Cloning of fragments of lambda dapB2 DNA and identification of the dapB gene product.
DNA of the specialized transducing phage lambda dapB2 has been digested with the restriction endonucleases Bam I, HindIII, or both together to generate fragments originating from the bacterial substitution on the phage. Seven such fragments ranging in size from 0.8 to 7.1 kilobases and encompassing the entire bacterial substitution of 18 kilobases of DNA have been covalently ligated to the vector pBR322. The recombinant plasmids so constructed have been tested for their ability to complement the dapB17 allele in Escherichia coli strain AT999. Only pGM4, which contains a 7.1 kilboase fragment generated by Bam I cleavage of lambdadapB2 inserted into pBR322, relieves this strain's requirement for DL-diaminopimelic acid and restores dihyrodipicolinic acid reductase activity to wild type levels. Deletions were obtained in pGM4 by two methods. None of the resultant shortened plasmids were proficient in complementation of the dapB17 allele. The proteins encoded by the parental plasmid and by those of seven deletions derived from it have been identified by coupled transcription and translation of plasmid DNA templates in vitro or by the stimulation of protein synthesis promoted by these plasmids in an ultraviolet irradiated host. The parent encodes four proteins unique to the 7.1 kilobase insert whose apparent molecular weights are 48,000, 36,000, 32,000, and 17,000. Of these, the protein of 32,000 is consistently missing when noncomplementing pasmids harboring deletions are used as templates. This protein is tentatively identified as the product of the dapB gene. The role of the other three proteins whose genes are closey linked to the dapB gene is unknown. There appear to be at least two transcriptional units within this cluster of genes, however, suggesting independent regulation and, possibly, function.
D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics* / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics*; D050746:Dihydrodipicolinate Reductase; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D005796:Genes; D010088:Oxidoreductases / Q000096:biosynthesis; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010848:Picolinic Acids / Q000096:biosynthesis; D010957:Plasmids; D014161:Transduction, Genetic; D014466:Ultraviolet Rays
D004274:DNA, Recombinant; D004279:DNA, Viral; D010848:Picolinic Acids; D010088:Oxidoreductases; D050746:Dihydrodipicolinate Reductase; D044925:Oxidoreductases Acting on CH-CH Group Donors; D004262:DNA Restriction Enzymes