Nonsense and insertion mutants in the relA gene of E. coli: Cloning relA

Abstract We have made use of lysogens of a specialized transducing bacteriophage, λ pyr G + relA + , to select nonsense ( relA non ) and insertion ( relA ins ) mutations in the relA gene. Three independent relA non mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup 6. Three independent relA ins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet light-killed host cells revealed that no stringent factor was coded for by either the relA ins or relA non phages (the latter in a sup + cell); stringent factor was detected when the relA non phages were used in a similar experiment with supD or supE host cells. The relA non and relA ins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.