Nutrient availability coordinates global patterns of bacterial gene expression as well as discrete pathway-specific induction or derepression. These complex networks ultimately involve regulation of the genomic repertoire and the participation of many regulatory mechanisms. This project focuses on the role of two analogs of GDP and GTP containing an esterified pyrophosphate on the ribose 3hydroxyl and abbreviated as (p)ppGpp collectively. Intracellular levels of (p)ppGpp increase during the stress of starving for amino acids, phosphate, nitrogen or energy sources and are accompanied by complex regulatory effects on metabolism, gene expression and cell physiology. The net result is slowing of bacterial growth, restricting energetically superfluous processes and maximizing chances of surviving the nutritional stress. In most bacteria a single bifunctional enzyme seems responsible for synthesizing and degrading (p)ppGpp whereas in E. coli and enteric relatives, one enzyme is dedicated to synthesis and another homolog is dedicated to degradation. This year we have mutationally delimited both catalytic domains of a Streptococcal bifunctional enzyme and demonstrated monofunctional catalytic activities for isolated nonoverlapping peptides. This constitutes proof of separate catalytic sites for synthesis and for degradation. We have also demonstrated the existence of distinct, multiple regulatory domains in the noncatalytic C-terminal half of RelA, a monofunctional (p)ppGpp synthetic enzyme. Finally, substrate specificity differences of gene fragments encoding (p)ppGpp synthetic activity have been matched with engineered changes in expression of an enzyme capable of converting pppGpp to ppGpp to allow nearly exclusive production of one or the other isomer in otherwise growing cells. Titrations of growth limiting effects of the two nucleotides surprisingly reveals ppGpp to be about eight-fold more potent a regulator than pppGpp. - E. coli ppGpp pppGpp starvation RNA polymerase stringent response
