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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2019 Annual Report of the Division of Intramural Research

Global Regulation of Bacterial Gene Expression by ppGpp

Michael Cashel
  • Michael Cashel, MD, PhD, Head, Section on Molecular Regulation
  • Llorenc Fernandez-Coll, PhD, Postdoctoral Intramural Research Training Award Fellow
  • Krishma M. Tailor, PhD, Volunteer

Our continuing research goals are to understand the emerging fundamental and widespread regulatory functions in bacteria of a special class of purine ribonucleotide analogs of GTP and ATP. The only function of these analogs is regulation because their ribose 3′ and 5′ hydroxyl groups are blocked, which prevents their participation in all metabolic reactions except their hydrolysis. There are now three structural categories of these 3′–5′ blocked nucleotide analogs: mono-cyclics, di-cyclics, and polyphosphorylated ribonucleotides. We focus on the last category, present in nearly all bacteria and plant chloroplasts but not in eukaryotes. Fifty years ago, we discovered (p)ppGpp, the Escherichia coli analogs of GTP and GDP. Two years ago, with collaborators, we added (p)ppApp as new ATP and ADP analog members of this subset. All bind to E. coli RNA polymerase (RNAP), and the regulatory effects of (p)ppApp in vitro suggest that it is an antagonist to (p)ppGpp. Our overarching objective is to understand sources and functions of both analogs, abbreviated collectively as ppApp or ppGpp.

Current research efforts of many laboratories led to a consensus that (p)ppGpp functions as the first responder to virtually all sources of physiological stress in bacteria, ranging from nutritional and physical to metabolic stress. Cellular defenses through (p)ppGpp serve to ensure bacterial adaptation and survival, including survival against host efforts to kill pathogens. We focus on E. coli as a model enteric organism, for which much is still to be learned regarding regulation at the transcriptional level, which arises from ppGpp binding at two sites on RNA polymerase. Our past work and that of several others has defined structural and regulatory features of both sites, which, in the presence of high levels of ppGpp, lead to activation as well as inhibition of nearly one third of the genome. Currently, our first goal is to examine the effects of very modest changes in (p)ppGpp basal levels on cellular physiology. This subtle regulation arises from responses to otherwise imperceptible perturbations in amino-acid, carbon, lipid, or energy metabolism that change the balance between ppGpp synthesis and hydrolysis. Our second goal is to understand roles for ppApp, a very newly discovered analog. The evidence that pathogenicity is enhanced by (p)ppGpp is now pervasive from the behavior of ppGpp0 cells that completely lack ppGpp. The profligate misuse of antibiotics coupled with very few new drugs makes fundamental studies of ppGpp acutely relevant if we are not to enter an era in which all antibiotics are ineffective.

Global regulation of bacterial gene expression by ppGpp

We have made progress towards our first goal of understanding physiological effects of low basal ppGpp levels. As classically defined, growth rates are said to be "balanced" when they are determined by the efficacy of use of different nutrients present in excess rather than by limited abundance. This caveat allows cells to fine-tune gene expression, which contrasts starkly with starvation, when adjustments to prevent death are very different from the fine-tuning during growth. Our commentary last year reviewed markedly different effects of starvation [Reference 1]. Classically, a large range of balanced growth rates are correlated with changes in the cellular content of protein, RNA, and DNA; the slower the growth, the lower the macromolecular content. We established early on that (p)ppGpp determines rates of balanced exponential bacterial growth [Mechold U, et al. Nucleic Acids Res 2013;41:6175]. Macromolecular content of ppGpp0 strains does not vary but remains at high levels even during slow growth. Hydrolase mutants that elevate ppGpp basal levels four- or eight-fold without stress, as well as RNAP suppressor mutants whose gene expression mimic effects of ppGpp in a ppGpp0 strain, were used to establish that ppGpp not only determines growth rates but is also both necessary and sufficient to account for growth-rate changes of RNA and protein content.

Recently we submitted a manuscript describing current experiments that allow a similar conclusion to be reached regarding the effects of ppGpp on the variation in initiation rates of chromosomal DNA replication. Bacteria divide in as quickly as every 30 minutes, but it takes an hour for cells to duplicate their circular chromosome. This disparity is resolved by multiple bidirectional initiation forks from a single origin (ori) region, which occur before DNA replication is completed at a single terminator (ter) sequence. Measurements of ori/ter DNA ratios by PCR provide accurate estimates of even small differences of initiation frequencies. It is also known that ppGpp mildly inhibits DNA elongation through primase. Our genomic sequencing results reinforce ori/ter ratio differences for slow-growing ppGpp0 strains and show that elongation inhibition sites are random without affecting ori/ter. The ori/ter ratios of ppGpp–containing cells drop about three-fold when comparing fast and slow growth, while the ori/ter ratios of ppGpp0 cells remain constantly high, even when their rate of balanced growth is slow. Parallel measurements of ori/ter ratios with suppressor mutants of ppGpp hydrolase and of RNAP again led to the conclusion that ppGpp is necessary and sufficient for initiation of DNA replication.

While this work was under way, a report appeared [Kraemer JA, et al. MBio 2019;10:pii e01330] proposing that ppGpp inhibition of ribosomal RNA operon (rrn) transcription from seven operons, comprising more than half of genomic transcripts, resulted in transmission of topological changes to the ori region that inhibit initiation. We tested this hypothesis with a strain deleted for all seven chromosomal rrn operons. The strain remains viable because two high-copy plasmids carry either a single rrnB operon or several essential tRNA genes with the rrn deletions. Thus, topological changes resulting from ppGpp inhibition of chromosomal rrn transcription are not possible. Inhibition of ori/ter ratios at slow growth rates persists in this strain as well as in its ppGpp0 derivative, a prediction not sustained by the hypothesis. A direct inhibitory mechanism is suggested by PCR showing three-fold more gyrase transcripts in wild-type but not in ppGpp0 cells. A manuscript describing this work manuscript has been submitted.

Our second goal is to characterize ppApp. In collaboration with the lab of Katarzyna Potrykus, we have now established the biological and structural relevance of ppApp binding to RNA polymerase. Bioinformatics identified a gene in Methylobacterium extorquens encoding SAHMex, a small ortholog of the MESH-1 eukaryotic ppGpp hydrolase discovered by Katarzyna Potrykus in our lab, which also degrades ppApp. The ppGpp/ppApp hydrolase activities of the small SAHMex protein were confirmed in vitro. This led us to seek the source of ppApp in this organism. It is a large RSHMex enzyme commonly associated with ppGpp synthesis in all Gram-positive bacteria. The pure enzyme synthesizes ppApp and ppGpp in vitro, providing the first rigorous biochemical proof that ppApp is the catalytic product of this enzyme in bacteria. Induced ectopic expression of RSHMex in E. coli led to appreciable (p)ppApp formation, providing evidence of its regulatory activity. Surprisingly, E. coli controls without ectopic RSHMex protein also revealed traces of (p)ppApp, constituting the first observation of (p)ppApp in E. coli. [Reference 2]. Searches are under way in E. coli for physiological conditions provoking (p)ppApp to determine whether cellular (p)ppApp might function to antagonize (p)ppGpp regulation, as suggested in vitro. Interestingly, not all RSH enzymes synthesize or hydrolyze (p)ppApp. Synthesis and hydrolase activities of a streptococcal RSHSeq are limited to ppGpp but not to ppApp. A future goal will be to exploit the RSHMex and RSHSeq proteins to achieve preferential accumulation of either ppApp or pppApp in order to assign specific functions for each, as we did for ppGpp and pppGpp [Mechold U, et al. Nucleic Acids Res 2013;41:6175]. It is noteworthy that the RNAP binding sites for (p)ppApp are found in both Gram-positive and -negative bacteria, whereas direct (p)ppGpp–regulatory RNAP interactions are absent from Gram-positives, suggesting that another level of diversity exists for roughly half the bacterial kingdom.

Advances this year build on past work. Work described for the first goal, resulting in a very recent submission, has been under way for more than two years and as noted, was influenced by Katarzyna Potrykus's studies as a postdoctoral fellow in this lab [Mechold U, et al. Nucleic Acids Res 2013;41:6175] and well as by our recent peer-reviewed commentary on starvation for sources of carbon, nitrogen, and phosphate [Reference 1]. Collaborative work on the second goal also originated from another postdoctoral discovery by Katarzyna Potrykus, namely that MESH-1 hydrolase cleaves both ppGpp and ppApp. Our collaborative work on this topic was published earlier [Reference 5]. Similarly, our characterization of adaptive growth on different sugars in last year's report [Reference 6] included development of an innovative high-throughput ppGpp isotope assay method, which was published as a video this year [Reference 4]. Finally, Llorenc Fernández-Coll's experience with RNAP secondary channel-binding proteins led to a peer-reviewed commentary this year on oxidation of zinc finger cysteines that inactivate DksA, a RNAP–binding protein co-factor for ppGpp regulation [Reference 3].


  1. Potrykus K, Cashel M. Growth at the best and worst of times. Nat Microbiol 2018;3:862-863.
  2. Sobala M, Bruhn-Olszewska B, Cashel M, Potrykus K. Methylobacterium extorquens RSH enzyme synthesizes (p)ppGpp and pppApp in vitro and in vivo, and leads to discovery of pppApp synthesis in Escherichia coli. Front Microbiol 2019;10:859.
  3. Fernández-Coll L, Potrykus K, Cashel M. Puzzling conformational changes affecting proteins binding to the RNA polymerase. Proc Natl Acad Sci USA 2018;115:12550-12552.
  4. Fernandez-Coll L, Cashel M. Using microtiter dish radiolabeling for multiple In vivo measurements of Escherichia coli (p)ppGpp followed by thin layer chromatography. J Vis Exp 2019;148:doi: 10.3791/59595.
  5. Bruhn-Olszewska B, Molodtsov V, Sobala M, Dylewski M, Murakami KS, Cashel M, Potrykus K. Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro. Biochim Biophys Acta Gene Regul Mech 2018;1861(8):731-742.
  6. Fernández-Coll L, Cashel M. Contributions of SpoT hydrolase, SpoT synthetase, and RelA synthetase to carbon source diauxic growth transitions in Escherichia coli. Front Microbiol 2018;9:1802.


  • Bozena Bruhn-Olszewska, PhD, University of Gdansk, Gdansk, Poland
  • Katarzyna Potrykus, PhD, University of Gdansk, Gdansk, Poland
  • Michal Sobala, PhD, University of Gdansk, Gdansk Poland


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