Figure 1

Western blotting and PCR analysis are consistent with the integration of an ampicillin resistance cassette into the chromosomal pgdx gene of the acatalasaemic H. influenzae strain AB2593 and the virulent hktE H. influenzae type b strain Eagan mutant. (A) Western blot autoradiogram of PGdx; the preliminary SDS-PAGE experiment was conducted on samples of crude extracts derived from the following cultures; lane 1, E. coli, overexpressing PGdx (0.1 μg of total protein); lane 2, hktE^- pgdx^- H. influenzae Rd (1 μg of total protein); lane 3; hktE^- pgdx^- H. influenzae Rd (50 μg of total protein); lane 4, hktE^- pgdx^- H. influenzae type b strain Eagan (1 μg of total protein); lane 5, hktE^- pgdx^- H. influenzae type b strain Eagan (50 μg of total protein); lane 6, wild-type H. influenzae Rd (1 μg of total protein). Monomeric PGdx has a polypeptide mass of 26.6 kDa. Note that the recombinant PGdx protein oxidizes under aerobic conditions to form a disulfide bonded dimer of about 53 kDa. (B) PCR analysis of the pgdx gene applied on chromosomal DNA from the following strains; lane 1, wild-type H. influenzae Rd; lane 2, hktE^- pgdx^- H. influenzae Rd; lane 3, hktE^- pgdx^- H. influenzae type b strain Eagan. The pgdx amplicon of 1,071 bp is enlarged by 1,021 bp due to the insertion of the ampicillin resistance cassette.