FIG. 2.

Construction and restriction map of plasmids used in this study. A 1-kb DNA fragment containing the crh1 allele and part of yvcN was cloned between the BamHI and EcoRI sites of pHT315 (1) to give plasmid pRC23. A 1.5-kb ClaI DNA fragment containing the kanamycin resistance gene aphA3 was inserted in yvcN at the unique BstBI restriction site, providing plasmid pRC33. Plasmid pRC35 was constructed as follows. An EcoRI-ClaI and a ClaI-BamHI fragment, corresponding to the 5′ and 3′ ends of hprK, respectively, were amplified by PCR. These two fragments were cloned into the integrative vector pJH101 digested with EcoRI and BamHI, thus reconstituting an hprK gene deleted from codons 26 to 232. pRC37 was obtained by insertion of the 1.5-kb kanamycin cassette into the newly created ClaI site of hprK.