FUNCTIONAL-ANALYSIS OF A RELA SPOT GENE HOMOLOG FROM STREPTOCOCCUS-EQUISIMILIS/

We examined the functional attributes of a gene encountered by sequenc ing the streptokinase gene region of Streptococcus equisimilis H46A. T his gene, originally called rel, here termed rel(S.equisimilis), is ho mologous to two related Escherichia coli genes, spoT and relA, that fu nction in the metabolism of guanosine 5',3' polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the h ighly expressed rel(S.equisimilis) gene encodes a strong (p)ppGppase a nd a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absen ce of the relA gene. We verified that the Rel(S.equisimilis) protein, purified from an E. coli relA spoT double mutant, catalyzed a manganes e-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel(S.equisimilis) protein preparation also w eakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was res tricted by the absence of manganese ions. An analogous activity has be en deduced for the SpoT protein from genetic evidence. In addition, th e Rel(S.equisimilis) protein displays immunological cross-reactivity w ith polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel(S.equisimilis) gene function in E. coli as being sim ilar to that of the native spoT gene, disruptions of rel(S.equisimilis ) in S. equisimilis abolish the parental (p)ppGpp accumulation respons e to amino acid starvation in a manner expected for relA mutants rathe r than spoT mutants.