Article Figures & Data
Figures
- Fig. 1.
Genetic organization of the choE region and transcriptional analysis of choE. (A) Physical map of the 3.9-kb chromosomal region encompassing the choE locus ofR. equi. The location of the putative choEpromoter and stem-loop transcription terminator is indicated. (B) Northern blot analysis of choE in strains R. equiMAD (left) and 103− (right). In both strains, a single 1.9-kb transcript was detected.
- Fig. 2.
Comparison of amino acid sequences of the cholesterol oxidases ChoE from R. equi (R. eq.; accession no. AJ242746 ) and ChoA from Streptomyces spp. strain SA-COO (S. sp.; accession no. A32260 ), and related ChoD polypeptides from M. leprae (M. le.; accession no. S72824 ), M. tuberculosis(M. tu.; accession no. F70736 ), and S. coelicolor (S. co.; accession no. AL161755 ). Identical amino acids are shaded in black and similar amino acids are in gray. The putative cleavage site of the signal peptide of ChoE is indicated by an arrowhead.
- Fig. 3.
Schematic diagram of the procedure for targeted mutagenesis of choE by homologous recombination and a physical map of the choE locus in the parent strain (WT) and the recombinants (SCR-1 and SCR-2, single crossover recombinants in sites 1 and 2, respectively, and DCR, double crossover recombinant). The recombinogenic cassette in the suicide plasmid, pRHE3, includes theaacC4 apramycin resistance gene, shown in black, surrounded by the flanking choE target sequences, shown in dark gray. The wild-type choE allele is light gray. The crossover target sequences in each type of recombinant are dashed in light and dark gray. CoN-CoC and CoIN-CoP1 primer pairs were used for PCR mapping, and M indicates the position of the MluI restriction sites used in Southern blot analysis of the recombinants (Fig. 4).
- Fig. 4.
PCR mapping and Southern blot analysis ofchoE recombinants. (A) PCR mapping with CoN-CoC (upper panel) and CoIN-CoP1 (lower panel) primer pairs. See text and Fig. 3legend for details. (B) Southern blot analysis. Genomic DNA from representative mutants of each recombinational type were cut withMluI and hybridized against the CoEN-CoEC probe covering the entire choE gene. Hybridization patterns of R. equi (WT), DCRs, and the two types of SCRs are shown. In R. equi, choE is flanked by two MluI sites separated by 2.3 kb; thus, the WT displays a single 2.3-kb hybridization band. pRHE3 has a single MluI site located at the middle of theaacC4 gene (Fig. 3); therefore, DCR shows two bands of 2.6 and 1.3 kb. In SCR-1, there were two bands of 2.6 and 5.7 kb. The 2.6-kb band is equivalent to the band of the same size observed in DCR and corresponds to the left side of the aacC4-disruptedchoE allele; the 5.7-kb band includes the right side of theaacC4-disrupted choE allele, pUC19, and the intact copy of choE (Fig. 3). In SCR-2, two hybridization bands of 7.0 and 1.3 kb were observed (Fig. 3). When membranes were rehybridized with a pUC19 probe, hybridization signals were detected in SCR-1 and SCR-2, confirming the presence of the complete pRHE3 plasmid integrated at the R. equi choE locus as the result of an SCR event (data not shown).
- Fig. 5.
Cooperative hemolysis assays with the sphingomyelinase-producing indicator species L. ivanovii(horizontal streaks). (A) Wild-type R. equi103− (WT) and its isogenic derivatives RHE3-19 (choE/pRHE5 mutant) and RHE3-19 (choE mutantchoE). The choE mutant has lost the capacity to produce a shovel-shaped CAMP-like reaction and this property is recovered upon complementation with choE. (B) E. coli K-12 with the control vector pRHE2 (vector) and with thechoE-containing plasmid pRHE1 (+choE); complementation with choE confers CAMP-like activity similar to that of wild-type R. equi.
Tables
- Table 1.
Bacterial strains and plasmids
Bacterial strain or plasmid Description Source or reference R. equi ATCC 6939 Type strain 30 MAD Cholesterol oxidase-hyperproducing strain 42 103+ Clinical isolate with virulence plasmid 8 103− 103 derivative lacking virulence plasmid 8 RHE3-15+ choE knockout mutant of 103+ This work RHE3-19 choE knockout mutant of 103− This work L. ivanovii ATCC 19119 Type strain Collection E. coli DH5α Cloning host strain Our laboratory HB101 Cloning host strain Our laboratory TG1 Cloning host strain Our laboratory Plasmids pUC19 Cloning vector 54 pGEM-Te T-vector for cloning of PCR products Promega pPE207 E. coli-Mycobacterium shuttle vector containing apramycin resistance marker 37 pVK173-T E. coli-Mycobacterium shuttle vector containing hygromycin resistance marker 37 pRE7 E. coli-R. equi shuttle vector 55 pRHE1 pUC19 inserted with choE This work pRHE2 pUC19 inserted with accC4 This work pRHE3 pUC19 with choE::accC4 mutant allele (suicide vector for choE mutagenesis by gene replacement) This work pRHE4 pGEM-Te inserted withchoE and its natural promoter This work pRHE5 pVK173-T inserted with choE and its natural promoter This work pRHE6A pRE7 inserted withaccC4 This work - Table 3.
Recombination frequencies of the suicide plasmid pRHE3 (containing choE target sequences) or pRHE2 (nochoE target sequences) in R. equi
Suicide plasmid Recombination frequencya in strain: 103+ 103− pRHE3 1.9 × 10−3 7.5 × 10−3 pRHE3ss 11.5 × 10−3 47.5 × 10−3 pRHE2 <10−5 <10−6 pRHE2ss <10−5 <10−6 ↵a Recombination frequencies were calculated as the ratio between transformation frequencies (expressed as Aprr colonies per microgram of DNA) obtained with the suicide plasmids and with the pRHE6A vector, which replicates inR. equi.
