THE RELA SPOT-HOMOLOGOUS GENE IN STREPTOMYCES-COELICOLOR ENCODES BOTHRIBOSOME-DEPENDENT (P)PPGPP-SYNTHESIZING AND (P)PPGPP-DEGRADING ACTIVITIES/

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to t he RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp met abolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribos ome associated, and its activity in vitro is ribosome dependent. Analy sis in vivo of the active recombinant protein in well-defined Escheric hia coli relA and reLA/spoT mutants provides evidence that S, coelicol or Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-ind ependent (p)ppGpp formation, in the same manner as the E. coli truncat ed RelA protein (1 to 455 amino acids). An E. coli relA spoT double de letion mutant transformed with S. coelicolor rel gene suppresses the p henotype associated with (p)ppGpp deficiency. However, in such a strai n, a rel-mediated (p)ppGpp response apparently occurs after glucose de pletion, but only in the absence of amino acids. Analysis of ppGpp dec ay in E. coli expressing the S. coelicolor rel gene suggests that it a lso encodes a (p)ppGpp-degrading activity. By deletion analysis, the c atalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degra dation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coel icolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nuc leotides in antibiotic biosynthesis.