm F. Arigoni
 pT7Blue bla lacZαNovagen, Madison, Wis.
 pUC19 bla lacZαNew England Biolabs, Beverly, Mass.
 pDYD11pBSII-SK+::kanupstreamdef This study
 pDYD12pBAD/Myc-HisB::def; PBAD -def fusionThis study
 pDYD13pKO3::upstreamdef kan araCΔPdef::PBAD -def This study
 pVCRΔtolCpKO3::ΔtolC This study
 pPV16-1pBS-SK::defB This study
 pPV45-1pET20b+::defA; PT7-defB fusionThis study
 pPV54-2pT7Blue::defA This study
 pPV58-1pET20b+::defB; PT7-defA fusionThis study
 pPV46-1pT7Blue::repF(Ts)This study
 pPV72-2pAW9::repF(Ts); shuttle vector, temperature sensitive for replication in S. aureus This study
 pPV77-1 lacG disruption plasmid derived from pPV72-2This study
 pPV92-3 gusA disruption plasmid lacking S. aureus homology derived from pPV72-2This study
 pPV120-1 defB disruption plasmid derived from pPV72-2This study
 pPV150-1pT7Blue::PdefAΔdefA fmt This study
 pPV158-1pAW8::PdefAΔdefA fmt; fmt complementationThis study
 pPV171-1pBS-KS+::erm repF(Ts) shuttle vector, temperature sensitive for replication inS. aureus This study
 pPV172-4pPV46-1::erm; shuttle vector, temperature sensitive for replication in S. aureus This study
 pPV179-1pPV171-1::PdefAΔdefA::aac-aph fmt This study
 pPV188-1pT7Blue::PdefA defAΔfmt This study
 pPV214-1pT7Blue::erm repF(Ts) PdefA defA This study
 Δfmt::aac-aph