FIG. 4.

Construction of PDO354 with mucD217 expressing MucD-S217A with a defect in the conserved protease motif in single copy from the chromosome. Oligonucleotide mutagenesis as described previously (20) was used with pLW1 (pUC19 with “mucB mucC mucD lep” [2.5 kb]) to form pLW63 with the mucD217 allele. Suicide plasmid pLW63 was transferred to PDO350 (ΔmucD::aacCIΩ, mucoid), with selection for carbenicillin (bla) resistance to mediate integration into the chromosome by homologous recombination. The resulting strain, PDO354, was verified by PCR for correct insertion in the chromosomal operon, and production of MucD-S217A was confirmed by Western analysis.