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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2020 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, Visiting Fellow

Our research goal is to understand the emerging fundamental regulatory functions in bacteria of the (p)ppGpp (guanosine pentaphosphate or tetraphosphate) alarmone, which is produced by RSH enzymes) and a new candidate alarmone, (p)ppApp (adenosine pentaphosphate or tetraphosphate), which are analogs of GTP, GDP, ATP and ADP. The (p)ppGpp pair are present in nearly all bacteria and many plant chloroplasts but, so far, have not been found in eukaryotes. The distribution of (p)ppApp is broad, but the details of its kinetic constants are uncertain. All appear to bind to E. coli RNA polymerase (RNAP), with (p)ppApp binding at a site opposite to the catalytic center of (p)ppGpp. Regulatory effects of (p)ppApp in E.coli in vitro suggest that the alarmone functions as an antagonist to (p)ppGpp. Our overarching objective is to learn, for both analogs, their enzymatic sources, functions, and whether there is regulatory cross talk. Current research efforts of many laboratories led to a consensus that (p)ppGpp functions as a first responder to most sources of physiological stress in bacteria, ranging from nutritional and physical to metabolic stress. Cellular defenses triggered by ppGpp operate to ensure bacterial adaptation and survival, including survival against host efforts to kill pathogens. We usually focus on E. coli as a model organism and on comparisons with other strains. Our past work and that of many others defined structural and regulatory features for both ppGpp–binding sites on RNAP, which, in the presence of high levels of ppGpp, can lead to activation as well as inhibition of nearly one third of the genome. Our first goal is to examine the spectrum of effects on cellular physiology, ranging from very modest to massive changes in (p)ppGpp basal levels. Subtle and intricate regulation responses result in almost imperceptible perturbations in amino-acid, carbon, lipid, or energy metabolism, which alter the balance between ppGpp synthesis and hydrolysis. Such behavior contrasts with effects of massive increases, or spikes, of ppGpp, which others recently associated with metabolic destabilization of ribosomes, rRNA, and tRNA, thereby requiring re-evaluation of a sixty-year central dogma, which we published in a recent review and which documents the rapid changes in the field [Reference 5]. 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 (p)ppGpp acutely relevant, if we are not to enter an era in which all antibiotics are ineffective.

(p)ppGpp is necessary and sufficient for initiation of chromosomal DNA synthesis.

E. coli growth can be determined by the different efficiencies of use of nutrients present in excess or by limiting abundance of efficiently used substrates, allowing cells to fine-tune gene expression, which contrasts starkly with starvation, where adjustments to prevent death are very different from the fine-tuning during growth. Classically, the Copenhagen school established that the range of balanced growth rates when nutrient efficiency is varied is correlated with changes in the cellular content of protein, RNA, and DNA: the faster the growth, the higher the macromolecular content. When ppGpp is deleted, the correlation is abolished: macromolecular content remains at the high levels typical of fast growth even during slow growth. The same is true for hydrolysis-suppressor mutants that elevate ppGpp under all growth conditions. Apparently ppGpp is necessary and sufficient for this phenotype, regardless of stress. RNAP suppressor-mutant phenocopies of spoT behave similarly in ppGpp0 backgrounds, indicating that the phenotype is mimicked entirely by transcriptional changes. Such an approach can establish that ppGpp not only determines growth rates but is also both necessary and sufficient to account for RNA and protein content changes.

This year, we published a paper [Reference 1] indicating that ppGpp0 strains can be used with DNA to reach a similar conclusion, i.e., that ppGpp is necessary and sufficient for chromosomal DNA initiation. Bacteria can divide as quickly as every 30 minutes, but it takes an hour to duplicate their circular chromosome. The 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 in initiation frequencies. It is also known that ppGpp mildly inhibits primase-mediated DNA elongation. Our genomic sequencing results reinforce ori/ter ratio differences for 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. As before, parallel measurements of ori/ter ratios with suppressor mutants of ppGpp hydrolase and of RNAP mutants 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 proposing that ppGpp inhibition of ribosomal RNA operon (rrn) transcription from seven operons, comprising more than half of genomic transcripts, results in transmission of topological changes to the ori region that inhibit initiation. We tested the hypothesis with a strain deleted for all seven chromosomal rrn operons. The deletion strain remains viable because two high-copy plasmids carry either a single rrnB operon or several essential tRNA genes with the rrn deletions. Thus, indirect topological changes of ori/ter resulting from ppGpp inhibition of chromosomal rrn transcription are not possible. Inhibition of ori/ter ratios at slow growth rates persist 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, which shows three-fold more gyrase transcripts in wild-type than in ppGpp0 cells.

Is (p)ppApp a regulator or a toxin in bacteria?

The Potrykus lab approached this question by asking whether synthetic (p)ppApp has regulatory activity towards (p)ppGpp–sensitive E. coli promoters. Weak regulatory activities were encouraging, but the finding of strong specific binding near the RNAP catalysis center across from one of the (p)ppGpp binding sites was exciting and amplified by a similar sequence in B. subtilis RNAP. This led them to use bioinformatics to look for ppApp synthetase and a Mesh-1 like enzyme that could hydrolyze it. In Methylobacterium extorquens (Mex) they found a (p)ppGpp and (p)ppApp synthetase present on a purified Rel-like protein catalytic fragment as well as a (p)ppApp hydrolase on the Mesh-like protein. Clearly, bacterial enzymes can synthesize (p)ppApp and ppGpp in vitro, providing the first biochemical proof that ppApp is the catalytic product of this enzyme in bacteria, as well as proof that (p)ppApp exists along with (p)ppGpp in both E.coli and B. subtilis [Reference 2]. 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 native (p)ppApp in E. coli. Searches are now under way in E. coli for synthetase and hydrolase combinations that allow incremental accumulation of (p)ppApp or (p)ppGpp alone as well as together.

GreA is a well-studied RNA polymerase ancillary protein with complex functions, all involving ppGpp, that relate to fixing problems arising during transcription elongation arrest. The functions involve restoring proper reading frame and proof-reading when the polymerase slips out of phase. Katarzyna Potrykus discovered that GreA regulates its own synthesis by a unique stuttering mechanism, in a region termed GraL, rather than at a unique stop-site, during elongation [Reference 3]. GraL may be among the new functions found in a random mutant GreA protein library [Reference 4].


  1. Fernandez-Coll L, Maciag-Dorsznska M, Tailor K, Vadia S, Levin PA, Szalewska-Palasz A, Cashel M. The absence of (p)ppGpp renders initiation of Escherichia coli chromosomal DNA synthesis independent of growth rates. mBio 2020;11(2):e03223-19.
  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. Dylewski M, Fernandez-Coll L, Brun-Olszewska B, Balsalobre C, Potrykus K. Autoregulation of GreA expression relies on GraL expression rather than the GreA promoter region. Int J Mol Sci 2019;20(20):5224.
  4. Fernandez-Coll L, Potrykus K, Cashel M, Balsombre C. Mutational analysis of Escherichia coli GreA protein reveals new functional independent of antipause and lethal when overexpressed. Sci Rep 2020;32999370.
  5. Fernandez-Coll L. Cashel M. Possible roles for basal levels of (p)ppGpp: growth efficiency vs surviving stress. Front Microbiol 2020 11:592718.


  • 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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