Our published work in the Section on Molecular Regulation reflects broad interests in molecular approaches to bacterial genetics, biochemistry and physiology. Emphasis is on regulation of stress responses mediated by nucleotide regulators (p)ppGpp and (p)ppApp. We especially like to use bacteria and bacteriophage to ask research questions because experimental outcomes are apparent in a day or two, not weeks or months. This allows easy hypothesis testing and rapid progress in attempts to unravel fundamental biological mechanisms. It has led to dissecting out mechanisms that lead to accumulation of the ppGpp due to nutrient limited or to biological damage from a variety of physical stresses. The resulting accumulation of ppGpp becomes a signal that then leads to adjustments of global gene expression that lead to curtailing rapidly growing cellular synthetic processes that are wasteful in stress damaged slow or nongrowing cells. At the same time, global ppGpp adjustments preserve a small fraction of residual synthetic capacity that allows adapting to specific sources of stress and survival. Examples for nutrient limitation are to induce motility to find new nutrients, induce transport processes to get them into cells and induce metabolic processes to consume them. Overall, we are attempting to understand how ppGpp achieves an exquisite balance of these processes in a manner that tailors the most efficient specific response to each of many sources of stress. This understanding has turned out to be of fundamental importance for microbial pathogenesis, carrier states, antibiotic function, microbiome interrelationships and for therapy. 

Our publications during the past ten years reflect broad interests. They include discoveries that ppGpp is the signal that links bacterial growth rate to the cellular ability to use available nutrients with maximal efficiency and documenting that these effects are exerted at several levels. One is regulation of transcription by RNA polymerase to limit synthesis of ribosomes as well as selective effects on gene expression to induce synthesis of specific enzymes that ensure survival. Genetic manipulation of biochemical pathways for ppGpp synthesis and degradation without stress allow us to achieve incremental levels of either ppGpp or pppGpp during growth without stress. This trick allows us to evaluate concentration-dependent functions of each regulator, completely independent of stress. Accumulation of each nucleotide is sufficient to inhibit growth even in rich media as if it were poor; also ppGpp is a more potent inhibitor than pppGpp. Conversely, genetic constructs made to completely eliminate (p)ppGpp fail to make the normal compensatory adjustments to slow growth, even on nutrient poor media, as if the presence of (p)ppGpp is not only sufficient but also necessary to control growth. These studies go far to establish that (p)ppGpp is the key regulatory link between growth rate and medium composition. Several decades of work in many labs has led to understanding of how ppGpp regulates transcription by binding to RNA polymerase at two sites. Our current and past work with collaborators has contributed to a structural and functional understanding of both sites. We also have evidence that ppGpp regulates initiation of chromosomal DNA synthesis by criteria similar to those described for controlling growth. A very different topic involving ppGpp comes from finding, again with collaborators, that ppGpp is needed to efficiently repair DNA damage due to attachment of bulky chemicals or ultraviolet irradiation. Both events stop transcription in its tracks. We have recently found that ppGpp is involved in in post-translational modification of proteins by acetylation and the changeover from cells exhausting glucose as a carbon-energy source to use of other sugars. Current collaborative studies have led to the discovery that (p)ppApp, the adenine analog of (p)ppGpp, can coexist in cells with (p)ppGpp. Suggestive biochemical experiments prompt us to now excitedly pursue the possibility that (p)ppApp provides regulatory functions that might balance those of (p)ppGpp.

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