Cloning the spoT gene of Escherichia coli: identification of the spoT gene product.
We have isolated five specialized transducing lambda bacteriophages (lambda dpyrE spoT) carrying the pyrE and spoT genes of Escherichia coli. A fragment from one of these phages was used as the source of DNA to clone the spoT and pyrE genes on a multicopy plasmid, pBR322. Insertions and deletions in this plasmid were obtained. These plasmids were used to transform a minicell-producing strain, and the gene products synthesized were determined. Our experiments demonstrate that the spoT and pyrE genes are separated by about 4 magadaltons and suggest that the spoT gene product is a protein whose molecular weight is 80,000. The strain in which the spoT+ allele is carried on a plasmid produced nine times more spoT gene activity than a normal spoT+ strain when assayed in crude extracts. This strain was used to prepare partially purified gene product, guanosine 5'-diphosphate, 3'-diphosphate pyrophosphatase. The enzyme has the following characteristics. (i) It hydrolyzes pyrophosphate from the 5'-pyrophosphate of guanosine 5'-diphosphate, 3'-diphosphate, yielding GDP and pyrophosphate. (ii) Its activity is strongly stimulated by Mn2+ and slightly stimulated by salt. (iii) Its activity is inhibited by uncharged tRNA. There are also two additional activities in the cell extract which degrade guanosine in 5'-diphosphate, 3'-diphosphate in vitro but which are not specified by the spoT gene.
D000483:Alleles; D003090:Coliphages / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D010957:Plasmids; D011755:Pyrophosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014161:Transduction, Genetic
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D006159:Guanosine Tetraphosphate; D011755:Pyrophosphatases