Cloning and characterization of a bifunctional RelA SpoT homologue from Mycobacterium tuberculosis

A 2.2 kb relA/spoT homologue was isolated from Mycobacterium tuberculosis (Mtb) genomic DNA by PCR-amplification. The Mtb gene encodes a protein of 738 amino acid residues, and is flanked upstream by an ORF that is highly similar to the apt gene, and downstream by an ORF that is highly similar to the cypH gene. This dual function Mtb homologue belongs to the relA/spor family of genes that mediate the stringent response by regulating the synthesis and degradation of guanosine 3',5'-bis (diphosphate) (pPGPP) and pppGpp. In vitro biochemical data indicate that purified Rel(Mtb) is a ribosome- and tRNA-independent ATP:GTP/GDP/ITP 3'-pyrophosphoryltransferase. Additionally, purified Rel(Mtb) is an Mn2+-dependent, ribosome and tRNA-independent, (p)ppGpp 3'-pyrophosphohydrolase. These reactions were also assessed in vivo in E. coli deleted in both the relA and spoT genes, which generates a (p)ppGpp(0) phenotype. Rel(Mtb) can suppress this phenotype and can generate more (p)ppGpp than relA in the wild type E, coli control. (C) 1999 Published by Elsevier Science B.V. All rights reserved.