ppGpp couples transcription to DNA repair in
            <i>E. coli</i>

Kamarthapu, Venu; Epshtein, Vitaly; Benjamin, Bradley; Proshkin, Sergey; Mironov, A. S.; Cashel, Michael; Nudler, Evgeny

doi:10.1126/science.aad6945

Cited by 112 publications

(156 citation statements)

References 42 publications

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“…To date, the starvation signaling molecule guanosine tetraphosphate [(p)ppGpp] is the only known widely conserved regulator of cell cycle exit in bacteria and functions by a variety of mechanisms in different bacterial species, ranging from inhibiting replication initiation (7,8) to modulating replication elongation rates (9,10) and promoting septation (11). (p)ppGpp was also recently shown to play a role in transcription-coupled DNA repair (12). A related molecule, polyphosphate (polyP), is an inorganic polymer consisting simply of phosphoryl groups and can be hundreds of protomers in length.…”

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confidence: 99%

Polyphosphate granule biogenesis is temporally and functionally tied to cell cycle exit during starvation in Pseudomonas aeruginosa

Racki

Tocheva

Dieterle

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

112

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Polyphosphate (polyP) granule biogenesis is an ancient and ubiquitous starvation response in bacteria. Although the ability to make polyP is important for survival during quiescence and resistance to diverse environmental stresses, granule genesis is poorly understood. Using quantitative microscopy at high spatial and temporal resolution, we show that granule genesis in Pseudomonas aeruginosa is tightly organized under nitrogen starvation. Following nucleation as many microgranules throughout the nucleoid, polyP granules consolidate and become transiently spatially organized during cell cycle exit. Between 1 and 3 h after nitrogen starvation, a minority of cells have divided, yet the total granule number per cell decreases, total granule volume per cell dramatically increases, and individual granules grow to occupy diameters as large as ∼200 nm. At their peak, mature granules constitute ∼2% of the total cell volume and are evenly spaced along the long cell axis. Following cell cycle exit, granules initially retain a tight spatial organization, yet their size distribution and spacing relax deeper into starvation. Mutant cells lacking polyP elongate during starvation and contain more than one origin. PolyP promotes cell cycle exit by functioning at a step after DNA replication initiation. Together with the universal starvation alarmone (p)ppGpp, polyP has an additive effect on nucleoid dynamics and organization during starvation. Notably, cell cycle exit is temporally coupled to a net increase in polyP granule biomass, suggesting that net synthesis, rather than consumption of the polymer, is important for the mechanism by which polyP promotes completion of cell cycle exit during starvation.ost of our understanding of bacterial physiology comes from laboratory studies of bacteria growing under nutrientrich conditions. However, in many environments, bacteria face dramatic fluctuations in nutrient conditions, including long periods of scarcity when they survive in a nonproliferative stationary state. Although eukaryotic cells and some bacteria have discrete cell cycle checkpoints, many fast-growing bacterial species have uncoupled DNA replication and cell division. They use multifork DNA replication to achieve a doubling time that is faster than the time required to copy the chromosome, giving them a competitive edge in nutrient-replete conditions. This strategy requires distinct regulatory mechanisms and comes at a considerable cost when there is a rapid downshift in nutrient availability: stalled open DNA replication forks are vulnerable to potentially lethal double-stranded DNA breaks (1). Therefore, the ability to reallocate resources under such conditions to prioritize completion of DNA replication is critical for survival. Prioritizing chromosome remodeling and compaction by starvation-specific nucleoid structural proteins is also important during such transitions because resources for DNA repair become limited in deep starvation (2-4). Operating an uncoupled cell cycle, where growth, DNA replication, and cell ...

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confidence: 99%

Polyphosphate granule biogenesis is temporally and functionally tied to cell cycle exit during starvation in Pseudomonas aeruginosa

Racki

Tocheva

Dieterle

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

112

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“…Alarmones can dramatically influence transcription during the stringent response in different bacteria (26,27). Recent publications show that ppGpp is robustly, but transiently, induced in response to DNA damage so as to allow efficient nucleotide excision repair in E. coli (28). Alarmones have been shown to induce the stringent response upon nitrogen starvation (29).…”

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confidence: 99%

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

Juengert

Borisova

Mayer

et al. 2017

Appl Environ Microbiol

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In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033-8044, 2012, https://doi.org/ 10.1128/AEM.01693-12) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutropha. IMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha. This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a highperformance liquid chromatography-mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha. This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and mas-

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“…Backtracking by UvrD requires the high concentrations of the protein that are produced during the bacterial response to DNA damage that is termed the SOS response. It is also enhanced by a small-molecule alarmone, (p)ppGpp, which is transiently induced following exposure of cells to UV light (16,19,20). In addition to mapping the rates of repair across the Their analysis used a technique called eXcision repair sequencing (XR-seq) (2), which involves the extraction, immunoprecipitation, and sequencing of all of the short lesion-containing oligonucleotides that are produced by UvrABC (Fig.…”

Section: Understanding Bias In Dna Repairmentioning

confidence: 99%