Table 1.

Strains and plasmids used in this work

Strain or plasmidRelevant characteristicsSource or reference
Strains
E. coli
  VECO2068F araD139 Δ(ara, leu)7697 galE15 galK16Δ(lac)X74 rpsL hsdR2 (rk mk +) mcrA mcrB1ΔtolCΔPdef::PBAD= def; PBAD-regulated defgene13
  BL21(DE3)/pLysSF ompT hsdSB (rB mB ) dcm gal λ(DE3) (pLysS); expression strain for PT7-regulated gene expressionNovagen, Madison, Wis.
S. pneumoniae
  R6xUnencapsulated strain for laboratory genetics24
  ATCC 49619American Type Culture Collection, Manassas, Va.
  VSPN6501ATCC 49619defB(Q172K); actinonin-resistantThis study
  VSPN6503ATCC 49619 defB(A123D); actinonin-resistantThis study
  VSPN6504ATCC 49619defB(A123D); actinonin-resistantThis study
  VSPN7011R6x defA::pR326defA; defA disruptionThis study
  VSPN7035R6xdefB +::pPV302-7; expresses wild-typedefB This study
  VSPN7036R6xdefB::pPV303-7; expresses defB(Q172K)This study
  VSPN7037R6xdefB::pPV304-5; expresses defB(A123D)This study
Plasmids
 pET20b(+)PT7; overexpression vectorNovagen, Madison, Wis.
 pET20bSpn defBo p t (C-His) pET20b(+) carrying Spn defB with optimized codon usage; encodes C-terminally His-tagged PDF (wild type)This study
 pET20bSpn defBo p t (C-His, Q172K) pET20bSpn defBo p t (C-His) mutagenized to introduce Q172K mutation This study
 pET20bSpn defBo p t(C-His, A123D) pET20bSpn defBo p t (C-His) mutagenized to introduce A123D mutation This study
 pGEX-5X-3Ptac; overexpression vector, GST fusion proteinAmersham Pharmacia, Piscataway, N.J.
 pGEXSpn defA pGEX-5X-3::defA; Ptac -gst-defA fusionThis study
 pGEXSpn defB pGEX-5X-3::defB; Ptac -gst-defB fusionThis study
 pR326 cat; E. coli, S. pneumoniae shuttle vector6
 pR326rafE pR326::'rafE'a 23
 pR326defA pR326::'defA'This study
 pR326defB pR326::'defB'This study
 pR326fmt pR326::'fmt'This study
 pPV302-7pR326::'defB +; downstream end of wild-type defB ORFThis study
 pPV303-7pR326::'defB(Q172K); downstream end of mutated defB ORFThis study
 pPV304-5pR326::'defB(A123D); downstream end of mutated defB ORFThis study
  • a Apostrophes indicate the truncation of the gene at the upstream and/or downstream end, as indicated.