Organization and subcloning of the dacA-rodA-pbpA cluster of cell shape genes in Escherichia coli

Abstract

The transducing bacteriophage lambda pBS10 carries a small cluster of Escherichia coli penicillin-binding protein/cell shape genes, including pbpA, rodA, and dacA. Deletion mapping and subcloning showed that these genes, and the gene for a cytoplasmic membrane protein of molecular weight 54,000, are located within a 5.6-kilobase region and are probably contiguous. The dacA gene, which codes for penicillin-binding protein 5, was cloned on a 1.5-kilobase fragment into a low-copy-number plasmid vector, but insertion into high-copy-number plasmids produced deleterious effects on bacterial growth, and the plasmids could not be stably maintained. The direction of transcription of dacA was determined. The rodA gene was cloned on a 1.6-kilobase fragment into both low- and high-copy-number plasmids, and the identification of its gene product is described in the accompanying paper (Stoker et al., J. Bacteriol. 155:854-859). The pbpA gene, which codes for penicillin-binding protein 2, was cloned on a 3.7-kilobase fragment in low-copy-number plasmids, but insertion of the fragment into high-copy-number plasmids resulted in deleterious effects on bacterial growth, and the plasmids could not be stably maintained.