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.