The overall purpose of this project is to understand cellular mechanisms which coordinate the gene expression during transitions between balanced growth and nutritional impoverishment. We are particularly interested in the regulatory role played by the nucleotide, guanosine 3',5' bispyrophosphate (ppGpp) as an intracellular signal to control these processes in E. coli. We have dissected the genetic elements controlling ppGpp synthesis (the relA gene) and ppGpp degradation (the spoT gene). Each gene in plasmids was localized, sequenced, precisely deleted, and replaced by an antibiotic resistance gene. These null alleles were placed in the chromosome by manipulations involving crossing the modified genes into a phage lambda, preparing and curing phage lysogens to allow quantitation of recombinational replacement. The results are both surprising and complex. Two routes of ppGpp synthesis have been proven. One route was previously known to be associated with the RelA protein operating on a ribosome paused on a mRNA codon for lack of an available aminoacylated tRNA. When a relA deletion eliminated this route, a second route of synthesis persisted that responds only to energy source deprivation. Unexpectedly, the second route of ppGpp synthesis was abolished when the spoT gene was deleted, along with the relA gene, yielding the first prokaryotic cell devoid of detectible ppGpp. Thus, the spoT gene is either bifunctional and also encodes a ppGpp synthetic function or represses a second (still unidentified) synthetic gene. We have reasons to believe that a bifunctional protein is more likely. Cells lacking ppGpp give clues to the functions of ppGpp. Such cells are found to be vulnerable to physiological stress; normally innocuous transient deprivations for either energy sources or amino acids become lethal as does thermal shock. In contrast, transitions from nutritionally poor to enriched conditions occur normally. Surprisingly, these cells also have cell division defects as well growth requirements for as many as 15 of the 20 amino acids. Single step revertants that grow in the absence of amino acids have been localized to the RNA polymerase beta subunit (rpoB), showing that the auxotrophic phenotype can be overcome by modification of transcription and that RNA polymerase is a likely target of ppGpp regulation.
