Characterization of the relA/spoT gene from Bacillus stearothermophilus

By use of degenerate primers, we amplified a fragment of a relAlspoT homolo gous gene from Bacillus stearothermophilus. Chromosomal walking enabled us to sequence the entire gene and its flanking regions. The primary sequence of the gene product is 78% identical to the RelA/SpoT homologue of Bacillus subtilis, and both gene loci share a similar genetic organization. The B. stearothermophilus rel gene was analyzed in vivo by heterologous expression in the B. subtilis relA deletion strain TW30, and is shown to complement t he growth defects of TW30. The recombinant Rel(Bst) protein was detected by Western immunoanalysis, and synthesizes guanosine-3'-diphosphate-5'-(tri)d iphosphate ((p)ppGpp) after amino acid stress and carbon starvation. These in vivo data, the genetic organization, and the primary structure compared to other RelA/SpoT homologues provide circumstantial evidence that the iden tified gene encodes the only (p)ppGpp synthetase in B. stearothermophilus, presumed to serve also as (p)ppGpp hydrolase. (C) 2000 Federation of Europe an Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.