Figure 2.

The three-plasmid expression system for eryA genes. In each vector, the eryA gene is expressed under the control of the upstream actI promoter and actII–ORF4 gene as previously described (16, 17). To facilitate construction of the various eryA mutations, a SpeI site (ACTAGT) was introduced at nt 10366–10371 of the eryAI ORF by making D3455T and A3456S mutations (27) in pKOS025-179 (Q.X., unpublished data). This change enables insertion of the mutated gene segment between the PacI site and the SpeI site of pKOS025-179 (16, 17). After the replacement of the 6-kb fragment between the AscI sites in eryAII (nucleotides 1213 and 7290) with a 6-kb AscI fragment containing a specific AT substitution in an intermediate plasmid, the resulting PacI–XbaI fragment containing the mutant eryAII gene was inserted into pKOS025-143. All eryAIII mutants were constructed by replacing the segment in pKOS010-153 between the unique BglII site at nucleotide 251 and the EcoRI site (nucleotide 9290) that overlaps the stop codon.