CLONING AND CHARACTERIZATION OF A RELA SPOT HOMOLOG FROM BACILLUS-SUBTILIS/

A PCR-amplified DNA fragment of the relA gene from genomic Bacillus su btilis DNA was used to isolate the entire relA/spoT homologue and two adjacent open reading frames (ORFs) from a lambda ZAP Express library. The relA gene, which encodes a protein of 734 amino acid residues (aa ), is flanked by an ORF (170 aa) that shares high similarity to adenin e phosphoribosyltransferase genes (apt), and downstream by an ORF (131 aa) of unknown function. This genetic organization is similar to that in Streptomyces coelicolor A3(2) and Streptococcus equismilis H46A. r elA shows significant similarity to the Escherichia coli relA and, spo T genes, which are responsible for the synthesis and degradation of th e highly phosphorylated guanosine nucleotides (p)ppGpp, triggering the stringent response, Deletion of the relA gene generated a (p)ppGpp(0) phenotype that demonstrated its essential role in the response to ami no acid deprivation and resulted in impaired/lowered induction of prot eins involved in stress response as well as amino acid biosynthesis, a s judged by two-dimensional gel electrophoresis. The same effects of i mpaired induction of some sigma(B)-independent proteins could also be shown in a sigB/relA double mutant, supporting the role of relA in der epression/induction of catabolic and anabolic genes during stringent r esponse.