Molecular biology and enzymology of the hemD locus of Escherichia coli K-12

A genetically engineered E.coli strain, ST10-46, has been shown to carry the hemD locus, encoding uroporphyrinogen III synthase (cosynthase) using biochemical and genetic techniques. The strain was produced originally by subcloning a 2.89kb Sau3A fragment from E.coli K-12 chromosomal DNA, maintained within the Carbon and Clarke plasmid pLC41-4, into the BamHI site of pBR322. Direct assay of sonicated extracts of strain ST1046 showed the level of uroporphyrinogen III synthase activity to be 200 times higher than that of the wild type control. The region of DNA mapping the hemD structural gene was delineated as being closely linked to hemC. Subsequently the hemD gene was cloned into phage M13 derived vectors from which the nucleotide sequence was determined using the DNA chain termination sequencing method of Sanger et al. (1977). An open reading frame (ORF) of 738 nucleotides, which could code for a protein of molecular weight 27,333 daltons, was identified for the hemD gene. The hemD initiation codon ATG overlapped with the stop codon TGA of hemC. Purification to homogeneity, molecular weight determination and N-terminal amino acid sequencing of E.coli uroporphyrinogen III synthase from the overproducing strain, ST1046, provided conclusive evidence that the sequenced ORF was indeed the hemD gene. Subsequently, the hemD locus was subcloned into several other expression vectors and the levels of uroporphyrinogen III synthase were determined. One of the strains generated, BM2, in which the recombinant plasmid, a derivative of the pUC plasmid vector, carried the hemD gene in a 1.75kb XhoII-PstI DNA fragment, showed a level of synthase activity 600 times that of the wild type control. Two ORF's, encoding two unidentified proteins, were detected at the 3' flank of hemD and were sequenced. They consisted of 1179bp (hem3) and 1194bp (hem4) respectively. It seems that hemD and the two ORFs at its 3' flank may also be under the control of the putative hemC promoter. Data presented in this thesis have permitted the relative position of cvaA, hemC and hemD and the two ORFs on the E. coli linkage map to be defined.