Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis.
We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants.
Mechold
U
U
Institute for Molecular Biology, Jena University, Germany.
Cashel
M
M
Steiner
K
K
Gentry
D
D
Malke
H
H
D003429:Cross Reactions; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006158:Guanosine Pentaphosphate / Q000378:metabolism*; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D011755:Pyrophosphatases / Q000187:drug effects / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*
D006159:Guanosine Tetraphosphate; D006158:Guanosine Pentaphosphate; D008345:Manganese; C027627:guanosine-3',5'-bis(diphosphate) 3'-pyrophosphatase; D011755:Pyrophosphatases; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases