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Systemic characterization of pppGpp, ppGpp and pGpp targets in Bacillus reveals NahA converts (p)ppGpp to pGpp to regulate 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]

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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 biosynthesis and control of

8 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 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.

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17 Introduction 18 Organisms from to humans rely on timely and appropriate responses to

19 survive various environmental challenges. The stress signaling guanosine

20 tetraphosphate (ppGpp) and (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 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

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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 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 (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

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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 regulatory GTPases

72 (HflX, Der, Obg, RbgA, TrmE, Era); and 3) nucleotide hydrolytic , 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 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

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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). This suggests that either YvcI requires a co-factor present in the lysate to

106 bind to (p)ppGpp, or YvcI may rapidly hydrolyze the (p)ppGpp and release the product.

107 Therefore, we incubated purified YvcI with [5’-α-32P]-(p)ppGpp and ran the reaction

108 product using TLC (Figure S3). We found that YvcI can hydrolyze both ppGpp and

109 pppGpp. In addition to (p)ppGpp, we also tested the ability of YvcI to hydrolyze GTP and

110 8-oxo-GTP to sanitize guanosine nucleotide pool26. YvcI failed to hydrolyze either GTP

111 or 8-oxo-GTP (data not shown). The inability of YvcI to hydrolyze GTP and the strong

112 structural similarity between GTP and (p)ppGpp suggest that NahA is a specific

113 (p)ppGpp hydrolase which requires its substrate to have pyrophosphate group on the 3′

114 end. However, unlike B. subtilis RelA, which hydrolyzes the 3’-pyrophosphate group

115 from ppGpp and pppGpp to produce GDP and GTP, NahA hydrolyzed ppGpp and

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116 pppGpp to yield a single nucleotide species that migrated differently than GTP (Figure

117 S3a) or GDP (Figure S3b). To determine the identity of YvcI’s (p)ppGpp hydrolysis

118 product, we analyzed the sample by liquid chromatography coupled with mass

119 spectrometry (LC-MS), and compared to a pGpp standard produced by E. faecalis SAS

120 (RelQ) in vitro 22. The LC-MS profile revealed a peak of the same mass over charge ratio

121 (m/z) as GTP but with a different retention time (11.75 min versus 11.15 min for GTP).

122 The retention time is the same as the pGpp standard (Figure 2b), suggesting that the

123 hydrolysis product of YvcI is pGpp.

124 The production of pGpp from pppGpp and ppGpp agrees with the NuDiX

125 hydrolase function, inferring that pppGpp and ppGpp are hydrolyzed between the 5’-α

126 and 5’-β phosphate groups to produce guanosine-5’-monophosphate-3’-diphosphate

127 (pGpp). Therefore, we renamed the enzyme NahA (NuDiX alarmone hydrolase A).

128 It is possible, although unlikely, that NahA hydrolyzes the 3′-phosphate to

129 produce ppGp, which would run at the same retention time as pGpp in LC-MS. To

130 distinguish these two possibilities, we analyzed the NahA cleavage products of [3′-β-32P]-

131 pppGpp. If NahA cleaves the 5’-γ and β phosphates, the reaction would yield [3′-β-32P]-

132 pGpp. In contrast, if NahA cleaves the 3’-β-phosphate and the 5’-γ-phosphate, the

133 reaction would yield free 32P-phosphate. TLC analysis revealed that the radioactive 32P

134 after NahA hydrolysis of [3′-β-32P]-pppGpp co-migrates with the pGpp nucleotide rather

135 than the free phosphate that migrates to the very end of TLC plate (Figure 2c). This

136 result showed that the product has an intact 3’-pyrophosphate group, confirming the

137 product to be pGpp rather than ppGp. Finally, quantification of [3′-β-32P]-pppGpp

138 hydrolysis by NahA showed that the decrease of substrate radioactivity mirrored the

139 increase of the single product radioactivity (Figure 2d), demonstrating the product is

140 exclusively pGpp.

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141 As a NuDiX hydrolase, NahA has been reported to have a modest activity in

142 removing the 5’-phosphate of mRNA 27. We found that NahA is far more efficient at

143 hydrolyzing (p)ppGpp than at decapping mRNA. Enzymatic assays revealed that NahA

-1 144 hydrolyzes ppGpp following Michaelis-Menten kinetics, with a kcat of 1.22 ± 0.17 s and a

145 Km of 7.5 ± 2.3 μM (Figure 2e, Table 1). NahA also effectively and cooperatively

-1 146 hydrolyzes pppGpp, with a Hill coefficient of 2.78 ± 0.47, kcat of 10.0 ± 0.5 s and Km of

147 177.4 ± 0.4 μM (Figure 2e, Table 1). In contrast, its kcat to decap RNA is around 0.0003

-1 27 148 s (estimation based on published figure) . The difference in kcat in vitro suggest that

149 NahA’s major function is to regulate (p)ppGpp rather than to decap the 5’ cap of mRNA.

150

151 NahA hydrolyzes (p)ppGpp to produce pGpp in vivo

152 NahA was previously identified as a constitutively expressed protein with ~600

153 copies per cell 28. To examine its impact on (p)ppGpp in vivo, we engineered a nahA

154 deletion strain, and developed an LC-MS metabolome quantification for pppGpp, ppGpp

155 and pGpp in B. subtilis cells (see Material & Methods). LC-MS measurement of cell

156 extracts showed that ΔnahA mutant cells accumulate more pppGpp and ppGpp than

157 wild type cells during both log phase and stationary phase, in agreement with NahA’s

158 ability to hydrolyze (p)ppGpp (Figure 3a). In contrast, ΔnahA mutant has much less

159 pGpp than wild type cells (Figure 3a). Specifically, during log phase, pGpp can hardly

160 be detected in ΔnahA (Figure S4a). When we complement ΔnahA with an

161 overexpressed copy of nahA, the pGpp levels increased to more than wild type levels

162 (Figure S4). These results support the function of NahA in producing pGpp.

163 We also used the drug arginine hydroxamate (RHX) which mimics amino acid

164 starvation to induce accumulation of (p)ppGpp12. Using both LC-MS and TLC, we

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165 observed rapid accumulation of (p)ppGpp after RHX treatment, with ΔnahA cells

166 showing stronger (p)ppGpp accumulation than wild type cells (Figure S5a-d). We still

167 observe the accumulation of pGpp in ΔnahA cells, although to a much less extent than

168 wild type cells (Figure S5e), this remaining pGpp in ΔnahA cells is likely due to the

169 function of the enzyme SAS1, which can synthesize pGpp from GMP and ATP 22.

170

171 pGpp can be hydrolyzed efficiently by RelA in vitro

172 The strong reducing effect of NahA on (p)ppGpp levels suggests that (p)ppGpp

173 are efficiently hydrolyzed to pGpp. However, pGpp must be eventually degraded. In B.

174 subtilis, pppGpp and ppGpp are both hydrolyzed by RelA, which has a functional

175 (p)ppGpp hydrolase domain. Therefore, we performed in vitro assays using purified B.

176 subtilis RelA and pGpp, ppGpp or pppGpp. We found that all three nucleotides can be

177 hydrolyzed efficiently by RelA, furthermore, pGpp was hydrolyzed more rapidly than

178 (p)ppGpp (Figure 3b).

179

180 Protein binding spectrum of pGpp is distinct from (p)ppGpp

181 To understand whether pGpp is just an intermediate of (p)ppGpp hydrolysis or is

182 a bona fide alarmone with its own regulatory targets, we used DRaCALA to

183 systematically screen the B. anthracis library for pGpp-binding targets (Figure 1a-b,

184 Table S1). Our screen showed that pGpp binds strongly to multiple purine nucleotide

185 synthesis enzymes (Figure 1c), but binds to none of the (p)ppGpp-binding ribosome and

186 translation regulation GTPases (Figure 1d). We then purified selected pGpp and

187 (p)ppGpp binding targets and tested with [5′-α-32P]-labeled pGpp, ppGpp and pppGpp

188 using DRaCALA (Figure 3c-d). These results confirmed strong pGpp binding to

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189 guanosine nucleotide synthesis proteins (Hpt1, Gmk, Xpt). In contrast, GTPases

190 involved in ribosome biogenesis (HflX, Obg, Der) bind ppGpp strongly, but do not bind

191 pGpp (Figure 3d). Titration analysis of these GTPases showed their strong affinity to

192 ppGpp, modest affinity to pppGpp and lack of affinity to pGpp (Figure 3e-g, Table S2).

193 We conclude that among the two main groups of conserved interaction targets of

194 (p)ppGpp, pGpp exclusively regulates the purine pathway, but not the GTPases, thus

195 can serve as a specialized signal (Figure 3h).

196

197 nahA mutant exhibits stronger inhibition of translation upon amino acid starvation,

198 delayed outgrowth and loss of competitive fitness

199 Finally, we analyzed the effects of nahA on cellular growth and metabolism. We

200 first compared the key metabolites (NTPs, NDPs, NMPs, nucleosides and nucleobases)

201 between wild type cells and the nahA mutant, using LC-MS analyses of expontential

202 growth and stationary phase cells (Figure 4a, Table S3). Despite much higher (p)ppGpp

203 levels and much lower pGpp levels in the nahA mutant, most purine nucleotides exhibit

204 very little difference, corroborating with the observation that pGpp regulates purine

205 metabolism similarly to (p)ppGp.

206 Next, we examined the effect of nahA on protein translation. The fact that pGpp

207 does not directly regulate ribosome biogenesis and translation implicates an in vivo

208 function of NahA: reducing (p)ppGpp levels to alleviate translation inhibition upon stress,

209 while still keeping purine biosynthesis in check. To test this hypothesis, we measured

210 total protein translation rate of wild type and ΔnahA B. subtilis by pulsed incorporation of

211 35S-methionine (Figure 4b). We found that after amino acid starvation, the rate of 35S-

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212 methionine incorporation is significantly further reduced in ΔnahA than in wild type

213 (P<0.05), indicating a stronger inhibition of translation (Figure 4b).

214 Finally, we examined the impact of nahA on growth fitness of B. subtilis. We

215 performed a growth competition assay in which a mixture of ΔnahA and wild type cells

216 were grown to saturation and then diluted repeatedly for days. The proportion of ΔnahA

217 rapidly decreased, indicating significant loss of fitness. The calculated relative fitness of

218 ΔnahA is 0.875 ± 0.008 (mean ± S.D.) compared to wild type cells (Figure 4c). We

219 found that ΔnahA has a similar doubling time as wild type cells (Figure 4d), but has a

220 longer lag phase in adjusting to starvation (Figure 4e), likely due to excessive inhibition

221 of translation (Figure 4b). Together, these results suggest that NahA tunes alarmone

222 composition and alarmone levels to promote B. subtilis adjustment to nutrient fluctuation

223 and optimizes growth and fitness.

224

225 Discussion

226 Alarmones are universal stress signaling nucleotides in bacteria, however, the

227 repertoire of alarmones and the target spectrums for each alarmone are incompletely

228 understood. In this work, we have comprehensively characterized the interactomes of

229 the related alarmones pppGpp and ppGpp and established the in vivo presence of pGpp

230 as a closely related alarmone in Gram-positive Bacillus species. We characterized the

231 direct targets of (p)ppGpp by screening an open reading frame expression library from B.

232 anthracis. From this screen, we identified an enzyme NahA that converts (p)ppGpp to

233 pGpp as efficient means to produce pGpp and to reduce (p)ppGpp concentrations, thus

234 regulating the composition of the alarmones. We demonstrated that pGpp binds a

235 distinct subset of protein receptors of (p)ppGpp. We also identified a key role of NahA in

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236 nutrient adaptation, suggesting that regulating alarmone composition may serve as a

237 separation-of-function strategy for optimal adaptation.

238

239 Conservation of pppGpp and ppGpp regulation of purine biosynthesis and

240 ribosome biogenesis pathways across different species of bacteria

241 (p)ppGpp regulates diverse cellular targets that differ between different bacteria.

242 For example, (p)ppGpp directly binds to RNA polymerase in E. coli to control the

243 of ribosomal and tRNA yet the (p)ppGpp binding sites on RNA

244 polymerase are not conserved beyond proteobacteria4–8. Instead, (p)ppGpp

245 accumulation in firmicutes strongly down-regulates synthesis of GTP, the exclusive

246 transcription initiating nucleotides of rRNA and tRNA operons in firmicutes, to achieve a

247 similar transcription control with different direct targets12,29,30. Therefore, identifying

248 whether certain aspects of (p)ppGpp regulation are conserved among bacterial species

249 is important for understanding bacterial survival and adaptation.

250 Our DRaCALA screen with a B. anthracis library revealed many ppGpp and

251 pppGpp binding targets in this pathogenic Gram-positive bacterium. Comparison to

252 ppGpp-binding targets in B. anthracis to S. aureus18 and E. coli15,20 identified novel

253 targets but more importantly, revealed a clear theme of conservation. Most notably,

254 (p)ppGpp in all three species binds to multiple proteins in two key pathways: 1) purine

255 nucleotide synthesis, 2) translation-related GTPases including ribosome biogenesis

256 factors.

257 The regulation of purine nucleotide synthesis in firmicutes includes well

258 characterized targets Gmk and HprT whose regulation by (p)ppGpp protects Bacillus

259 subtilis against nutrient changes like amino acid starvation and purine fluctuation,

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260 preventing cells from accumulating toxic high levels of intracellular GTP12,14,31. These

261 also include new-found likely targets such as enzymes GuaC and PurA and the de novo

262 pathway transcription factor PurR. Intriguingly, in the evolutionarily distant E. coli, the

263 ppGpp targets in the purine biosynthesis pathway are different. For example, Gmk is not

264 regulated by (p)ppGpp in E. coli. On the other hand, (p)ppGpp directly targets the E. coli

265 de novo enzyme PurF15, a target that is not conserved in firmicutes. Therefore, despite

266 differences in precise targets, (p)ppGpp extensively regulates the purine biosynthesis

267 pathway in evolutionarily diverse bacteria (Figure 1c), highlighting this critical

268 physiological role of (p)ppGpp.

269 We also found that (p)ppGpp interacts with essential GTPases that are

270 implicated in ribosome biogenesis and translation in B. subtilis and B. anthracis (Figure

271 1d). (p)ppGpp’s targets in GTPases from E. coli to firmicutes are conserved16–19: HflX,

272 Obg, Era, are also (p)ppGpp-binding proteins in E. coli and S. aureus15,18,20; Der and

273 TrmE were identified in E. coli although not in S. aureus as (p)ppGpp-binding

274 proteins15,20; RbgA doesn’t exist in E. coli and it is identified as a (p)ppGpp-binding

275 protein in S. aureus18. HflX is a ribosome-spliting factor which may rescue stalled

276 under stressed conditions32, and mediates the dissociation of hibernating

277 100S ribosome to resume normal translation33. Obg16, Der34 and RbgA35,36 participate in

278 the maturation of the 50S subunit of ribosome. Era functions in the assembly of the

279 functional 70S ribosome complex37. TrmE functions in the maturation of tRNA to

280 facilitate translation38. (p)ppGpp can be produced by amino acid starvation-induced

281 translational stress or by defects in tRNA maturation39. Thus the conservation of

282 (p)ppGpp regulation of GTPase targets highlights the key function of (p)ppGpp in quality

283 control of ribosome biogenesis and regulation of translation.

284

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285 The third major alarmone pGpp in Gram positive species and its specific

286 regulatory effect

287 In addition to pppGpp and ppGpp, it was long suspected that in B. subtilis, there

288 are additional alarmones such as pppApp, pGpp, and ppGp accumulating during the

289 stringent response40,41. Here we detected pGpp in B. subtilis cells, characterized an

290 enzyme for its production in vivo and in vitro, and identified its targets. In contrast to our

291 DRaCALA screens for pppGpp and ppGpp which identify similar targets and

292 functionalities between these two alarmones, DRaCALA screen for pGpp displays a

293 strong difference from (p)ppGpp with regarding to two conserved pathways. The affinity

294 of pGpp for purine nucleotide synthesis proteins is equivalent or higher than that of

295 (p)ppGpp. In contrast, pGpp’s affinity to GTPases involved in translational regulation is

296 much lower, or absent, compared to (p)ppGpp. The distinct profile of target receptors

297 distinguish pGpp as a different alarmone from (p)ppGpp, potentially allowing fine tuning

298 of bacterial stress response. We propose a model for the function of NahA in growth

299 recovery and competitive fitness by its role in transforming the alarmones. In wild type

300 cells, (p)ppGpp produced in response to amino acid starvation will be hydrolyzed in part

301 by NahA to pGpp. (p)ppGpp concentration in wild type cells during amino acid starvation

302 can reach >1 mM. Given the copy number of NahA as ~600 copies per cell and its

303 maximum velocities for (p)ppGpp hydrolysis, (p)ppGpp concentrations observed in wild

304 type cells is a balance of RelA synthesis and NahA hydrolysis. Thus, in ΔnahA cells,

305 (p)ppGpp accumulates higher than in wild type cells during amino acid starvation.

306 Stronger inhibition of translation due to overly high level of (p)ppGpp in ΔnahA leads to

307 slower growth recovery after culture enduring nutrient downshift and thus leads to fitness

308 loss when co-cultured with wild type strain. In addition, because pGpp appears to be

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309 hydrolyzed more efficiently by RelA (Figure 3b), another role of NahA is perhaps to

310 speed up (p)ppGpp removal to promote growth recovery.

311 In E. coli, (p)ppGpp binding proteins also include NuDiX hydrolases NudG and

312 MutT20. Like NahA, NudG and MutT also hydrolyze (p)ppGpp. Unlike NahA, NudG and

313 MutT produce guanosine 5’-monophosphate 3’-monophosphate (pGp) rather than pGpp.

314 Similarly, in the bacterium Thermus thermophilus, the NuDiX hydrolase Ndx8 is also

315 found to hydrolyze (p)ppGpp to produce pGp42. Sequence alignment shows that their

316 homology with NahA is mostly restricted in the NuDiX box that is shared by NuDiX

317 hydrolase family (Figure S1). Therefore, NudG, MutT and Ndx8 are considered

318 alternative (p)ppGpp hydrolysis pathways to remove (p)ppGpp and promote growth42,

319 rather than an alarmone-producer.

320 In E. coli, the enzyme GppA converts pppGpp to ppGpp, which may also regulate

321 the composition of its alarmones21. In the absence of GppA, the alarmone pppGpp

322 accumulates to a higher level than ppGpp43. It is possible that a similar separation-of-

323 function regulation exists in E. coli by tuning pppGpp vs ppGpp levels. This can be

324 addressed by examining pppGpp interactome in E. coli, which may reveal a differential

325 theme than ppGpp. Ultimately, discovery of more enzymes that interconvert signaling

326 molecules may reveal a universal theme of optimization through fine-tuning in signal

327 transduction.

328

329 Acknowledgement

330 We thank Mona Orr for technical help. We thank José Lemos for the gift of pGpp

331 standard. This work is supported, in part, by an R35 GM127088 from NIGMS (to JDW),

15 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.

332 a GRFP DGE-1256259 from NSF (to BWA) and R01AI110740 from NIAID and NIDDK

333 (to VTL).

334

335 Materials and Methods

336 Bacillus anthracis ORFeome Library Construction

337 Bacillus anthracis Gateway® Clone Set containing plasmids bearing B. anthracis open

338 reading frames was acquired from BEI Resources and used for Gateway cloning

339 (Invitrogen protocol) into overexpression vectors pVL791 (10xHis tag ampicillin-resistant)

340 and pVL847 (10xHis-MBP tag, gentamycin-resistant) and transformed into Escherichia

341 coli BL21 lacIq to produce two open reading frame proteome over-expression libraries

342 (ORFeome library). The resulting ORFeome library contains 5139 ORFs from the

343 genome of B. anthracis str. Ames (91.2% of 5632 ORFs in the genome). The

344 corresponding proteins were expressed in E. coli and the cells were lysed to prepare the

345 overexpression lysates for the downstream analysis.

346 Plasmid and Strain Construction and Growth Conditions

347 Plasmid for NahA purification was constructed as follows: nahA encoding sequence was

348 PCR amplified and incorporated into pE-SUMO vector by Golden Gate assembly

349 method (New England BioLabs), and into pVL847 (His-MBP-tag overexpression vector)

350 by Gateway cloning method (Invitrogen). ΔnahA mutant of Bacillus subtilis was

351 constructed by CRISPR/Cas9 editing method (Ref). Primers for plasmid construction

352 and mutation verification are listed in (Table S4). nahA deletion was confirmed by DNA

353 sequencing.

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354 If not specifically mentioned, B. subtilis strains were grown in S7 defined media 44 with

355 modifications (50 mM MOPS instead of 100 mM, 0.1% potassium glutamate, 1%

356 glucose, no additional tryptophan or methionine) , with shaking at 250 rpm 37℃. The

357 modified S7 defined media with 20 amino acids contains 50 μg/mL alanine, 50 μg/mL

358 arginine, 50 μg/mL asparagine, 50 μg/mL glutamine, 50 μg/mL histidine, 50 μg/mL lysine,

359 50 μg/mL proline, 50 μg/mL serine, 50 μg/mL threonine, 50 μg/mL glycine, 50 μg/mL

360 isoleucine, 50 μg/mL leucine, 50 μg/mL methionine, 50 μg/mL valine, 50 μg/mL

361 phenylalanine, 500 μg/mL aspartic acid, 500 μg/mL glutamic acid, 20 μg/mL tryptophan,

362 20 μg/mL tyrosine, and 40 μg/mL cysteine.

363 Nucleotide Preparation

364 (p)ppGpp was synthesized in vitro using RelSeq1-385 and GppA, purified and quantified

365 as demonstrated21. pGpp was synthesized, purified and quantified as demonstrated22.

366 Radiolabeled (p)ppGpp was synthesized in vitro using RelSeq1-385 and GppA as

367 demonstrated21 with modifications. We replaced non-radiolabeled GTP with 750 μCi/mL

368 32P-α-GTP (3000 mCi / mmol; PerkinElmer) for production of [5′-α-32P]-(p)ppGpp. We

369 replaced non-radiolabeled ATP with 750 μCi/mL 32P-γ-ATP (3000 mCi/mmol;

370 PerkinElmer) for production of [3′-β-32P]-ppGpp. After the reaction was done, we 1:10

371 diluted the reaction mix in Buffer A (0.1 mM LiCl, 0.5 mM EDTA, 25 mM Tris pH 7.5).

372 The mixture was loaded onto a Buffer-A-equilibrated 1 mL HiTrap QFF column (GE

373 Healthcare). Then the column was washed sequentially by 10 column volumes of Buffer

374 A, and 10 column volumes of Buffer A with 170 mM LiCl (for ppGpp purification, the LiCl

375 concentration was 160 mM) with 1 mL fractions. Radiolabeled (p)ppGpp was eluted by 5

376 column volumes of Buffer A with 500 mM LiCl with 1 mL fractions. The radioactivity and

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377 purity of radiolabeled (p)ppGpp were analyzed by thin-layer chromatography and

378 phosphorimaging.

379 Radiolabeled pGpp was synthesized in vitro as demonstrated above for radiolabeled

380 ppGpp synthesis with modifications. We replaced non-radiolabeled GTP with 750

381 μCi/mL 32P-α-GTP (3000 mCi / mmol; PerkinElmer) for production of [5′-α-32P]-pGpp. We

382 replaced GppA with NahA for production of [5′-α-32P]-pGpp. After the reaction was done,

383 we 1:10 diluted the reaction mix in Buffer A (0.1 mM LiCl, 0.5 mM EDTA, 25 mM Tris pH

384 7.5). The mixture was loaded onto a Buffer-A-equilibrated 1 mL HiTrap QFF column (GE

385 Healthcare). Then the column was washed by 10 column volumes of Buffer A, and

386 eluted by 10 column volumes of Buffer A with 155 mM LiCl with 1 mL fractions. Purified

387 radiolabeled pGpp often come out in the 5th to 9th column volumes during elution. The

388 radioactivity and purity of radiolabeled pGpp were analyzed by thin-layer

389 chromatography and phosphorimaging.

390 Overexpression and Purification of NahA

391 pE-SUMO-NahA was transformed into E. coli BL21(DE3) lacIq by chemical

392 transformation. A single colony of the corresponding strain was grown in LB with 30

393 μg/mL kanamycin overnight. Overnight culture was 1:100 diluted in LB-M9 media (7 g/L

394 Na2HPO4, 2 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 2 g/L glucose, 1 g/L sodium

395 succinate dibasic hexahydrate; 10 g/L tryptone, 5 g/L yeast extract and 3 mM MgSO4)

396 with 30 μg/mL kanamycin and grown at 30℃ with shaking for 4 hr. After adding 1 mM

397 IPTG for induction, the culture was further grown at 30℃ for 4 hr. Then the culture was

398 centrifuged at 4000 g for 30 min at 4℃ and the supernatant was discarded. The pellet

399 was stored at -80℃ before cell lysis. All the following steps were performed at 4℃.

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400 Pellet was re-suspended in Lysis Buffer (50mM Tris-HCl pH 8, 10% sucrose w/v, 300mM

401 NaCl) and lysed by French press. Cell lysate was centrifuged at 15000 rpm for 30 min,

402 and the supernatant was collected, filtered through 0.45 μm pore-size cellulose syringe

403 filter. His-SUMO-NahA filtered supernatant was purified using a 5 mL HisTrap FF

404 column (GE Healthcare) equipped on an ÄKTA FPLC apparatus (GE Healthcare).

405 SUMO Buffer A (50 mM Tris-HCl, 25 mM imidazole, 500 mM NaCl, 5% glycerol v/v) was

406 used as washing buffer, and SUMO Buffer C (50 mM Tris-HCl, 500 mM imidazole, 500

407 mM NaCl, 5% glycerol v/v) was used as the elution buffer. Fractions containing most

408 abundant His-SUMO-NahA were combined with 100 μg SUMO Protease and dialyzed

409 twice against SUMO Protease Buffer (50 mM Tris-HCl, 500 mM NaCl, 5% glycerol v/v, 1

410 mM β-mercaptoethanol) overnight. His-SUMO tag was removed by flowing through

411 HisTrap FF column and collecting the flow-through. Purified NahA was analyzed by

412 SDS-PAGE and the concentration was measured by the Bradford assay (Bio-rad).

413 Differential Radial Capillary Action Ligand Assay (DRaCALA)

414 Cell lysate with overexpressed protein and purified protein were used for DRaCALA as

415 described24. 10 μL cell lysate or diluted, purified protein was mixed with 10 μL diluted [5′-

416 α-32P]-(p)ppGpp or [5′-α-32P]-pGpp (~0.2 nM) in a buffer containing 10 mM Tris pH 7.5,

417 100 mM NaCl and 5 mM MgCl2, incubated at room temperature for 10 min. ~2 μL

418 mixture was blotted onto nitrocellulose membrane (Amersham; GE Healthcare) and

419 allowed for diffusion and drying. The nitrocellulose membrane loaded with mixture was

420 exposed on phosphor screen, which was scanned by a Typhoon FLA9000 scanner (GE

421 Healthcare). Fraction of (p)ppGpp or pGpp binding was analyzed as described24.

422 Quantification of Intracellular Nucleotides by Thin-Layer Chromatography

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423 Quantification of ATP, GTP, and (p)ppGpp levels by thin-layer chromatography was

424 performed as described before 45,46 with modifications. Cells were grown in low

425 phosphate S7 defined media (0.5 mM phosphate instead of 5 mM), labeled with 50

426 μCi/mL culture decontaminated 32P-orthophosphate (9000 mCi/mmol; Perkin-Elmer)

427 (decontamination was performed as demonstrated previously47) at an optical density at

428 600 nm (OD600nm) of ~0.01 and grown for two to three additional generations before

429 sampling. Nucleotides were extracted by adding 100 μL of sample into 20 μL 2 N formic

430 acid on ice for at least 20 min. Extracted samples were centrifuged at 15,000 g for 30

431 min to remove cell debris. Supernatant samples were spotted on PEI cellulose plates

432 (EMD-Millipore) and developed in 1.5 M or 0.85 M potassium phosphate monobasic

433 (KH2PO4) (pH 3.4). TLC plates were exposed on phosphor screens, which were scanned

434 by a Typhoon FLA9000 scanner (GE Healthcare). Nucleotide levels were quantified by

435 ImageJ (NIH). Nucleotide levels are normalized to ATP level at time zero.

436 Kinetic Assay of (p)ppGpp Conversion by NahA

437 100 μL reaction mix was prepared with 40 mM Tris HCl pH 7.5, 100 mM NaCl, 10 mM

32 438 MgCl2, non-radioactive and P-radiolabeled (p)ppGpp (concentration as required by

439 specific experiments), 100 nM purified NahA (add enzyme last). Before adding enzyme,

440 reaction mix was prewarmed at 37℃ for at least 10 min. After adding enzyme, 10 μL

441 reaction mix was aliquoted into 10 μL ice-chilled 0.8 M formic acid to stop the reaction

442 and preserve nucleotides at every time point. Samples at each time point were resolved

443 by thin-layer chromatography on PEI-cellulose plates with 1.5 M KH2PO4 (pH 3.4).

444 Nucleotide levels were quantified as mentioned above and the phosphorimager counts

445 of substrate and product were used to calculate the concentration of product by a

446 formula:

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  ͤ ·  

447 Here was the concentration of product, was the concentration of substrate before

448 the reaction starts, was the phosphorimager count of product, and was the

449 phosphorimager count of substrate. Initial rates of hydrolysis () were calculated using

450 the slope of the initial linear part of over time curve at different initial substrate

451 concentrations. Michaelis-Menten constant () and catalytic rate constant () were

452 obtained by fitting the data of - to the model

)  ·/ ·ͤ ͤ ) ) ( ͤ

453 by MATLAB, where was the concentration of NahA, was the initial concentration

454 of substrate, and was the Hill’s coefficient (for ppGpp hydrolysis, fix to 1).

455 Kinetic Assay of (p)ppGpp Hydrolysis by RelA

456 100 μL reaction mix was prepared with 25 mM Tris HCl pH 7.5, 1 mM MnCl2, 100 μM

457 non-radioactive and ~0.2nM 32P-radiolabeled (p)ppGpp, 200 nM purified RelA (add

458 enzyme last). Before adding enzyme, reaction mix was prewarmed at 37℃ for at least

459 10 min. After adding enzyme, 10 μL reaction mix was aliquoted into 10 μL ice-chilled 0.8

460 M formic acid to stop the reaction and preserve nucleotides at every time point. Samples

461 at each time point were resolved by thin-layer chromatography on PEI-cellulose plates

462 with 1.5 M KH2PO4 (pH 3.4). Nucleotide levels were quantified as mentioned above and

463 the phosphorimager counts of substrate and product were used to calculate the

464 concentration of product by a formula:

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  ͤ ·  

465 Here was the concentration of product, was the concentration of substrate before

466 the reaction starts, was the phosphorimager count of product, and was the

467 phosphorimager count of substrate.

468 LC-MS Quantification of Metabolites

469 Cells were grown to designated OD600nm. 5 mL of cultures were sampled and filtered

470 through PTFE membrane (Sartorius) at time points before and after 0.5 mg/mL arginine

471 hydroxamate treatment. Membranes with cell pellet were submerged in 3 mL extraction

472 solvent mix (on ice 50:50 (v/v) chloroform/water) to quench metabolism, lyse the cells

473 and extract metabolites. Mixture of cell extracts were centrifuged at 5000 g for 10 min to

474 remove organic phase, then centrifuged at 20000 g for 10 min to remove cell debris.

475 Samples were frozen at -80℃ if not analyzed immediately. Samples were analyzed

476 using an HPLC-tandem MS (HPLC-MS/MS) system consisting of a Vanquish UHPLC

477 system linked to heated electrospray ionization (ESI, negative mode) to a hybrid

478 quadrupole high resolution mass spectrometer (Q-Exactive orbitrap, Thermo Scientific)

479 operated in full-scan selected ion monitoring (MS-SIM) mode to detect targeted

480 metabolites based on their accurate masses. MS parameters were set to a resolution of

481 70,000, an automatic gain control (AGC) of 1e6, a maximum injection time of 40 ms,

482 and a scan range of 90-1000 mz. LC was performed on an Aquity UPLC BEH C18

483 column (1.7 μm, 2.1 X 100 mm; Waters). Total run time was 30 min with a flow rate of

484 0.2 mL/min, using Solvent A (97:3 (v/v) water/methanol, 10 mM tributylamine pH~8.2-

485 8.5 adjusted with approximately 9 mM acetic acid) and 100% acetonitrile as Solvent B.

486 The gradient was as follows: 0 min, 5% B; 2.5 min, 5% B; 19 min, 100% B; 23.5 min

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487 100% B; 24 min, 5% B; 30 min, 5% B. Raw output data from the MS was converted to

488 mzXML format using inhouse-developed software, and quantification of metabolites

489 were performed by using the Metabolomics Analysis and Visualization Engine

490 (MAVEN) software suite48,49. Normalized ion count was defined and calculated as the ion

491 count per OD600 per unit volume (5 mL) of the culture.

492 Growth Competition Assay

R 493 nahA∷erm mutant and wild type cells were mixed in LB broth to an OD600nm of 0.03 and

494 grown at 37℃ with vigorous shaking. After every 24 hour period, the stationary phase

495 culture was back-diluted in fresh LB broth to an OD600nm of 0.02, for a total period of 7

496 days. Each day, the culture was sampled, serially diluted and spread over LB agar and

497 LB agar containing 0.5 μg/mL erythromycin/12.5 μg/mL lincomycin to obtain the CFU of

498 total bacteria and erythromycin-resistant strain, respectively. Relative fitness was

/ v ĘĥĠċ ĘĥĠċ 499 calculated by the formula50: ħ t , where w means relative fitness Č / Č v ĘĥĠ ħ ĘĥĠ t

ċ 500 of the erythromycin-resistant strain to the erythromycin-sensitive strain; t and

ċ 501 ħ means the total CFU of erythromycin-resistant strain before and after

Č Č 502 competition, respectively; t and ħ means the total CFU of erythromycin-

503 sensitive strain before and after competition, respectively.

504 [35S]-Methionine Incorporation Assay

505 [35S]-Methionine (10.2 mCi/mL, PerkinElmer) was diluted to 0.5 μCi/μL and aliquoted into

506 1.5 mL Eppendorf tubes (10 μL per tube, heated at 37). Cells were grown to OD600nm at

507 around 0.2 (record the exact OD). Aliquots of 200 μL culture were taken at time points

508 before and after 0.5 mg/mL RHX treatment, added into tubes of aliquoted [35S]-

509 Methionine, incubated at 37℃ for 5 min and fixed by adding 200 μL ice-chilled 20% (w/v)

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510 trichloroacetic acid. Fixed samples were chilled before filtration. Glass fiber filters (24mm,

511 GF6, Whatman) were prewetted with 3 mL ice-chilled 5%(w/v) trichloroacetic acid and

512 applied with fixed samples. Filters were then washed three times by 3 x 10 mL ice-

513 chilled 5%(w/v) trichloroacetic acid and dried by ethanol. Dried filters were put in

514 scintillation vials, mixed with 5 mL scintillation fluid and then sent for scintillation count in

515 the range of 2.0-18.6 eV. Counts per minutes (CPM) measured by scintillation counter

516 were used as the representative of translation rate. The ratio of the CPM of one sample

517 relative to the CPM at time zero was calculated as the relative translation rate.

518 Growth recovery from nutrient downshift

519 Cells were grown in defined rich medium (S7 Glucose + 20 amino acids) to mid log

520 phase, and then washed in poor medium (S7 Glucose without amino acid). Washed

521 cultures were diluted into fresh rich medium and the growth was monitored by a plate

522 reader (Synergy 2, Biotek) at 37℃ under shaking.

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523 Figure 1. Proteome-wide DRaCALA screen identifies both conserved categories of

524 binding targets and novel targets.

525 a) The Bacillus anthracis ORF donor vector library was recombined by Gateway cloning

526 into overexpression vectors to generate ORFs with an N-terminal His-tag or His-MBP tag.

527 The plasmids were transformed into E. coli for overexpression of recombinant proteins.

528 Lysates of each ORF overexpressed in E. coli were assayed for binding to pppGpp,

529 ppGpp and pGpp using DRaCALA. b) List of identified (p)ppGpp binding targets in E.

530 coli, S. aureus, and B. anthracis and pGpp binding targets in B. anthracis. c-d)

531 Schematics of pathways differentially regulated by pGpp and (p)ppGpp: c) Enzymes in

532 purine nucleotide synthesis, including HprT, Xpt, Gmk and GuaC, bound both pGpp and

533 (p)ppGpp; d) GTPases, involved in ribosome biogenesis and translational control (Obg,

534 HflX, Der, RbgA and Era), bound (p)ppGpp, but not pGpp.

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535 Figure 2. NahA (YvcI) produces pGpp via (p)ppGpp hydrolysis.

536 a) DRaCALA of [5′-α-32P]-ppGpp binding to B. subtilis HisMBP labeled -YvcI (NahA)

537 overexpressed in E. coli cell lysate. Unlablled GTP (100 uM), unlabeled ppGpp (100 μM)

538 and EDTA (10 mM) were added as indicated. b) Ion count vs. retention time curves from

539 LC-MS of GTP and pGpp standards and NahA-catalyzed hydrolysis products from

540 ppGpp and pppGpp. c) TLC analysis of NahA activity over time with [3′-β-32P]-pppGpp.

541 Expected NahA-catalyzed conversion of [3′-β-32P]-pppGpp to [3′-β-32P]-pGpp is shown

542 on the right. Radiolabeled [3′-β-32P]-phosphorus atom is highlighted in red. d)

543 Quantitation of pppGpp and pGpp in the hydrolysis of pppGpp in (c). e) Initial velocity vs.

544 pppGpp or ppGpp concentration curves for NahA synthesis of pGpp. Data were obtained

545 by kinetic assay using radiolabels (see Methods). Curves represent the best fit of the

546 data from three independent experiments. Error bars represent standard error of the

547 mean.

548

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549

550 Figure 3. pGpp is produced by NahA in vivo and has a distinct binding spectrum

551 compared to (p)ppGpp.

552 a) LC-MS analyses of pGpp, ppGpp and pppGpp of wild type and ΔnahA in log phase

553 and stationary phase. Normalized ion count is ion count per OD600 per unit volume of the

554 culture. Error bars represent standard error of the mean of three biological replicates. A

555 two-tailed two-sample equal-variance Student’s t test was performed between samples

556 indicated by asterisks. Asterisks indicate statistical significance of differences (* P≤0.05,

557 ** P≤0.01, *** P≤0.001). b) RelA hydrolysis of pGpp, ppGpp and pppGpp over time. For

558 each nucleotide, 200 nM RelA was incubated with a mix of 100 μM unlabeled alarmone

559 and a small amount of 5′-α-32P-labeled version. The degradation of the alarmone was

560 analyzed by TLC to measure the decreased radioactivity. Error bars represent standard

561 error of the mean of three replicates. c) DRaCALA of purified His-MBP-tagged B.

562 anthracis proteins (1 μM) with less than 0.1 nM of identical amount of 5′-α-32P-labeled

563 pGpp, ppGpp, and pppGpp. d) Quantification of DRaCALA in (c). e-g) Titrations of B.

564 anthracis GTPases and quantification of their binding to pGpp, ppGpp and pppGpp

565 using DRaCALA. GTPases HflX e), Obg f) and translation elongation factor G (EF-G) g).

566 Error bars represent standard errors of the mean. h) Schematic showing the relationship

567 between pGpp binding targets and (p)ppGpp binding targets.

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568 Figure 4. The effect of NahA on purine nucleotides, translation, fitness and growth

569 recovery of B. subtilis cells. a) Hierarchical clustering of selected metabolites in

570 exponential growth and stationary phase wild type and ΔnahA cells. Metabolites were

571 measured by LC-MS. Normalized ion count is ion count per OD600 per unit volume of the

572 culture. b) Protein translation rate of wild type and ΔnahA cells in a time course of

573 arginine hydroxamate (RHX) treatment. Translation rate was measured by a two minutes

574 pulse of [35S]-methionine incorporation into TCA-precipitable fraction. Error bars

575 represent standard errors of the mean from three replicates. c) Relative fitness of ΔnahA

576 and wild type strains obtained from a 7 day serial-dilution competition assay. Error bar

577 represents standard deviation from 3 repeats. d) Doubling times of ΔnahA and wild type

578 cells in log phase. A two-tailed two-sample equal-variance Student’s t test was

579 performed between samples indicated by asterisks. Asterisk indicates statistical

580 significance of difference (* P≤0.05, *** P≤0.001, n.s. P>0.05). e) Growth recovery from

581 nutrient downshift. Log phase cultures of wild type, ΔnahA and nahA complementation

582 (ΔnahA IPTG-nahA) strains in rich media were firstly downshifted in minimum media

583 without amino acids for 10 min, and then diluted in rich media for outgrowth. Error bars

584 in (d-e) represent standard errors from 6 replicates.

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585 Figure S1. Protein sequence alignment of NahA (YvcI) homologs in different

586 species.

587 Multiple sequence alignment of YvcI homologs from B. subtilis (Bsub), B. anthracis (Ban),

588 B. halodurans (Bhalo), Thermus thermophilus (TTherm) and E. coli (Ec). Alignment was

589 obtained with MAFFT. Red shading indicates identical residues among Bacillus and

590 other species, and orange shading indicates identical residues only in Bacillus species.

591 The Bacillus halodurans YvcI structure (PDB ID: 3FK9) shows its conserved structure as

592 a NuDiX hydrolase. Red dots label the key residues for catalytic activity. Numbers on the

593 sides of the sequences indicate the residue numbers in the corresponding proteins.

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594 Figure S2. Cell lysate containing YvcI, but not purified YvcI, binds (p)ppGpp.

595 DRaCALA of cell lysate containing His-MBP-tagged B. subtilis YvcI(NahA) and purified

596 His-MBP-tagged B. subtilis YvcI(NahA) with 5’-α-32P-labeled (p)ppGpp.

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597 Figure S3. NahA hydrolyzes (p)ppGpp in vitro.

598 a) TLC analysis of pppGpp and ppGpp hydrolysis by NahA over time. The same assay

599 was also performed using GTP. b) TLC analysis of ppGpp hydrolysis by RelA and NahA

600 over time. Substrate and product identities are labeled on the left side of the figure.

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601 Figure S4. nahA complementation rescues pGpp production in vivo.

602 a) Intensity profile of pGpp from LC-MS measurement of mid log phase wild type, ΔnahA

603 and nahA complementation strain (ΔnahA IPTG-nahA). x-axis: retention time in LC-MS;

604 y-axis: ion counts. For each strain, a representative curve was shown. Dashed lines are

605 the shoulders of the GTP signal peak. b) Normalized ion count (ion count per OD600 per

606 unit volume of the culture) of corresponding pGpp levels, obtained as the average of 3

607 biological replicates in a). Error bars represents standard error of the mean.

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608 Figure S5. nahA mutant displayed higher (p)ppGpp and lower pGpp in vivo in B.

609 subtilis in a time course of amino acid starvation.

610 a-b) pppGpp (a) and ppGpp (b) levels in wild type and ΔnahA B. subtilis cells treated

611 with the nonfunctional arginine analog RHX (arginine hydroxamate). Nucleotide levels

612 were assayed via TLC and are normalized to the ATP level at time zero (ATPt=0). Error

613 bars represent standard errors of mean of three independent experiments. c-e) LC-MS

614 analyses of pppGpp (c), ppGpp (d) and pGpp (e) of wild type and ΔnahA in a time

615 course of RHX treatment. Normalized ion count is ion count per OD600 per unit volume of

616 the culture. Error bars represent standard error of the mean of two biological replicates.

617 Asterisks indicate statistical significance of differences (* P≤0.05, ** P≤0.01).

33 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.

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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 Fig. 1 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

B. anthracis (This Work) E. coli 15,20 S. aureus 18 a b Name pppGpp ppGpp pGpp (p)ppGpp ppGpp

Purine Synthesis MBP ORF DRaCALA His-tag pGpp Pathway HprT + + + +15,20 + Transformation Xpt + + + NA ppGpp 5341 vectors Gmk + + + + 15 ORF PurA + + + + Gateway Overexpression ×58 Plates pppGpp GuaC NT + + Lysis PurR + + + Cloning His-MBP-tagged His-MBP-tagged PurB +15 Overexpression Overexpression 5341 vectors 15 Vector Lysate PurC + PurD +15 PurF +15 His-tag ORF DRaCALA Gpt NA NA NA +15,20 B. anthracis ORF pGpp BEI Donor Vector Transformation Ribosome and

5341 vectors Translation ppGpp Regulation 20 Overexpression ×58 Plates HflX + + + + Lysis pppGpp Der + + +15,20 His-tagged His-tagged Obg + + +15,20 + Overexpression Overexpression Vector Lysate RbgA + + NA + TrmE + + +15,20 Era + + +15,20 +

(p)ppGpp

Metabolism (p)ppGpp synthetase + + + +20 NuDiX hydrolase + + +(MutT)20 5′-nucleotidase + + + +(UshA)15

c d Hypoxanthine PurF PurD HprT Obg 100S PurC Der PurB PurA RbgA HflX PRPP IMP ATP 50S GuaB Xpt 70S Xanthine XMP GuaC 30S Translation GuaA Era HprT Gmk Guanine GMP GDP GTP Purine nucleotide synthesis Ribosome biogenesis and translation regulation

Red: bound by pGpp, ppGpp and pppGpp Blue: bound by ppGpp and pppGpp, but not by pGpp Figure 1. Proteome-wide DRaCALA screen identifies both conserved categories of binding targets and novel targets. a) The Bacillus anthracis ORF donor vector library was recombined by Gateway cloning into overexpression vectors to generate ORFs with an N-terminal His-tag or His-MBP tag. The plasmids were transformed into E. coli for overexpression of recombinant proteins. Lysates of each ORF overexpressed in E. coli were assayed for binding to pppGpp, ppGpp and pGpp using DRaCALA. b) List of identified (p)ppGpp binding targets in E. coli, S. aureus, and B. anthracis and pGpp binding targets in B. anthracis. c-d) Schematics of pathways differentially regulated by pGpp and (p)ppGpp: c) Enzymes in purine nucleotide synthesis, including HprT, Xpt, Gmk and GuaC, bound both pGpp and (p)ppGpp; d) GTPases, involved in ribosome biogenesis and translational control (Obg, HflX, Der, RbgA and Era), bound (p)ppGpp, but not pGpp. 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 Fig. 2 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a b GTP pGpp 32P-ppGpp + + + + GTP Standard GTP - + - - 4.8×107 EDTA - - + - pGpp Standard ppGpp - - - + 3.2×107 YvcI/NahA 1.6×107

0

O 4.8×107 ppGpp c N hydrolysis product O NH pGpp POH O N 7 O N NH2 3.2×10 OH

O O OH 7

Ion Count 1.6×10 OH P O OHP OH O [3′-β-32P]-pGpp 0 PPi 6.4×107 NahA pppGpp O hydrolysis product H2O N 7 NH 4.8×10 O O O OH P PO PO O N O N NH2 OH OH OH 7 pppGpp 3.2×10 O O OH OH P O OHP 1.6×107 0 10 20 40 80 0 10 20 40 80 OH O Time (min) [3′-β-32P]-pppGpp NahA No Enzyme 0 10.85 11.25 11.65 12.05 Retention Time (min)

d e pGpp production by NahA Hydrolysis of pppGpp and synthesis of pGpp by NahA 1000 1000 pGpp 800 800 pppGpp

600 600 (nM/s) 0 V 400 400

Radioactivity (a.u.) 200 200 ppGpp pppGpp 0 0 10 20 30 40 50 60 70 80 90 0 Time (min) 0 200 400 900 1000 Substrate concentration (μM)

Figure 2. NahA (YvcI) produces pGpp via (p)ppGpp hydrolysis. a) DRaCALA of [5′-α-32P]-ppGpp binding to B. subtilis HisMBP labeled -YvcI (NahA) overexpressed in E. coli cell lysate. Unlablled GTP (100 uM), unlabeled ppGpp (100 μM) and EDTA (10 mM) were added as indicated. b) Ion count vs. retention time curves from LC-MS of GTP and pGpp standards and NahA-catalyzed hydrolysis products from ppGpp and pppGpp. c) TLC analysis of NahA activity over time with [3′-β-32P]-pppGpp. Expected NahA-catalyzed conversion of [3′-β-32P]-pppGpp to [3′-β-32P]-pGpp is shown on the right. Radiolabeled [3′-β-32P]-phosphorus atom is highlighted in red. d) Quantitation of pppGpp and pGpp in the hydrolysis of pppGpp in (c). e) Initial velocity vs. pppGpp or ppGpp concentration curves for NahA synthesis of pGpp. Data were obtained by kinetic assay using radiolabels (see Methods). Curves represent the best fit of the data from three independent experiments. Error bars represent standard error of the mean. 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 Fig. 3 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. a WT pppGpp 6 * 6×10 ∆nahA d DRaCALA of purified B. anthracis protein e 80 HflX 4×106 60 pGpp ppGpp pGpp pppGpp ppGpp 2×106 60 pppGpp 40 0 ppGpp 40 1.5×107 * 20 1×107 20

5×106 * 0 0 Fraction of Radiolabel Binding (%) Fraction of Radiolabel Binding (%) Fraction

Normalized Ion Count 0 0.01 0.1 1 10 pGpp Ctrl Xpt Der *** HprT Gmk HflX Obg 6×105 *** Purine nucleotide Ribosome Protein concentration (µM) synthesis regulation 4×105 f g 2×105 60 Obg 15 EF−G 0 0 pGpp pGpp Mid Log Phase Stationary Phase ppGpp ppGpp pppGpp pppGpp b Hydrolysis of pGpp and (p)ppGpp by RelA 40 10 100 pGpp 80 ppGpp 20 5 pppGpp 60 0 0

40 of Radiolabel Binding (%) Fraction of Radiolabel Binding (%) Fraction 0.01 0.1 1 10 0.01 0.1 1 10

Concentration ( μ M) Concentration 20 Protein concentration (µM) Protein concentration (µM)

0 0 5 10 15 20 h (p)ppGpp targets c Time (min)

pGpp ppGpp pppGpp pGpp ppGpp pppGpp Hpt1 HflX pGpp targets Ribosome and translation Gmk Obg Purine nucleotide regulation GTPases synthesis proteins Xpt Der

Ctrl Ctrl

Figure 3. pGpp is produced by NahA in vivo and has a distinct binding spectrum compared to (p)ppGpp. a) LC-MS analyses of pGpp, ppGpp and pppGpp of wild type and ΔnahA in log phase and stationary phase.

Normalized ion count is ion count per OD600 per unit volume of the culture. Error bars represent standard error of the mean of three biological replicates. A two-tailed two-sample equal-variance Student’s t test was performed between samples indicated by asterisks. Asterisks indicate statistical significance of differences (* P≤0.05, ** P≤0.01, *** P≤0.001). b) RelA hydrolysis of pGpp, ppGpp and pppGpp over time. For each nucleotide, 200 nM RelA was incubated with a mix of 100 μM unlabeled alarmone and a small amount of 5′-α-32P-labeled version. The degradation of the alarmone was analyzed by TLC to measure the decreased radioactivity. Error bars represent standard error of the mean of three technical replicates. c) DRaCALA of purified His-MBP-tagged B. anthracis proteins (1 μM) with less than 0.1 nM of identical amount of 5′-α-32P-labeled pGpp, ppGpp, and pppGpp. d) Quantification of DRaCALA in (c). e-g) Titrations of B. anthracis GTPases and quantification of their binding to pGpp, ppGpp and pppGpp using DRaCALA. GTPases HflX e), Obg f) and translation elongation factor G (EF-G) g). Error bars represent standard errors of the mean. h) Schematic showing the relationship between pGpp binding targets and (p)ppGpp binding targets. 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 Fig. 4 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a b Protein translation d Exponential growth ADP n.s. 100 n.s. UDP 50 UMP PRPP 50 WT 40 XMP ∆nahA CTP UTP 25 30 CDP IMP 20 GTP 12.5 S]−met inco rporation (%)

ATP 35 PEP * (min) Time Doubling 10 Inosine 6.25 Hypoxanthine 0 Guanine Rate of [ 0 20 40 60 WT ∆nahA ∆nahA pGpp IPTG-nahA AMP Time after RHX treatment (min) GDP GMP c Competition growth assay e CMP Growth recovery from nutrient downshift Guanosine 1.2 Cytidine *** 1.000 Uridine 1.0 Adenine pppGpp 0.8 0.500 ppGpp

e fitness 0.6 600nm WT WT 0.250

∆nahA ∆nahA OD 0.4

Relativ WT ∆nahA 0.2 0.125 ∆nahA IPTG-nahA Mid log phase 0.0 Stationary Phase WT ∆nahA 0 100 200 300 400 500

log2(Normalized ion count) Time (min)

-6 -4 -2 0 2 4

Figure 4. The effect of NahA on purine nucleotides, translation, fitness and growth recovery of B. subtilis cells. a) Hierarchical clustering of selected metabolites in exponential growth and stationary phase wild type and

ΔnahA cells. Metabolites were measured by LC-MS. Normalized ion count is ion count per OD600 per unit volume of the culture. b) Protein translation rate of wild type and ΔnahA cells in a time course of arginine hydroxamate (RHX) treatment. Translation rate was measured by a two minutes pulse of [35S]-methionine incorporation into TCA-precipitable fraction. Error bars represent standard errors of the mean from three replicates. c) Relative fitness of ΔnahA and wild type strains obtained from a 7 day serial-dilution competition assay. Error bar represents standard deviation from 3 repeats. d) Doubling times of ΔnahA and wild type cells in log phase. A two-tailed two-sample equal-variance Student’s t test was performed between samples indicated by asterisks. Asterisk indicates statistical significance of difference (* P≤0.05, *** P≤0.001, n.s. P>0.05). e) Growth recovery from nutrient downshift. Log phase cultures of wild type, Δ nahA and nahA complementation (ΔnahA IPTG-nahA) strains in rich media were firstly downshifted in minimum media without amino acids for 10 min, and then diluted in rich media for outgrowth. Error bars in (d-e) represent standard errors from 6 replicates. 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.

ppGpp pppGpp

-1 kcat (s ) 1.22 ± 0.17 10.0 ± 0.5

KM (μM) 7.5 ± 2.3 177.4 ± 0.4

Hill coefficient 1 2.78 ± 0.47

Table 1. Kinetic parameters of (p)ppGpp hydrolysis by NahA. 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 Fig. S1 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Bsub_NahA 1 MTYLQRVTNCVLQTDDKVLLLQKP----RRGWWVAPGGKMESGESVRDSVIREYREETGIYIINPQLKGVFTFIIKD-GDHIVSEWMMFT 85 2 Ban_YvcI(BA5385) 1 ---MQRVTNCVLIRDNEVLLLQKP----RRNWWVAPGGKMERGETVRDSVVREYREETGIYLKNPALKGVFTFVIQE-GDKVVSEWMMFS 82 3 Bhalo_YvcI(BH3570) 1 ---MQRVTNCIVVDHDQVLLLQKP----RRGWWVAPGGKMEAGESILETVKREYWEETGITVKNPELKGIFSMVIFD-EGKIVSEWMLFT 82 1 Bsub_NahA 1 M-TYLQRVTNCVLQTDDKVLLLQKP----RRGWWVAPGGKMESGESVRDSVIREYREETGIYIINPQLKGVFTFIIKDGDHIVSEWMMFT 85 4 EcMutT 1 MKKLQIAVGIIRNENNEIFITRRAADAHMANKLEFPGGKIEMGETPEQAVVRELQEEVGITPQHFSLFEKLEYEFPDRHITLWFWLVERW 90 2 Ban_YvcI(BA5385) 1 ----MQRVTNCVLIRDNEVLLLQKP----RRNWWVAPGGKMERGETVRDSVVREYREETGIYLKNPALKGVFTFVIQEGDKVVSEWMMFS 82 5 EcNudG 1 MK-MIEVVAAIIERDGKILLAQRPAQSDQAGLWEFAGGKVEPDESQRQALVRELREELGIEATVGEYVASHQREVSGRIIHLHAWHVPDF 89 3 Bhalo_YvcI(BH3570) 1 ----MQRVTNCIVVDHDQVLLLQKP----RRGWWVAPGGKMEAGESILETVKREYWEETGITVKNPELKGIFSMVIFDEGKIVSEWMLFT 82 4 TTherm_Ndx8 1 MRREILVAAAILLDSRGRVLLVGNDWGRRGRVRYTLPGGTVEPGETAVEALVREVREETGLRVRSVEHLA-YVIQVEDRRKN-ERTLAMA 88 5 EcMutT 1 M-KKLQIAVGIIRNENNEIFITRRAADAHMANKLEFPGGKIEMGETPEQAVVRELQEEVGITPQHFSLFEKLEYEFPDRHITLWFWLVER 89 6 EcNudG 1 M--KMIEVVAAIIERDGKILLAQRPAQSDQAGLWEFAGGKVEPDESQRQALVRELREELGIEATVGEYVASHQREVSGRIIHLHAWHVPD 88 NuDiX Box 1 Bsub_NahA 86 FVADSYTGQNV-SESEEGKLQWHDVNDIQNLPMAPGDGHILDFMMKGQGLLHGTFTYTPEFELLSYRLDPQ-HIK--- 158 2 Ban_YvcI(BA5385) 83 FLATDFAGENK-LESEEGIIGWHTFDKIDDLAMAPGDYHIIDYLIKGNGIIYGTFVYTPDFELLSYRLDPS------152 1 Bsub_NahA 86 FVADSYTGQNVSESEEGK-LQWHD--VNDIQNLPMAPGDGHILDFMMKGQGLLHGTFTYTPEFELLSYRL------DPQHI----K 158 3 Bhalo_YvcI(BH3570) 83 FKATEHEGEML-KQSPEGKLEWKKKDEVLELPMAAGDKWIFKHVLHSDRLLYGTFHYTPDFELLSYRLDPEPQMKKGV 159 2 Ban_YvcI(BA5385) 83 FLATDFAGENKLESEEGI-IGWHT--FDKIDDLAMAPGDYHIIDYLIKGNGIIYGTFVYTPDFELLSYRL------DPS------152 4 EcMutT 91 EGEPWGKEGQP------GEWMSLVGLNADDFP--PANEPVIAKLKR------L------129 3 Bhalo_YvcI(BH3570) 83 FKATEHEGEMLKQSPEGK-LEWKK--KDEVLELPMAAGDKWIFKHVLHSDRLLYGTFHYTPDFELLSYRL------DPEPQMKKGV 159 5 EcNudG 90 HGTLQAHEHQA------LVWCS--PEEALQYPLAPADIPLLEAFMA------LRAAR--PAD------135 4 TTherm_Ndx8 89 FRAT-YEGLLNPRDPDGHIVEARFFTLEEVEERL--KGHLPLLEPLRA------Y-----LRGERGRFYAYAGWHAPGVV----V 155 5 EcMutT 90 WEGE------PWGKEGQPGEWMSLVGLNADDFP--PANEPVIAKLKR------L------129 6 EcNudG 89 FHGT------LQAHEHQALVWCS--PEEALQYPLAPADIPLLEAFMA------LRAAR------PAD------135 Figure S1. Protein sequence alignment of NahA (YvcI) homologs in different species. Multiple sequence alignment of YvcI homologs from B. subtilis (Bsub), B. anthracis (Ban), B. halodurans (Bhalo), Thermus thermophilus (TTherm) and E. coli (Ec). Alignment was obtained with MAFFT. Red shading indicates identical residues among Bacillus and other species, and orange shading indicates identical residues only in Bacillus species. The Bacillus halodurans YvcI structure (PDB ID: 3FK9) shows its conserved structure as a NuDiX hydrolase. Red dots label the key residues for catalytic activity. Numbers on the sides of the sequences indicate the residue numbers in the corresponding proteins. 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 Fig. S2 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

ppGpp His-MBP-YvcI lysate pppGpp

ppGpp Purified YvcI pppGpp

Figure S2. Cell lysate containing YvcI, but not purified YvcI, binds (p)ppGpp. DRaCALA of cell lysate containing His-MBP-tagged B. subtilis YvcI(NahA) and purified His-MBP-tagged B. subtilis YvcI(NahA) with 5’-α-32P-labeled (p)ppGpp. 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 Fig. S3 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a

pGpp

GTP

ppGpp

pppGpp

0 10 20 40 80 120 160 0 10 20 40 80 120 160 0 10 20 40 80 120 160 Time (min) ppGpp pppGpp GTP YvcI(NahA)

b

GDP

pGpp

ppGpp

0 10 20 40 80 0 10 20 40 80 Time (min) RelA YvcI(NahA)

Figure S3. NahA hydrolyzes (p)ppGpp in vitro. a) TLC analysis of pppGpp and ppGpp hydrolysis by NahA over time. The same assay was also performed using GTP. b) TLC analysis of ppGpp hydrolysis by RelA and NahA over time. Substrate and product identities are labeled on the left side of the figure. 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. Fig. S4

a b pGpp pGpp

5×105

WT 4×105

∆nahA 3×105

Intensity 2×105 105 Normalized Ion Count ∆nahA IPTG-nahA 1×105

0 0

WT 11.5 11.7 ∆nahA Retention Time (min) ∆nahA IPTG-nahA

Figure S4. nahA complementation rescues pGpp production in vivo. a) Intensity profile of pGpp from LC-MS measurement of mid log phase wild type, ΔnahA and nahA complementation strain (ΔnahA IPTG-nahA). x-axis: retention time in LC-MS; y-axis: ion counts. For each strain, a representative curve was shown. Dashed lines are the shoulders of the GTP signal peak.

b) Normalized ion count (ion count per OD600 per unit volume of the culture) of corresponding pGpp levels, obtained as the average of 3 biological replicates in a). Error bars represents standard error of the mean. Fig. S5 c a in a time course of RHX treatment. Normalized ion countisperOD ΔnahA inatimecourseofRHXtreatment.Normalized and experiments. independent Asterisks indicatestatisticalsignificanceofdifferences (*P≤0.05,**≤0.01). replicates. standard errorofthemeantwobiological of theculture.Errorbarsrepresent volume pppGpp (a) and ppGpp (b) levels in wild type and type wild in levels (b) ppGpp and (a) pppGpp a-b) ΔnahA time courseofaminoacidstarvation. Figure S5. nahAmutantdisplayedhigher (p)ppGpp and lowerpGpp normalized to the ATP level at time zero (ATP arginine analogRHX(argininehydroxamate). Nucleotidelevelswereassayedvia TLC and are Normalized Ion Count 1.5×10 2.5×10 5×10 2×10 1×10 bioRxiv preprint 0 7 8 8 8 8

pppGpp relative to ATPt=0 02 30 20 10 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Time afterRHXtreatment(min) 01010202030350 300 250 200 150 100 50 0 was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. doi: Time afterRHXtreatment(min) https://doi.org/10.1101/2020.03.23.003749 pppGpp pppGpp c-e) ∆nahA WT ** LC-MS analyses of pppGpp (c), ppGpp (d) and pGpp (e) of wild type of pppGpp(c),ppGpp(d)andpGpp(e) wild LC-MS analyses ΔnahA WT d Normalized Ion Count 2×10 4×10 6×10 8×10 0 7 7 7 7 02 30 20 10 0 Time afterRHXtreatment(min) ; b t=0 this versionpostedMay15,2020. ). Error bars representstandarderrorsof mean of three

ppGpp ppGpp relative to ATPt=0 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 01010202030350 300 250 200 150 100 50 0 Time afterRHXtreatment(min) B. subtilis ∆nahA WT * ppGpp The copyrightholderforthispreprint(which e

treated withthe nonfunctional Normalized Ion Count 2×10 4×10 6×10 in vivo in 0 5 5 5 ** 02 30 20 10 0 Time afterRHXTreatment (min) ΔnahA WT B. subtilis pGpp 600 perunit in a ∆nahA WT *