National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

Global Regulation of 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

The goal of our research continues to be to learn how bacteria use noncanonical nucleotides as regulatory signals, which they do in two ways. First, they sense sources of nutritional and physical stress that limit growth and then they either adjust host gene expression to counteract stress or become metabolically dormant until the stress has passed. The first example to be discovered was a cyclic nucleotide, 3′-5′ cAMP, which senses glucose limitation. We have a very long-standing research interest in sensing and response functions of noncyclic analogs of GTP and GDP that contain pyrophosphate residues on their ribosyl 3′ hydroxyl. These are pppGpp and ppGpp, respectively, abbreviated as (p)ppGpp (the extra phosphate termed the 5′ gamma phosphate). The (p)ppGpp nucleotides are ubiquitous in bacteria and plant plastids, and their regulatory scope is broad. Changes in gene expression affect about one third of bacterial and chloroplast genomes. The (p)ppGpp nucleotides sense and respond to both physical, nutritional, and pathogenic sources of stress. In bacteria these regulatory systems are fundamentally relevant to pathogenesis because the mechanisms of host defenses and bacterial antibiotics that are designed to eliminate invading bacteria trigger counteractive (p)ppGpp responses. Evidence for the role of (p)ppGpp in pathogenicity is pervasive, both with respect to antibiotic resistance and the reduction in host carrier states. The current crisis occasioned by the lack of new antibiotics coupled with emergence of multidrug-resistant pathogens has led to widespread interest in (p)ppGpp. Our lab is, however, too small to develop new antibiotics. Instead, we continue to study how structural features of (p)ppGpp contribute to its function, to discover new basic regulatory responses to (p)ppGpp, such as the role found for ppGpp in coupled transcription DNA repair, and to explore comparative studies with (p)ppApp, a new analog. Our specific objectives are to learn what regulatory effects are altered by changing individual residues of pppGpp, to pursue our discovery that (p)pGpp is necessary and sufficient for initiation of bacterial DNA synthesis, and to determine whether (p)ppGpp alters transcription during lytic T4 bacteriophage infection.

Regulatory effects of changing individual residues of (p)ppGpp

Most bacteria accumulate both ppGpp and pppGpp but the relative amounts differ in systematic ways. For example, Gram-negative bacteria such as E. coli accumulate both pppGpp and ppGpp in roughly equal proportions during the first few minutes of amino acid starvation, but thereafter the level of ppGpp greatly exceeds that of pppGpp. When the same bacteria are starved for a glucose or other carbon sources, ppGpp levels greatly exceed those of pppGpp. For Gram-positive bacteria the opposite happens, i.e., more pppGpp accumulates than ppGpp. Regulation is different for this class and responds instead to GTP limitation caused by ppGpp. The question arises as to whether the 5′ gamma phosphate alters regulatory activities, i.e., whether ppGpp regulates differently than does pppGpp. We have genetic constructs of E. coli cell lines that accumulate either ppGpp or pppGpp, but not both. In either case, accumulation can be varied artificially but not as a consequence of stress. These studies revealed that ppGpp is a more potent inhibitor of growth and of other regulatory events in vivo. We thus found ppGpp to be a more potent regulator in vitro for initiation of rRNA transcripts, which is how ppGpp regulates growth rate. RNA polymerase has two ppGpp–binding sites, and our structural studies revealed that both ppGpp and pppGpp bound to the same site (site 1) on RNA polymerase, a site that is distant from the catalytic center; however, binding constants were not obtained. Other labs found that ppGpp also binds at a second site near the catalytic center; however, pppGpp was not studied. So far, in E. coli, we conclude that ppGpp is a more potent regulator than pppGpp, i.e., the 5′-gamma phosphate matters.

Studies on pGpp allow us to test whether the 5′-beta phosphate in ppGpp also matters. We collaborated in the discovery that a (p)ppGpp synthetase (RelQ) from the Gram-positive Enterococcus faecalis can also use GMP as a substrate to make pGpp in vitro. Comparative studies of several in vitro reactions regulated by (p)ppGpp, including E. coli RNA polymerase, reveal that pGpp has relatively little regulatory activity. This allows us to come to a conclusion relating structure to function: altering ppGpp (the GDP derivative) by either adding a 5′-gamma phosphate (pppGpp, the GTP derivative) or by removing its 5′-beta phosphate (pGpp, the GMP derivative) compromises its regulatory potency.

Comparison of pppGpp with (p)ppApp, a new nucleotide regulator

Initial studies that led to a focus on (p)ppApp have been presented in our earlier annual reports. Our interest in (p)ppApp arose because of the discovery by Katarzyna Potrykus, while a postdoctoral fellow in our lab, that a (p)ppGpp hydrolase called MESH, present in animals, could hydrolyze (p)ppApp as well as (p)ppGpp. Potrykus returned to Gdansk, and her laboratory discovered a strain with a MESH–like enzyme that not only hydrolyzes (p)ppApp but also lacks the archetypical (p)ppGpp hydrolase activity. The discovery suggests that (p)ppApp is biologically significant. The group also has evidence that the regulatory interactions of (p)ppApp and (p)ppGpp are distinct. Evidently, exchanging the G nuclease in ppGpp for A also makes a difference.

(p)ppGpp plays a necessary and sufficient role in initiation of DNA replication.

Our interest in regulatory events related to glucose starvation led to the discovery that (p)ppGpp plays an important role in regulating the initiation of bacterial DNA synthesis. We first found that increasing basal (p)ppGpp slows growth, which is correlated with an increase in the levels of acetyl phosphate (Ac~P). We also learned that Agnieska Szalewska (also a former postdoctoral fellow) made an important discovery, which provided a clue to our observations. She found that a mutant of a gene (dnaA46), necessary for DNA initiation, was reactivated by deleting genes necessary for Ac~P formation, genes that are also related to ppGpp. The DnaA protein works to start DNA replication by forming multimers attached to each other in a DNA sequence–dependent manner at the chromosomal start region, called ori. This first step in the process requires ATP in order to phosphorylate a specific amino acid (lysine 178) in the ATP–binding site of the DnaA protein. A recent report convincingly showed that acetylation of the crucial K178 can occur, and when it does, it is associated with reversible inhibition of DnaA oligomerization at chromosomal origins. Increased Ac~P concentrations can acetylate lysine 178 either non-enzymatically or enzymatically. We initially thought that non-enzymatic acetylation linked Ac~P levels to ppGpp, but we now have genetic evidence that DnaA activity is regulated instead by enzymatic acylation and enzymatic deacylation. We can show that elevated ppGpp exerts its inhibitory effects on initiation even in mutants that cannot form Ac~P. This suggests that elevated (p)ppGpp is sufficient for inhibition of chromosomal DNA initiation. Conversely, cells completely deficient in ppGpp do not inhibit DNA synthesis initiation despite slow rates of growth, suggesting that (p)ppGpp is also necessary for DNA replication inhibition. However, the observation that (p)ppGpp is necessary and sufficient does not reveal details of the mechanism by which this occurs. We are currently pursuing this question.

Publications

  1. Kamarthapu V, Epshtein V, Benjamin B, Proshkin S, Mironov A, Cashel M, Nudler E. ppGpp couples transcription to DNA repair in E. coli. Science 2016 352:993-936.
  2. Potrykus K, Cashel M. Preferential cellular accumulation of ppGpp or pppGpp in Escherichia coli. In: de Brujin FJ, ed. Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria. John Wiley and Sons, Inc. 2016 7:1-8.

Collaborators

  • Deborah Hinton, PhD, Gene Expression and Regulation Section, NIAID, Bethesda, MD
  • Katsuhiko S. Murakami, PhD, The Huck Institutes of the Life Sciences, Penn State University, University Park, PA
  • Evgeny Nudler, PhD, New York University School of Medicine, New York, NY
  • Katarzyna Potrykus, PhD, University of Gdansk, Gdansk, Poland
  • Agnieszka Szalewska-Palasz, PhD, University of Gdansk, Gdansk, Poland

Contact

For more information, email cashel@mail.nih.gov or visit http://smr.nichd.nih.gov.

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