Article Figures & Data
Figures
- Fig. 1.
Alignment of conserved domains of deformylase proteins. Partial sequences of the predicted products of the defA anddefB homologs of S. aureus and S. pneumoniae are shown aligned with consensus PDF domains (13). Residues that diverge from the consensus are highlighted. Modifications in the deformylase enzyme in the resistantS. pneumoniae mutants VSPN6501, VSPN6503, and VSPN6504 are indicated by arrows. The positions of the motifs in the S. pneumoniae DefB are as follows: box 1, G69 to Q76; box 2, A123 to S132; and box 3, Q172 to G182. The sequences of the S. pneumoniae def homologs from strain R6x have been submitted to GenBank (accession numbers: defA, AY014508; defB,AY014509). Sequences of the S. pneumoniae def andfmt homologs from S. pneumoniae ATCC 49619 were also submitted to GenBank (accession numbers: defA,AY014510; defB, AY014511; fmt, AY014512).
Tables
- Table 1.
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.
- Table 3.
Susceptibilities to actinonin of S. pneumoniaeR6x strains transformed with wild-type or mutated defB and inhibition of purified His-tagged PDF enzymes by actinonin
S. pneumoniae strain Expressed defB MIC (μg/ml) Enzyme IC50 (nM) VSPN7035 defB 16 Wild type 53 ± 6 VSPN7036 defB (Q172K) 64 Q172K 70 ± 8 VSPN7037 defB(A123D) ≥64 A123D 136 ± 16
