Cloning, mapping, and expression of the fumarase gene of Escherichia coli K-12.
Two classes of fumarase-transducing phages, lambda fumA and lambda fumB, were isolated from populations of recombinant phages containing HindIII fragments of Escherichia coli DNA; they were isolated by virtue of their ability to complement the metabolic lesion of a fumarase-negative mutant. The strongly complementing lambda fumA phages contained a 6.2-kilobase HindIII fragment encoding: the fumA gene, located at 35.5 min in the E. coli linkage map and expressing the major fumarase activity; the mannosephosphate isomerase gene, manA; and an unidentified gene, g48. The three genes were located relative to the restriction map of the cloned fragment and the genetic linkage map (terC-g48-fumA-manA-uidAoR), their transcription polarities were defined as anticlockwise in the chromosome, and the molecular weights of the corresponding gene products were established: fumA, 61,500; manA, 42,000; g48, 48,000. Organisms containing the fumA gene sub-cloned in multicopy plasmids overproduced fumarase up to 50-fold. The weakly complementing class of transducing phages, lambda fumB, contained several genes in an 8.2-kilobase HindIII fragment, including one (fumB) that determines a minor fumarase activity. Complementation by fumB was only observed in high-copy situations such as transduction plaques and in strains containing a multicopy plasmid in which 40% of normal fumarase activity was detected. The basis for the complementation by fumB was not defined.
D010582:Bacteriophage lambda / Q000235:genetics; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005649:Fumarate Hydratase / Q000235:genetics*; D015870:Gene Expression; D005796:Genes; D005798:Genes, Bacterial; D008242:Lysogeny; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics; D010957:Plasmids; D014161:Transduction, Genetic
D004262:DNA Restriction Enzymes; D005649:Fumarate Hydratase; D008359:Mannose-6-Phosphate Isomerase