bioRxiv preprint doi: https://doi.org/10.1101/2020.03.23.003749; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Systemic characterization of pppGpp, ppGpp and pGpp targets in Bacillus reveals NahA converts (p)ppGpp to pGpp to regulate alarmone composition and signaling Jin Yang1, Brent W. Anderson1, Asan Turdiev2, Husan Turdiev2, David M. Stevenson1, Daniel Amador-Noguez1, Vincent T. Lee2,*, Jue D. Wang1,* 1 Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA 2 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA *Correspondence: [email protected]; [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.23.003749; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Abstract 2 The alarmones pppGpp and ppGpp (collectively (p)ppGpp) protect bacterial cells from 3 nutritional and other stresses. Here we demonstrate the physiological presence of pGpp 4 as a third closely related alarmone in bacterial cells and also characterize and compare 5 the proteomic targets of pGpp, ppGpp and pppGpp in Gram-positive Bacillus species. 6 We revealed two regulatory pathways for ppGpp and pppGpp that are highly conserved 7 across bacterial species: inhibition of purine nucleotide biosynthesis and control of 8 ribosome assembly/activity through GTPases. Strikingly, pGpp potently regulates the 9 purine biosynthesis pathway but does not interact with the GTPases. Importantly, we 10 identified a key enzyme NahA that efficiently produces pGpp by hydrolyzing (p)ppGpp, 11 thus tuning alarmone composition to uncouple the regulatory modules of the alarmones. 12 Correspondingly, a nahA mutant displays significantly reduced pGpp levels and elevated 13 (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive 14 fitness. These cellular consequences for regulating alarmone composition strongly 15 implicate an expanded repertoire of alarmones in a new strategy of stress response in 16 Bacillus and its relatives. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.23.003749; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 17 Introduction 18 Organisms from bacteria to humans rely on timely and appropriate responses to 19 survive various environmental challenges. The stress signaling nucleotides guanosine 20 tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) are conserved across 21 bacterial species. When induced upon starvation and other stresses, they mediate 22 multiple regulations and pathogenesis by dramatically remodeling the transcriptome, 23 proteome and metabolome of bacteria in a rapid and consistent manner1–3. (p)ppGpp 24 interacts with diverse targets including RNA polymerases in Escherichia coli 4–8, 25 replication enzyme primase in Bacillus subtilis9–11, purine nucleotide biosynthesis 26 enzymes12–15, and GTPases involved in ribosome assembly16–19. Identification of 27 (p)ppGpp binding targets on a proteome-wide scale is one way to unravel a more 28 extensive regulatory network15,18,20. However, because binding targets differ between 29 different species and most interactomes have not been characterized, the conserved 30 and diversifying features of these interactomes remain incompletely understood. 31 Another understudied aspect of (p)ppGpp regulation is whether ppGpp and 32 pppGpp, while commonly referred to and characterized as a single species, targets the 33 same or different cellular pathways21. In addition, there is evidence for potential 34 existence of a third alarmone, guanosine-5′-monophosphate-3′-diphosphate (pGpp), 35 since several small alarmone synthetases can synthesize pGpp in vitro22,23. However, 36 the clear demonstration of pGpp in bacterial cells has been challenging. More 37 importantly, the regulation specificities and physiological importance of having multiple 38 closely-related alarmones in bacteria have not been systematically investigated. 39 Here we demonstrate pGpp as a third alarmone in Gram-positive bacteria by 40 establishing its presence in cells, systematically identifying its interacting targets, and 41 revealing a key enzyme for pGpp production through hydrolyzing (p)ppGpp. We also 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.23.003749; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 42 compare the targets of pGpp, ppGpp and pppGpp through proteomic screens in Bacillus 43 anthracis. We found that both pppGpp and ppGpp regulate two major cellular pathways: 44 purine synthesis and ribosome biogenesis. In contrast, pGpp strongly regulates purine 45 synthesis targets but does not regulate ribosome biogenesis targets, indicating a 46 separation of regulatory function for these alarmones. In B. subtilis and B. anthracis, 47 pGpp is efficiently produced from pppGpp and ppGpp by the NuDiX (Nucleoside 48 Diphosphate linked to any moiety “X”) hydrolase NahA (NuDiX alarmone hydrolase A), 49 both in vitro and in vivo. A ΔnahA mutant has significantly stronger accumulation of 50 pppGpp and decreased accumulation of pGpp, as well as slower recovery from 51 stationary phase and reduced competitive fitness against wild type cells. Our work 52 suggests a mechanism for the conversion and fine tuning of alarmone regulation and the 53 physiological production of the alarmone pGpp. 54 55 Results 56 Proteome-wide screen for binding targets of pppGpp and ppGpp from Bacillus 57 anthracis 58 To systematically characterize the binding targets of (p)ppGpp and identify novel 59 (p)ppGpp binding proteins in Bacillus species, we screened an open reading frame 60 (ORF) library of 5341 ORFs from the pathogen Bacillus anthracis (Figure 1a). Using 61 Gateway cloning, we placed each ORF into two expression constructs, one expressing 62 the ORF with an N-terminal histidine (His) tag and the other with an N-terminal histidine 63 maltose binding protein (HisMBP) tag. 64 We first charaterized the binding targets of ppGpp using the B. anthracis library. 65 To this end, each ORF in the His-tagged and HisMBP-tagged library was overexpressed 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.23.003749; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 66 and binding [5′-α-32P]-ppGpp was assayed using differential radial capillary action of 67 ligand assay (DRaCALA)24 (Figure 1a). The fraction of ligand bound to protein in each 68 lysate was normalized as a Z-score of each plate to reduce the influence of plate-to- 69 plate variation (Table S1). We found that the strongest ppGpp-binding targets in B. 70 anthracis can be categorized to three groups: 1) purine nucleotide synthesis proteins 71 (Hpt-1, Xpt, Gmk, GuaC, PurA, PurR); 2) ribosome and translation regulatory GTPases 72 (HflX, Der, Obg, RbgA, TrmE, Era); and 3) nucleotide hydrolytic enzymes, including 73 NuDiX hydrolases and nucleotidases (Figure 1b). We compared these targets to those 74 obtained from previous screens for ppGpp targets in E. coli and for an unseparated mix 75 of pppGpp and ppGpp in S. aureus18. Comparison of our results with these previous 76 screens yielded conserved themes (Figure 1b). Among the most conserved themes are 77 the purine nucleotide synthesis proteins (Figure 1c) and ribosome and translation 78 regulation GTPases (Figure 1d). 79 Next, we performed a separate screen to characterize the binding of the B. 80 anthracis proteome to pppGpp (Figure 1a). pppGpp is the predominant alarmone 81 induced upon amino acid starvation in Bacillus species, rising to a higher level than 82 ppGpp. However, despite potential differences in specificity between pppGpp and 83 ppGpp, the pppGpp interactome has not been systematically characterized in bacteria. 84 We found that pppGpp shares almost identical targets with ppGpp, with similar or 85 reduced binding efficacy for most of its targets compared to ppGpp (Table S1). By 86 sharing targets with ppGpp, pppGpp also comprehensively regulates purine synthesis 87 and ribosome assembly. We also find that several proteins bind to pppGpp but not 88 ppGpp, including the small alarmone synthetase YjbM (SAS1). This is expected for YjbM, 89 since it is allosterically activated by pppGpp, but not ppGpp25. 90 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.23.003749; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 91 NahA, a NuDiX hydrolase among the (p)ppGpp interactome in Bacillus, hydrolyzes 92 (p)ppGpp to produce pGpp in vitro 93 The putative NuDiX hydrolase, BA5385, was identified as a novel binding target 94 of (p)ppGpp. Protein sequence alignment showed that BA5385 has homologs in different 95 Bacillus species with extensive homology and a highly conserved NuDiX box (Figure 96 S1). We cloned its homolog, YvcI, from the related species Bacillus subtilis and showed 97 that overexpressed B. subtilis YvcI in cell lysate also binds ppGpp and pppGpp (Figure 98 2a). The binding is highly specific, as non-radiolabeled ppGpp effectively competes with 99 radiolabeled (p)ppGpp binding, whereas non-radiolabeled GTP failed to compete. EDTA 100 eradicated (p)ppGpp binding to His-MBP-YvcI cell lysate, which implies that the divalent 101 cations present in the reaction (Mg2+) is essential for (p)ppGpp binding to YvcI (Figure 102 2a). 103 We noticed that YvcI overexpression cell lysate showed strong and specific 104 binding to (p)ppGpp, the purified protein does not appear to bind (p)ppGpp in DRaCALA 105 (Figure S2).