Cloning and characterization of a relA/spoT homologue from Bacillus subtilis.
A PCR-amplified DNA fragment of the relA gene from genomic Bacillus subtilis 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 (170aa) that shares high similarity to adenine 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. relA shows significant similarity to the Escherichia coli relA and spoT genes, which are responsible for the synthesis and degradation of the highly phosphorylated guanosine nucleotides (p)ppGpp, triggering the stringent response. Deletion of the relA gene generated a (p)ppGpp0 phenotype that demonstrated its essential role in the response to amino acid deprivation and resulted in impaired/lowered induction of proteins involved in stress response as well as amino acid biosynthesis, as judged by two-dimensional gel electrophoresis. The same effects of impaired induction of some sigmaB-independent proteins could also be shown in a sigB/relA double mutant, supporting the role of relA in derepression/induction of catabolic and anabolic genes during stringent response.
Wendrich
T M
TM
Fachbereich Chemie/Biochemie, Philipps-Universität Marburg, Germany.
Marahiel
M A
MA
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D006158:Guanosine Pentaphosphate / Q000378:metabolism; D008025:Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D017931:DNA Primers; D006158:Guanosine Pentaphosphate; C027627:guanosine-3',5'-bis(diphosphate) 3'-pyrophosphatase; D011755:Pyrophosphatases; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases