Affinity-based capture and identification of protein effectors of the growth regulator ppGpp

Wang, Boyuan; Dai, Peng; Ding, David; Rosario, Amanda del; Grant, Robert A.; Pentelute, Bradley L.; Laub, Michael T.

doi:10.1038/s41589-018-0183-4

Cited by 159 publications

(227 citation statements)

References 42 publications

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“…A future area of investigation is to understand why (p)ppGpp is essential for R. palustris longevity and to identify genes and proteins whose activities may be modulated by (p)ppGpp. (p)ppGpp has recently been shown to bind to Era from Staphylococcus aureus and E. coli to inhibit its GTPase activity ( 39–41 ), and this may influence ribosome biogenesis and integrity. There is good evidence that (p)ppGpp is important for development of antibiotic persisters ( 42 ) and for long term survival of latent and growth-arrested M. tuberculosis ( 43, 44 ).…”

Section: Discussionmentioning

confidence: 99%

Bacterial longevity requires protein synthesis and a stringent response

Yin

Nakayasu

et al. 2019

Preprint

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Gram-negative bacteria in infections, biofilms and industrial settings often stop growing due to nutrient depletion, immune responses or environmental stresses. Bacteria in this state tend to be tolerant to antibiotics and are often referred to as dormant. Rhodopseudomonas palustris, a phototrophic α-proteobacterium, can remain fully viable for more than four months when growth is arrested. Here, we show that protein synthesis, specific proteins involved in translation and a stringent response are required for this remarkable longevity. Because it can generate ATP from light during growth arrest, R. palustris is an extreme example of a bacterial species that will stay alive for long periods of time as a relatively homogeneous population of cells and it is thus an excellent model organism for studies of bacterial longevity. There is evidence that other Gram-negative species also continue to synthesize proteins during growth arrest and that a stringent response is required for their longevity as well. Our observations challenge the notion that growth-arrested cells are necessarily dormant and metabolically inactive, and suggest that such bacteria may have a level of metabolic activity that is higher than many would have assumed. Our results also expand our mechanistic understanding of a crucial but understudied phase of the bacterial life cycle.IMPORTANCEWe are surrounded by bacteria; but they do not completely dominate our planet despite the ability of many to grow extremely rapidly in the laboratory. This has been interpreted to mean that bacteria in nature are often in a dormant state. We investigated life in growth arrest of Rhodopseudomonas palustris, a proteobacterium that stays alive for months when it is not growing. We found that cells were metabolically active; they continued to synthesize proteins and mounted a stringent response, both of which were required for their longevity. Our results suggest that long-lived bacteria are not necessarily inactive but have an active metabolism that is well adjusted to life without growth.

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Section: Discussionmentioning

confidence: 99%

Bacterial longevity requires protein synthesis and a stringent response

Yin

Nakayasu

et al. 2019

Preprint

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“…Various proteins reported to bind with ppGpp include an hypothetical protein YjgA, 2424 GTPase family (EFG, EF‐Tu, Obg, and PurA), nucleotide‐related proteins (DnaG, GuaB, Apt, Hpt, and HisG), metabolic enzymes (PgsA and GdhA) and amino acid decarboxylase family (LdcI, LdcC, SpeF, and SpeC) . Moreover, 298 proteins have been reported recently as ppGpp‐binding proteins and the newly identified targets and effects of ppGpp require a further attention …”

Section: Introductionmentioning

confidence: 99%

The Activation of Glycerol Dehydrogenase from Escherichia coli by ppGpp

Hoang

Tran

Jung

2019

Bulletin Korean Chem Soc

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Glycerol dehydrogenase (GldA) from Escherichia coli is a Zn2+‐containing alcohol dehydrogenase which catalyzes the NAD+‐dependent oxidation of glycerol to dihydroxyacetone. In this study, GldA has been cloned, over‐expressed, and isolated by an affinity and an ion‐exchange chromatography. GldA shows a strong intrinsic fluorescence at 320 nm, when excited at 280 nm. The fluorescence intensity decreases in the presence of NAD+, NADH, and dihydroxyacetone, the substrate and products for GldA, which allows us to determine the dissociation constants for those molecules as 110.6 ± 5.0 μM, 9,1 ± 0.6 μM, 33.3 ± 2.3 mM, respectively. The dissociation constant for NAD+ was similar to the kinetic constant, KM. Guanosine‐5′‐diphosphate 3′‐diphosphate (ppGpp), accumulated in E. coli when starved for amino acids, nutrients, and phosphate, serves as a global regulator in replication, transcription, and translation. In this study, the fluorescence intensity of GldA also decreases in the presence of ppGpp and the dissociation constant for ppGpp is calculated as 108.9 ± 8.6 μM. ppGpp increases GldA activity with the half maximal activation at 33.1 ± 3.1 μM. On the contrary, GTP and GDP inhibit GldA, with the inhibition constants of 16.1 ± 1.1 mM and 10.6 ± 0.3 mM, respectively. Tris(hydroxymethyl)aminomethane serves as a competitive inhibitor against glycerol. GTP and GDP also bind to GldA with the dissociation constants of 60.0 ± 0.8 and 61.0 ± 1.3 μM, respectively. These results suggest that GTP and GDP bind to GldA as strongly as ppGpp but only ppGpp activates GldA. This study shows that ppGpp binds to GldA and activates its activity for the first time. It is also suggested that the strong intrinsic fluorescence of enzymes and their changes in the presence of various ligands can be utilized to measure the binding affinities for those ligands. The method described here is especially effective for bindings with the relatively lower affinities.

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“…Our demonstration that point mutations in IF2 affect (p)ppGpp binding and subsequent in vitro (Figure 5) and in vivo function (Figure 7) suggests that introducing similar mutations into the highly conserved G1 motif of other GTPases might be informative. Interestingly, a similar mutagenic strategy was reported very recently for the E. coli PurF glutamine amido-phoaphoribotransferease where a single point mutation yielded a protein that was insensitive to ppGpp inhibition (Wang et al, 2019).…”

Section: Discussionmentioning

confidence: 87%

(p)ppGpp directly regulates translation initiation during entry into quiescence

Diez

Ryu

Caban

et al. 2019

Preprint

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12Many bacteria exist in a state of metabolic quiescence where they must minimize energy 13 consumption so as to maximize available resources over a potentially extended period of time. 14 As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be 15 an appropriate target for downregulation during the transition from growth to quiescence. We find 16 that when Bacillus subtilis exits growth, a subpopulation of cells emerges with very low levels of 17 protein synthesis dependent on synthesis of the nucleotides (p)ppGpp. We show that (p)ppGpp 18 inhibits protein synthesis in vivo and in vitro by preventing the allosteric activation of the essential 19 GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that IF2 is 20 an authentic in vivo target of (p)ppGpp during the entry into quiescence, thus providing a 21 mechanistic basis for the observed attenuation of protein synthesis. 22 al., 2016), DNA primase (Wang et al., 2007) and the GTP biosynthetic enzymes HprT and Gmk 51 (Kriel et al., 2012), respectively. 52 Overexpression of a truncated RelA protein that synthesizes (p)ppGpp in the absence of 53 amino acid limitation results in a rapid decrease in 35 S-methionine incorporation (Svitil et al., 54 1993), consistent with an inhibitory effect of (p)ppGpp on translation. However, further 55investigation of a direct effect of (p)ppGpp on translation has been complicated by several factors. 56 First, (p)ppGpp affects synthesis of ribosomal proteins in E. coli (Lindahl et al., 1976). Whether 57 this effect is direct has been difficult to assess given the well-established effect of (p)ppGpp on 58 transcription by E. coli polymerase. Second, studies examining the effect of (p)ppGpp produced 59 by RelA use conditions that produce uncharged tRNAs in order to stimulate RelA (Arenz et al., 60 2016; Brown et al., 2016; Haseltine and Block, 1973; Loveland et al., 2016; O'Farrell, 1978). Since 61 uncharged tRNAs directly arrest translation, it has been difficult to differentiate this effect from a 62 direct effect of (p)ppGpp on translation. 63 In vitro, (p)ppGpp inhibits translation in a manner similar to that observed with non-64 hydrolyzable GTP analogs (Wagner and Kurland, 1980), suggesting that (p)ppGpp is targeting a 65 translational GTPase. Consistently, (p)ppGpp inhibits the GTPase activity of IF2 and EF-Tu 66 (Hamel and Cashel, 1974) as well as of GTPases that mediate other aspects of translation such 67 as ribosome assembly (Corrigan et al., 2016; Pausch et al., 2018), by acting as competitive 68 inhibitors. (p)ppGpp is capable of binding translational GTPases including EF-Tu, EF-G (Rojas et 69 al., 1984), IF2 (Milon et al., 2006; Mitkevich et al., 2010) and the ribosome assembly GTPase 70 ObgE (Persky et al., 2009) at affinities that are commensurate with the in vivo levels of (p)ppGpp 71 observed following stringent response induction, consistent with the enzymes being in vivo 72 targets. Of note, the affinity of IF2 f...

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Affinity-based capture and identification of protein effectors of the growth regulator ppGpp

Cited by 159 publications

References 42 publications

Bacterial longevity requires protein synthesis and a stringent response

Bacterial longevity requires protein synthesis and a stringent response

The Activation of Glycerol Dehydrogenase from Escherichia coli by ppGpp

(p)ppGpp directly regulates translation initiation during entry into quiescence

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