This project continues to involve attempting to understand how changes in gene expression are coordinated when cell growth is limited by nutrient availability. Escherichia coli has been studied with specific focus on amino acid and energy source availability as signals triggering cellular accumulation of analogs of GDP and GTP that bear 3'-pyrophosphate residues, abbreviated as ppGpp and pppGpp, respectively, or (p)ppGpp collectively. Estimates of regulatory effects of (p)ppGpp on gene expression and cell physiology gained from either the ability to manipulate (p)ppGpp concentrations at will or constructing mutant strains lacking the ability to form (p)ppGpp. This year we have learned that our interests must be extended to include stationary phase and polyphosphate metabolism. A monoclonal antibody, obtained from the Burgess laboratory, has been used to demonstrate that (p)ppGpp activates accumulation of a stationary phase- specific sigma (sigma-S) factor encoded by the rpoS gene. A strong dependence on (p)ppGpp was shown when growth was limited for either amino acids, glucose, phosphate, or when complex media was exhausted. Conversely, induction of (p)ppGpp, without nutrient limitation, rapidly induced sigma-S levels at least 50-fold. Since many stationary phase- specific genes show expression dependent upon a functional rpoS gene, our finding implies that (p)ppGpp accumulation can also indirectly activate these genes. This year's project work has also led to discovering that the """"""""housekeeping"""""""" sigma-70 (encoded by rpoD) is less stably associated with core RNA polymerase (subunits encoded by rpoA-C) in the absence of (p)ppGpp. This property is reversed by rpoD mutants selected as suppressors of phenotypic features of (p)ppGpp-deficiency. Mutant behavior hints that (p)ppGpp might alter global transcription by affecting the competition between core and alternative sigma factors. This notion is strengthened by localization of the rpoD mutational lesions within the same conserved domain as found for heat shock sigma-32 mutants to affect sigma-core association in C. Gross's laboratory. A relation between enzymes affecting pppGpp metabolism and polyphosphate metabolism noticed in A. Kornberg's laboratory has been verified from in vivo experiments.
