Strains and plasmids used in this work
| Strain or plasmid | Relevant characteristics | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| VECO2068 | F− araD139 Δ(ara, leu)7697 galE15 galK16Δ(lac)X74 rpsL hsdR2 (rk −mk +) mcrA mcrB1ΔtolCΔPdef::PBAD= def; PBAD-regulated defgene | 13 |
| BL21(DE3)/pLysS | F− ompT hsdSB (rB −mB −) dcm gal λ(DE3) (pLysS); expression strain for PT7-regulated gene expression | Novagen, Madison, Wis. |
| S. pneumoniae | ||
| R6x | Unencapsulated strain for laboratory genetics | 24 |
| ATCC 49619 | American Type Culture Collection, Manassas, Va. | |
| VSPN6501 | ATCC 49619defB(Q172K); actinonin-resistant | This study |
| VSPN6503 | ATCC 49619 defB(A123D); actinonin-resistant | This study |
| VSPN6504 | ATCC 49619defB(A123D); actinonin-resistant | This study |
| VSPN7011 | R6x defA::pR326defA; defA disruption | This study |
| VSPN7035 | R6xdefB +::pPV302-7; expresses wild-typedefB | This study |
| VSPN7036 | R6xdefB::pPV303-7; expresses defB(Q172K) | This study |
| VSPN7037 | R6xdefB::pPV304-5; expresses defB(A123D) | This study |
| Plasmids | ||
| pET20b(+) | PT7; overexpression vector | Novagen, 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-3 | Ptac; overexpression vector, GST fusion protein | Amersham Pharmacia, Piscataway, N.J. |
| pGEXSpn defA | pGEX-5X-3::defA; Ptac -gst-defA fusion | This study |
| pGEXSpn defB | pGEX-5X-3::defB; Ptac -gst-defB fusion | This study |
| pR326 | cat; E. coli, S. pneumoniae shuttle vector | 6 |
| pR326rafE | pR326::'rafE'a | 23 |
| pR326defA | pR326::'defA' | This study |
| pR326defB | pR326::'defB' | This study |
| pR326fmt | pR326::'fmt' | This study |
| pPV302-7 | pR326::'defB +; downstream end of wild-type defB ORF | This study |
| pPV303-7 | pR326::'defB(Q172K); downstream end of mutated defB ORF | This study |
| pPV304-5 | pR326::'defB(A123D); downstream end of mutated defB ORF | This study |
↵a Apostrophes indicate the truncation of the gene at the upstream and/or downstream end, as indicated.