The expression of the genomic repertoire of Escherichia coli is coordinately regulated in response to nutrient availability. Collectively, these responses determine growth as well as the ability to survive prolonged periods of starvation. This project focuses on regulation of these processes in eubacteria by analogs of the common nucleotides (GDP and GTP) that possess ribosyl 3' pyrophosphate esters; this family of compounds is abbreviated as (p)ppGpp and occurs only in prokaryotes. Intracellular levels of (p)ppGpp respond to availability of amino acids, phosphate, nitrogen or energy as well as other stress conditions; the ensuing (p)ppGpp signal alters global patterns of gene expression. In addition, (p)ppGpp can induce other global regulators and thereby recruit and integrate gene expression effects on broad genomic domains. This year, we studied our isolates of mutants of RNA polymerase subunits that phenotypically mimic gene expression patterns shown by the presence of (p)ppGpp in strains genetically rendered devoid of (p)ppGpp, called M+ mutants. RNA polymerase holoenzymes reconstituted with either of two mutant sigma-70 subunits have been shown defective on the galP2 promoter with respect to abortive RNA product formation, promoter clearance and stability of open complexes. A total of 61 M+ mutant alleles of core enzyme subunit genes rpoB and rpoC, including 52 of our isolates, have been screened and assigned to different functional classes with respect to phenotypes sensitive to (p)ppGpp, test promoters known to be induced or repressed by (p)ppGpp and for functions regulated by other global regulators. Eubacterial genomic sequencing has reinforced our view that the relA and spoT genes of E. coli are paralogous genes devoted to (p)ppGpp synthesis and breakdown, respectively. A single ancestral rel/spo gene encoding a protein with these presumed bifunctional activities is more widely distributed among prokaryotes, excepted only by species closely related to E. coli. We have characterized one such ancestral protein from a species of Streptococcus and localized protein domains for synthetic and degradation activities within a protease-resistant core. A second pair of E. coli gene paralogs exists that is also related to (p)ppGpp metabolism. The gppA gene encodes the major catalytic route for the conversion of pppGpp to ppGpp while the ppx gene encodes the major source of polyphosphate (polyP) degradation activity. These apparently disparate activities are related since a potent inhibitor of polyP phosphatase activity is found to be pppGpp. This goes far to explain why stress conditions leading to (p)ppGpp accumulation also provokes polyP accumulation.
