The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a Delta relA Delta spoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26 degrees C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was > 1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5x10(4) CFU of the Delta relA Delta spoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5x10(5) CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0x10(4) CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the Delta relA Delta spoT mutant strain is a promising vaccine candidate to provide protection against plague.