Characterization of a new regulator, pGpp. The old literature provides fleeting observations of unusual (p)ppGpp-like nucleotides found in extracts of diverse bacteria when subjected to various sources of stress, such as pGpp, ppGp, ppApp, pppAp and A5p(pn)p5Gpp. The Gaca et al. publication reports that purified RelQ protein from Enterococcus faecalis (RelQEF) catalyzes pyrophosphate transfer from ATP to the ribosyl-3hydroxyl of GMP without need of additional supplements shown by RSH enzymes (ribosomes with an empty A site, codon-specified uncharged tRNA. This observation argues against the possibility that pGpp is a degradation product of (p)ppGpp formed by a nudix-like enzyme. It is notable that the cellular stability of pGpp, ppGpp or pppGpp has not been reported in firmicutes so there is a formal possibility that pGpp is metabolically stable, unlike ppGpp or pppGpp in other organisms studied. The kinetic rate constants for pGpp synthesis reveal RelQEF enzymatic efficiencies (Kcat/Km) within a factor of two of those for forming ppGpp or pppGpp, yet far RelQEF activities are far below those typical for RelEF, the RSH protein of E. faecalis. Both chemical and enzymatic labilities of pure pGpp (by RelEF) are similar to those of (p)ppGpp. A comparative survey was made comparing several regulatory activities known for ppGpp and pppGpp to those of pGpp including 6-hydroxypurine transport, GMP kinase, transcription initiation from an E. coli rRNA promoter and regulatory effects on ppGpp synthetases. Generally pGpp regulatory activity was found to exist as quantitatively different from ppGpp and pppGpp but unique qualitative regulatory differences were not found. A central question that remains unanswered is whether an unusual nucleotide such as pGpp has unique regulatory functions that might explain its continued existence. A rational approach to understand the structural basis of (p)ppNpp substrate specificity differences. We have attempted to construct enzymes with dramatically different ribosyl-3pyrophosphohydrolase specific activities to exploit for studies of novel nucleotide behavior. A designer mutant approach was taken because protein structures are available for two hydrolases, one G-specific and one with degenerate specificity for A- or G-nucleotides. Dr. K. Potrykus while in our laboratory verified the observations that the Mesh hydrolase is able to hydrolyze (p)ppGpp and discovered it could also hydrolyze (p)ppApp. Salient structural differences were visualized by Dr.Tamara James and mutants were predicted that might restrict the specificity of Mesh to hydrolyze only (p)ppApp or (p)ppGpp. A set of 9 mutant proteins were expressed, purified and catalytic properties assessed and their effects on catalytic properties used to generate 3 more mutants. The measurements of enzyme kinetic constants for hydrolysis were performed by Nathan Thomas. Several mutants were found that lowered the efficiency of catalysis (kcat /km) about ten-fold relative to wild type Mesh but equally for both substrates. Two mutants were found to have similar lowered efficiency for one substrate but a modest three-fold higher efficiency for (p)ppGpp in one case or a five-fold higher efficiency for (p)ppApp in the other. No mutants were found with a sufficiently altered substrate preference to be useful for our further studies. Dr. K. Potrykus, our collaborator in Gdansk, Poland, is pursuing these studies exploring naturally occurring bacterial enzymes with sequences that are more closely related to MESH sequences than to those of RSH hydrolase domains. A functional interaction network exists between ppGpp regulation, chaperones, the omega subunit of RNA polymerase and oxidative stress during rapid growth in rich media. Last year we described synthetic lethal phenotypes for different combinations of deletion alleles: 1) the ppGpp synthetase gene (relA); 2) chaperones (dnaK, dnaJ, tig, clpB, and rpoZ- the RNA polymerase omega subunit). Functional interactions are deduced if the phenotypes for combinations are more severe than the simple sum of the same deletions present singly. In this manner it is observed that relA + dnaK or dnaJ enhance temperature sensitivity, ie show conditional synthetic lethality, which reveals one deletion functionally exacerbates the deficiency of the other. Since dnaK phenotypes are suppressed by DksA, a partner for ppGpp regulation, this is a plausible relation. Similar interactions can be noticed that link triple deletions (relA and omega with either tig or clpB) or (relA, omega and groEsEl) further strengthening the links between ppGpp, chaperones and the omega portion of the ppGpp binding site of RNA polymerase. This work has been extended to include complementation assays, showing that transcription factor proteins (GreA and GreB) are able to complement the temperature sensitivity of a dnaK deletion. This is significant because GreA and GreB have superimposable protein structure with DksA mentioned earlier, which acts both for ppGpp regulation and complementation of dnaK. Importantly, missense mutants of GreA GreB or DksA abolish their classical activities in transcription but do not abolish their ability to complement the defective chaperone phenotype of dnaK. This suggests transcription factors might have chaperone activity as a moonlight function. The dksA mutant also complements the synthetic lethal triple mutant (relA, omega and tig), which seems to close the loop to include probably all secondary channel factors in the same functional network. We have preliminary findings that by screening single gene libraries leads to finding gene functions that complement the relA-dnaK or relA-dnaJ synthetic lethality and therefore warrant further exploration. Not only are additional complementing genes found whose functions relate to ppGpp regulation, chaperones and transcription but also somewhat surprisingly genes were found that mediate responses to oxidative stress, such as oxyR and catalases. Some catalases we previously found to be regulated by ppGpp indirectly through its activation of the RpoS alternative sigma factor. The interconnections of these functions can be viewed as physiologically plausible, since ppGpp functions to preclude damage from stress by adjustments to circumvent stress while chaperone activities renature proteins denatured by stress conditions. However these observations of synthetic lethality are made with cells growing rapidly in rich LB media at nonstressful physiological temperatures (32-37o C). While this is not anticipated to be a stressful growth condition, nevertheless these functions appear to prevail as an extensive functional network. This behavior may suggest that a balance between these activities occurs not only during stress but also during normal growth. It is possible the network may even be necessary for normal growth. A few years ago we have published evidence that we interpret as showing ppGpp is necessary and sufficient to control growth rate in rich as well as poor minimal media. Classically, this feature is usually interpreted in terms of ppGpp regulation of ribosome synthesis but our current work suggests other factors may well be involved.