A Grad-Seq View of RNA and Protein Complexes in Pseudomonas

Home , Degradosome

bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

2 A Grad-seq view of RNA and protein complexes in Pseudomonas

3 aeruginosa under standard and bacteriophage predation conditions

5 Milan Gerovaca, Laura Wickea,b, Kotaro Chiharac, Cornelius Schneidera,d,

6 Rob Lavigneb,#, Jörg Vogela,c,#

8 a Institute for Molecular Infection Biology (IMIB), University of Würzburg, 9 97080 Würzburg, Germany

10 b Laboratory of Gene Technology, KU Leuven, 3001 Leuven, Belgium

11 c Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for 12 Infection Research (HZI), 97080 Würzburg, Germany

13 d Department of Biochemistry and Cancer Therapy Research Center (CTRC), 14 Theodor Boveri-Institute, University of Würzburg, 97074 Würzburg, Germany

16 # Corresponding authors: [email protected], [email protected]

18 Keywords: Grad-seq, Pseudomonas, ΦKZ, bacteriophage, infection

20 Running title: Grad-seq in Pseudomonas and phage

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

22 ABSTRACT

23 The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause

24 of nosocomial infections but also serves as a model species of bacterial RNA biology. While

25 its transcriptome architecture and post-transcriptional regulation through the RNA-binding

26 proteins Hfq, RsmA and RsmN have been studied in detail, global information about stable

27 RNA–protein complexes is currently lacking in this human pathogen. Here, we implement

28 Gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to

29 comprehensively predict RNA and protein complexes, based on glycerol gradient

30 sedimentation profiles of >73% of all transcripts and ~40% of all proteins. As to

31 benchmarking, our global profiles readily reported complexes of stable RNAs of

32 P. aeruginosa, including 6S RNA with RNA polymerase and associated pRNAs. We observe

33 specific clusters of non-coding RNAs, which correlate with Hfq and RsmA/N, and provide a

34 first hint that P. aeruginosa expresses a ProQ-like FinO domain containing RNA-binding

35 protein. To understand how biological stress may perturb cellular RNA/protein complexes,

36 we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which

37 has a well-defined transcription profile during host takeover, displayed efficient translational

38 utilization of phage mRNAs and tRNAs, as evident from their increased co-sedimentation

39 with ribosomal subunits. Additionally, Grad-seq experimentally determines previously

40 overlooked phage-encoded non-coding RNAs. Taken together, the Pseudomonas protein and

41 RNA complex data provided here will pave the way to a better understanding of RNA-protein

42 interactions during viral predation of the bacterial cell.

43 IMPORTANCE

44 Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation

45 and physiology in any bacterium of interest. It is therefore crucial to globally determine these

46 complexes in order to identify and characterize new molecular players and regulation

47 mechanisms. Pseudomonads harbour some of the largest genomes known in bacteria,

48 encoding ~5,500 different proteins. Here, we provide a first glimpse on which proteins and

49 cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa.

50 We additionally performed this analysis with bacteria subjected to the important and

51 frequently encountered biological stress of a bacteriophage infection. We identified several

52 molecules with established roles in a variety of cellular pathways, which were affected by the

53 phage and can now be explored for their role during phage infection. Most importantly, we

54 observed strong co-localization of phage transcripts and host ribosomes, indicating the

55 existence of specialized translation mechanisms during phage infection. All data are publicly

56 available in an interactive and easy to use browser.

57 INTRODUCTION

58 Pseudomonas aeruginosa is a Gram-negative environmental γ-proteobacterium and a critical

59 life-threatening pathogen in humans with compromised immune defence (1). It is the main

60 cause of death in cystic fibrosis patients (2) and hard to treat with antibiotics due to its

61 diverse export capabilities and its ability to form strong biofilms (3). Unsurprisingly, the

62 medical challenges associated with nosocomial infections and drug resistance of

63 P. aeruginosa have prompted much effort to better understand the molecular biology of this

64 important human pathogen. With a size of 6.3 Mbp and ~5,570 predicted open reading

65 frames (ORFs), the genome of P. aeruginosa is one of the largest among prokaryotes (4).

66 These genes, many of which encode paralogous protein, endow the bacterium with a

67 remarkable functional diversity that allows it to thrive in different habitats. Gene expression

68 control also seems to be complex in P. aeruginosa: an unusually high 8.4% of all genes are

69 predicted to encode regulatory proteins, foremost transcription factors.

70 In addition to extensive gene regulation at the DNA level, there has been increasing

71 evidence for post-transcriptional regulation to play an important role in P. aeruginosa (5).

72 The bacterium possesses homologs of two general RNA-binding proteins (RBPs), CsrA and

73 Hfq, which work in conjunction with small regulatory RNAs (6). An important role of Hfq in

74 P. aeruginosa was first predicted almost 15 years ago when an hfq knockout strain was

75 observed to display altered levels for 5% of all transcripts (7). More recent work employing

76 global RNA interactome techniques (8-11) suggests that Hfq interacts with a large number of

77 coding and noncoding transcripts of P. aeruginosa. A distinct feature of Hfq-mediated

78 regulation in Pseudomonas is that Hfq inhibits translation of target transcripts by forming a

79 regulatory complex with the catabolite repression protein Crc (12, 13), likely by recognition

80 of nascent transcripts (9).

81 The other major regulatory RBPs include the CsrA-like proteins RsmA and RsmN,

82 which act as global translational repressors through recognizing GGA motifs in the 5' region

83 of mRNAs (14-18), partly so in a combinatorial fashion with Hfq (19). By contrast, much less

84 is known about a putative homolog of FinO/ProQ-like proteins, which is an emerging new

85 family of sRNA-related RBPs in Gram-negative bacteria (20-24). Judging by the presence of a

86 FinO domain, P. aeruginosa does possess a candidate protein (25), but whether it is expressed

87 and forms complexes with cellular transcripts remains unknown. Similarly, cold shock

88 proteins (CSPs), which interact with hundreds if not thousands of transcripts in some

89 bacteria (26), have not been studied in P. aeruginosa.

90 The past couple of years have witnessed the development of new methods to discover

91 RBPs and study RNA-protein complexes and interactions on a global level (27), one of which

92 is gradient profiling by sequencing, a.k.a. Grad-seq (22). Grad-seq partitions native cellular

93 lysates, including RNA–protein complexes, according to their molecular weight and shape on

94 a glycerol gradient. Subsequent fractionation and analysis by RNA-seq and mass

95 spectrometry of each fraction enables visualization of in-gradient distributions of, ideally, all

96 expressed RNAs and detectable soluble proteins from the growth condition of interest. The

97 method has been successfully applied to several different bacteria, revealing in Salmonella

98 enterica a previously unknown global activity of ProQ (22), new stable RNA and protein

99 associations with the ribosome in Escherichia coli (28); and a mechanism of sRNA

100 stabilization by 3'→5' exonucleolytic trimming in the Gram-positive human pathogen

101 Streptococcus pneumoniae (29). In all these studies, Grad-seq produced a previously

102 unavailable resource for the comprehensive prediction of protein complexes, with or without

103 RNA components, of a wide functional spectrum.

104 Despite its importance as a human pathogen and model bacterium, global data

105 informing on RNA/protein complexes are lacking for P. aeruginosa as well as other

106 pseudomonads. Here, we pioneer the application of bacterial complexomics in P. aeruginosa,

107 analysing exponentially growing cultures of this bacterium with Grad-seq. Our analysis

108 provides the first systems biology-based description of RNA-based housekeeping and

109 regulatory systems in this species, which includes a stable RNA polymerase (RNAP)-6S RNA

110 complex. We describe in-gradient distribution for thousands of mRNAs and sRNAs and

111 predict hundreds of protein complexes involving metabolic and signal transduction proteins.

112 In addition to performing Grad-seq on naïve bacteria, we apply the approach for the first time

113 to visualize transcript associations of an invading phage (ΦKZ) and to identify phage-

114 encoded regulatory RNAs. These data sets are provided along with an online browser that

115 helps to visualize the obtained sedimentation profiles in a searchable fashion and allows for

116 them to be compared with Grad-seq data from other Gram-negative and Gram-positive

117 species.

118

119 RESULTS

120 Gradient fractionation of P. aeruginosa in early exponential growth phase

121 As a prerequisite for Grad-seq of P. aeruginosa (see workflow in Fig. 1A), we established a

122 glycerol gradient analysis of RNA and proteins from a lysate of strain PAO1. We chose to

123 analyse samples from the exponential phase of growth (OD600 of 0.3) in a rich culture medium

124 to allow comparisons to previously established analyses under standard growth conditions.

125 In addition, the exponential phase is optimal for phage infection and replication of ΦKZ (30).

126 Phage predation represents a biotic stress for PAO1, relevant to the experimental design and

127 narrative below. A cleared lysate was separated on a 10-40% glycerol gradient, followed by

128 fractionation and gel-based analysis of either protein or RNA molecules in each of the 20

129 fractions obtained. In addition, the pellet was included in this analysis, as it contains

130 translating ribosomes as well as aggregated and unfolded proteins.

131 As a quality control, we determined the absorption profile of the gradient fractions at

132 260 nm (Fig. 1B). We observed the expected bulk peak at the top of the gradient, which

133 corresponds to free proteins and RNAs in low-molecular-weight (LMW) fractions. Also, two

134 pronounced peaks corresponding to the small (30S) and large (50S) ribosomal subunits in

135 high-molecular-weight (HMW) fractions were observed. Compared to previously obtained

136 profiles for Grad-seq of E. coli or S. enterica (22, 28), we observed much fewer 70S ribosomes.

137 In addition, the ribosomal subunits were shifted by one fraction towards the top of the

138 gradient, which might indicate species-specific disintegration of ribosomal subunit

139 complexes, or a relaxed rRNA configuration in Pseudomonas in the present growth condition.

140 SDS-PAGE showed general distributions of abundant soluble proteins and their

141 complexes within the gradient (Fig. 1C). As expected, translation factor EF-Tu is one of the

142 most abundant proteins in Pseudomonas (Table S1). The EF-Tu protein, which transiently

143 delivers transfer RNAs (tRNAs) to the ribosome (31), primarily sedimented in a ribosome-

144 free manner, occurring in the LMW fractions 1-3. RNAP subunits were primarily found in

145 fractions 5-7, whereas the major protein GroEL abounded in fractions 6-9 (i.e., complexes in

146 the ~400-800 kDa range). The 30S and 50S ribosomal subunits, with a molecular weight of

147 ~1 MDa and ~2 MDa, respectively, sedimented in fractions 9-13 or 14-16.

148 Similarly, we stained abundant RNA molecules in the individual gradient fractions

149 separated in a denaturing gel (Fig. 1D). As expected, tRNAs sedimented primarily in LMW

150 fractions 1-4, whereas the abundant 16S and 23S/5S rRNA molecules coincided with the 30S

151 or 50S proteins in fractions 9-13 or 14-17, respectively. As a benchmark, we also determined

152 the sedimentation profiles of several stable RNAs by northern blot analysis (Fig. 1E).

153 Transfer/messenger RNA (tmRNA, encoded by ssrA) forms a ribonucleoprotein particle

154 (RNP) with the SmpB protein to rescue stalled ribosome by a trans-translation mechanism

155 (32). Consistent with previous studies in E. coli and S. enterica, tmRNA sedimented in

156 fractions 3-7 (22, 28). The 180-nt 6S RNA (33) accumulated in fractions 5-7, as expected from

157 its reported association with RNAP in E. coli (34). In summary, these observations qualified

158 the gradient samples for high-throughput analysis by Grad-seq, with the aim to obtain global

159 sedimentation profiles of cellular RNA and protein molecules (Fig. 1F).

160

161 Global predictions of macromolecular complexes for P. aeruginosa

162 For a global view of stable complexes in the exponential growth phase, we profiled the

163 proteins and transcripts from each gradient fraction by mass spectrometry and RNA-seq,

164 respectively (Table S1). Protein and RNA abundance was normalized using external spike-

165 ins, enabling us to delimit the detection thresholds (Fig. S1). Targeted inspection of these

166 high-throughput data readily showed expected co-occurrences of stable RNA species with

167 their major protein partners (Figs. 1F, S2). For example, cDNA counts of the 16S/23S rRNAs

168 peaked in the same fractions as did peptide counts of the respective ribosomal proteins, and

169 4.5S RNA of the signal recognition particle (SRP) peaked in the same fractions as did the

170 major SRP protein Ffh. Another case in point is the 6S RNA-RNAP complex whose protein

171 components (RpoA/B/C/Z encoding the α/β/β'/ω subunits of RNAP) aligned well with 6S

172 RNA in gradient fractions 5-6 (Fig. 2A). Grad-seq even recovered the tiny pRNAs (35, 36)

173 produced by RNAP as it disentangles itself from the 6S RNA (Fig. 2B). These established

174 interactions provide additional confidence to the correlations described below. Mass

175 spectrometry recovered 2,182 proteins in total, i.e., ~40% of the annotated proteome of

176 P. aeruginosa strain PAO1 (37), with these proteins showing a wide range of distribution in

177 the gradient (Figs. 3A, S2A). While most of these proteins sedimented in LMW fractions 1-3,

178 indicating that they are part of either no or only small complexes, there were also protein

179 clusters in the HMW fractions, in addition to the expected two ribosomal subunit clusters. We

180 will return to some of these HMW clusters in the next section.

181 Viewing the RNA-seq results from individual fractions (Fig. 3B), we observed the

182 expected sedimentation of rRNAs in the HMW part of the gradient, whereas tRNAs were

183 found in the LMW top fractions, both of which recapitulated the preceding gel analysis

184 (Fig. 1D). However, most of the 5,084 transcripts detected in total sedimented in the first

185 eight fractions, and this equally applied to mRNAs and non-coding RNAs (ncRNAs). With

186 respect to mRNAs, this implies that most of these reads stem from transcripts that are not

187 undergoing translation. However, there is a group of mRNAs with abundant reads in the

188 pellet, indicating association with 70S ribosomes or even polysomes. These transcripts show

189 enrichment for functions in oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and

190 other metabolic pathways. In essence, these sedimentation profiles provide a glimpse of

191 translational utilization of cellular mRNAs in the exponential growth phase in rich media.

192

193 P. aeruginosa proteins and their complexes

194 Grad-seq will primarily recover cytosolic proteins as it is performed on a cleared lysate.

195 Indeed, membrane-associated proteins were underrepresented among the ~2,200 detected

196 proteins (Fig. 4A). For a quick overview of functional relationship of co-sedimenting

197 proteins, we clustered all detected proteins by principal component analysis (PCA) according

198 to their sedimentation profiles (Fig. 4B). Central regions in the plot correlate with

199 sedimentation somewhere in fractions 4-10, i.e., complexes in the 200 kDa-to 1-MDa range,

200 clear examples of which are the clusters formed by RNAP or ATP synthase subunits. With

201 respect to HMW fractions, ~6% of the detected proteins show their main peak anywhere

202 between fractions 8 and 20, where they are very unlikely to sediment on their own (Figs. 3A,

203 5A). In other words, most of these proteins are present in stable molecular complexes,

204 indicating that they are associated with a protein or RNA partner to fulfil their function.

205 Next, we evaluated the integrity of complex sedimentation by manual inspection of

206 established proteins, incorporating knowledge from other species such as E. coli. Many

207 metabolic processes involve multi-enzyme complexes, some of which are giant. Several such

208 large complexes can readily be discerned from our Grad-seq data (Fig. 5A), including all three

209 dehydrogenase complexes: the pyruvate dehydrogenase complex (PDC), the oxoglutarate

210 dehydrogenase complex and the branched-chain α-keto acid dehydrogenase complex

211 (Fig. 5B). Interestingly, all of these dehydrogenase multi-enzyme complexes sedimented as

212 much smaller complexes than by their suggested multimeric sizes. This observation is in

213 agreement with a recently determined salt-labile structure of the PDC (38). The succinyl-

214 CoA-synthetase α2β2 complex, formed by the SucC and SucD proteins (39) from the TCA cycle,

215 sedimented in fraction 3 (MW of ~140 kDa). The aspartate carbamoyltransferase, which

216 catalyses the first step of the pyrimidine biosynthetic process, builds a complex of ~310 kDa

217 and sedimented in fraction 5.

218 Examples of large complexes outside metabolic functions include the tetradecameric

219 840 kDa GroEL complex, here observed in fraction 7 (Fig. 5B). Interestingly, the functionally

220 associated GroES complex sedimented away from it, in LMW fractions, as previously

221 observed in S. enterica (22). A particularly large particle seems to be formed by the

222 membrane associated FtsH protease, which is a hexamer that goes on to form a 1 MDa

223 holoenzyme with the membrane complex HflKC (40); we observed it in fraction 11.

224 Importantly, we find dozens of additional proteins to sediment in HMW fractions (Fig. S2A).

225 Since many of these are functionally uncharacterized, this region of the gradient bears great

226 potential for discovery of new large protein complexes.

227

228 Topology of the Pseudomonas transcriptome

229 RNA-seq analysis of the gradient fractions revealed 4,643 transcripts above threshold (>100

230 reads in all fractions), covering 73.6% of the transcriptome (Fig. S1E). Quantitatively

231 speaking, 84% of all reads came from rRNA (Fig. 6A). The class of tRNA contributed 6.5%

232 whereas reads from mRNAs were represented by ~9%. The diverse class of ncRNAs

233 contributed 0.4% of all reads, i.e., markedly fewer than in previous Grad-seq of enteric

234 bacteria in early stationary phase (for example, ~5% in E. coli (28)). Note that in

235 P. aeruginosa, too, many ncRNA genes are only upregulated as the cells enter stationary phase

236 (41).

237 For a better overview of the general sedimentation of all detected RNA classes, we

238 reduced the complexity of the 21 fractions to six clusters (Fig. 6B). The mRNA reads were

239 mainly found in fractions 3-8, coinciding with RNAP, and in the 30S subunit fractions 9-12,

240 as well as in the pellet. This distribution differs from previous Grad-seq analyses in E. coli,

241 S. enterica and S. pneumoniae in which mRNAs generally co-sedimented with ribosomes (22,

242 28, 29).

243 Systematic annotation of ncRNAs in P. aeruginosa lags behind other model

244 γ-proteobacteria, with only 44 formally annotated ncRNAs (42) as compared to hundreds

245 each in E. coli, Salmonella and Vibrio species (43, 44). However, a total of 473 ncRNAs have

246 been predicted in P. aeruginosa (45, 46), 336 of which were detectable in our Grad-seq data.

247 Most of the P. aeruginosa ncRNAs and candidates thereof occur in fractions 3-8, thus showing

248 a general sedimentation profile similar to most mRNAs (Fig. 6B).

249 Stable non-coding RNA fragments overlapping with 5' or 3' untranslated regions

250 (UTRs) constitute a potentially large class of bacterial riboregulators (47-50). Recent

251 experimental transcriptome annotation (51-53) suggested that also in the Pseudomonas

252 strain used here, many such UTR-derived candidate ncRNAs exist and accumulate to

253 considerable levels. Here, we observe that such UTR fragments often show a different

254 sedimentation profile when compared to the respective coding sequence (CDS) of the same

255 mRNA (Fig. 6C). These include several UTR fragments that were recently observed to be

256 highly enriched by co-immunoprecipitation with Hfq (8), which is a good indicator of 12

257 potential cellular function as a base pairing small regulatory RNA (sRNA) (54). Obvious

258 examples are the mRNA 5'UTRs of phosphate transporter PA0450, and the uncharacterized

259 proteins PA4643 and PA4972, or the mRNA 3'UTRs of putative transcriptional regulator

260 PA2758, cyclic-guanylate-specific phosphodiesterase PA3825, and serine dehydratase SdaB,

261 all of which peak at least four fractions away from the CDS part of their parental mRNAs

262 (Fig. 6C). With respect to the published literature, RhlS is an activating 5'UTR-derived ncRNA

263 from the rhlI gene (55). While the precise mechanism whereby RhlS promotes expression of

264 the rhlI mRNA is currently unknown, we notice that the two RNAs occur in different fractions

265 (Fig. 6C), supporting the proposed model of an independent regulatory function of the 5'UTR.

266 To validate the Grad-seq based detection of UTR-derived fragments, we chose to

267 probe the 3'UTR of PA2758 mRNA on a northern blot. We readily detected a distinct ~50-nt

268 RNA species with the predicted sedimentation in fractions 1-4, distinct from the full-length

269 PA2758 mRNA (Fig. 6D). This 3'UTR RNA species was much shorter than the previously

270 predicted ncRNA candidate ncRNA0222/SPA0115 from this genomic region (46).

271 Importantly, however, it comprises the most conserved part of the 3'UTR in question, which

272 includes the predicted Rho-independent transcriptional terminator of PA2758 (Fig. 6E). In

273 addition, this ~50-nt RNA species was recently shown to be highly enriched in global Hfq

274 co-immunoprecipitation experiments ((8); Fig. 6E), indicating that it might function as a base

275 pairing sRNA.

276 In silico target prediction using the CopraRNA algorithm (56) supports the notion that

277 the 3'UTR of PA2758 might act in trans to regulate mRNAs by base pairing (Fig. S3).

278 Focussing on potential binding sites around the start codon of mRNAs, CopraRNA predicts as

279 the top target an extended interaction that might translationally inhibit the mRNA of napC,

280 which is the last gene in the napEFDABC operon encoding nitrate reductase. Importantly,

281 base pairing would crucially engage a 13-nt long single-stranded region of the PA2758-

282 3'UTR, which looks like a conserved seed sequence of this sRNA. Further studies are required

283 to validate this particular prediction and to determine the targets of the UTR-derived sRNAs

284 proposed here.

285

286 Major RNA-binding proteins and associated ncRNAs

287 P. aeruginosa is known to possess two large post-transcriptional networks that depend on

288 globally acting RBPs: one governed by Hfq, the other by two CsrA-like Rsm proteins. The

289 mRNA and ncRNA ligands of these RBPs have been mapped in a transcriptome-wide fashion

290 (8, 9, 17, 19), albeit not in the growth phase studied here.

291 The 190-nt PhrS ncRNA, known as a direct activator of the mRNA of the

292 transcriptional regulator PqsR (57), is a well-characterized target of Hfq. PhrS is readily

293 detected in the gradient, by both RNA-seq and northern blot, and shows a very similar

294 sedimentation profile to Hfq (Fig. 7A). Its absence from fractions 1-2 suggests that this

295 ncRNA is always present in complex with Hfq and/or other interactors.

296 Another well-characterized Hfq-related ncRNA is CrcZ. This ~400-nt ncRNA forms a

297 stable complex with Hfq and Crc (12), which prevents the Hfq/Crc complex from repressing

298 translation of amiE mRNA (58). The CrcZ RNA sediments in fractions 6-9 (Fig. 7A), in which

299 Hfq is also found. Indeed, the in-gradient profiles of both the PhrS and CrcZ ncRNAs are well-

300 correlated with that of Hfq (coefficient >0.7), suggesting a stable molecular interaction

301 (Fig. 7A). By contrast, the Crc protein peaks far away, in LMW fraction 2, suggesting that the

302 Hfq/Crc/RNA complex is unlikely to exist in the rich media conditions used here (as

303 predicted previously; (13)). Additional explanations include that the Hfq/Crc targets are

304 rapidly degraded, leading to disassembly of the Hfq/Crc/mRNA complexes, or that these

305 complexes are simply too labile in a glycerol gradient.

306 Regarding the Rsm network, the RsmA protein alone is known to regulate

307 —directly or indirectly— 9% of the transcriptome (7, 19, 59). RsmA blocks translation by

308 binding to GGA motifs in mRNAs; its own activity is also regulated at the RNA level, by the

309 ncRNAs RsmY/Z/V/W, which sponge RsmA by presenting multiple GGA motifs (60). The

310 homologous RBP RsmN acts and is acted upon in the same fashion (15, 17, 18). Here, we

311 observe well-correlated sedimentation of the two Rsm proteins and its four-known decoy

312 RNAs, suggesting that in exponentially growing Pseudomonas cells (Fig. 7B), these two RBPs

313 are largely kept inactive by their ncRNA antagonists. The more abundant RsmA also

314 sedimented in ribosomal fractions, perhaps in association with polycistronic mRNAs. In any

315 case, the RsmA/N profiles are clearly distinct from Hfq and its associated RNAs.

316 Other RBPs and candidates thereof with unknown ligands also show intriguing

317 sedimentation profiles. Proteomics detected the cold-shock-like protein CspA in the first

318 fractions (Fig. 7C), which is in agreement with Grad-seq profiles of CspA in E. coli and

319 Salmonella (22, 28). Cold-shock proteins, while interacting with many cellular transcripts,

320 bind their targets with low affinity (26), which may explain their accumulation in LMW

321 fractions. Intriguingly, while one of the three other CspA-like proteins of P. aeruginosa shows

322 a congruent sedimentation profile, the other two migrate towards the middle of the gradient.

323 Thus, these two putative RBPs, PA0961 and PA1159, represent strong candidates for cold-

324 shock proteins that engage in stable complexes with either RNA or other proteins (Fig. 7C).

325 Chromosomally encoded RBPs with a FinO domain such as ProQ have recently

326 emerged as the third major ncRNA-associated RBP in enteric bacteria (25). Based on a Pfam

327 prediction (61), we propose protein Q9I0Q4 as the putative ProQ homolog in P. aeruginosa.

328 Peptides of it can be detected in almost all gradient fractions, although the majority of them

329 are found in fractions 3-11 (Fig. 7C). This ProQ profile is broader than in E. coli or Salmonella,

330 especially with respect to P. aeruginosa ProQ appearing in 30S ribosome fractions. In any

331 case, this broad sedimentation argues for P. aeruginosa to possess functional FinO/ProQ-like

332 RBPs.

333 To obtain a focused overview of ncRNA sedimentation patterns for potential cross-

334 comparison with RBPs, we clustered all 365 detected and candidate ncRNAs according to

335 their sedimentation profile, yielding ten clusters (Fig. 7D). Approximately three-quarter of

336 these ncRNAs end up in cluster 9, which includes the well-characterized Hfq-associated

337 ncRNA PhrS and PrrF1 (57, 62), and PhrX (63). In essence, many of these ncRNAs sediment

338 like mRNAs (Fig. 6B, cluster 4). This is particularly pronounced with the five members of

339 cluster 10, challenging the previous annotation of these transcripts as noncoding RNAs. For

340 example, OsiS (PA0611.1, pant67, (45)) has been suggested to act as a regulatory RNA to link

341 oxygen level sensing to the production of quorum sensing molecules (64). However, the

342 co-migration of these transcripts with 30S ribosomes suggests the possibility that they

343 possess overlooked small ORFs. Note that a putative 30S association recently guided the

344 discovery of a functional small ORF in the seemingly noncoding RyeG RNA of E. coli (28, 65).

345 In the present case, recent P. aeruginosa Ribo-seq data (66) provide additional support for

346 our prediction that OsiS is translated into a protein (Fig. S4).

347

348 Phage-induced stress affects cellular organization of the RNome

349 The in-gradient distributions of RNA and protein described above reflect the complexome of

350 P. aeruginosa under optimal growth conditions, characterized by rich supply of nutrients and

351 rapid growth. To determine how this molecular organization is perturbed by a biotic stress,

352 we subjected P. aeruginosa to infection by a phage. Specifically, the lytic giant phage ΦKZ

353 which is a well-established model phage of pseudomonads and known to usurp many cellular

354 functions of its host (67-69) (Fig. 8A).

355 Performing Grad-seq on P. aeruginosa cells after 10 min of infection by phage ΦKZ, we

356 found that phage transcripts accounted for ~3% of all reads, while the host mRNAs remained

357 at ~7% (Fig. S5). Nonetheless, a paired t-test showed that the sum of all host CDS and ncRNAs

358 at this early infection time point was approximately fivefold lower compared to uninfected

359 cells, notwithstanding a few prominent outliers such as the ncRNAs CrcZ, PrrH, and RnpB

360 (Fig. 8B). At the same time, the individual ΦKZ transcript exceeded the typical host transcript

361 in abundance by one order of magnitude (Fig. 8C).

362 While altered abundance of host transcripts is one consequence of phage predation,

363 we were particularly interested in the possibility that the phage infection might also perturb

364 host transcriptome organization, altering the association of host transcripts with cellular

365 factors to promote viral replication. Such changes, which should be visible as shifts in the

366 gradient, could point out stress response mechanisms and pathways that remain invisible in

367 standard RNA-seq experiments.

368 Focussing on transcripts with a <3-fold change in total level, we indeed observed

369 hundreds of transcripts with negative or positive shifts in their sedimentation profile

370 (Fig. 9A,B). An excellent example is given by the mRNAs of the uncharacterized proteins

371 PA4441 and PA2746a, which both showed redistribution towards fractions 10-11, possibly

372 reflecting increased translational initiation by 30S ribosomes. Another example is liuR,

373 coding for the transcriptional regulator of the of metabolic liu genes (70) (Fig. 9A,C).

374 Similarly, we predict several host ncRNAs to reassociate with different partners in the early

375 phase of phage infection, illustrative examples of which are ErsA, pant512 and pant253

376 (Fig. 9D). By contrast, with the exception of tRNALeu, transfer RNAs generally stayed in the

377 same fraction after ΦKZ infection (Fig. 9A-C).

378 ErsA is a well-characterized small RNA of P. aeruginosa, which has been proposed to

379 serve a dual regulatory role in biofilm development, activating the mRNA of transcriptional

380 regulator AmrZ while at the same time repressing the mRNA of AlgC, an important virulence-

381 associated enzyme for the production of sugar precursors (71, 72). In addition, ErsA acts as

382 a translational repressor of the mRNA of outer membrane porin OprD (73). ErsA is well-

383 expressed during the exponential growth phase (51) and bound by Hfq (8), making it an

384 excellent candidate for validation by an independent technique of the infection-dependent

385 changes seen in Grad-seq. Using an oligonucleotide probe for ErsA, we probed all 20 gradient

386 fractions plus the pellet by northern blotting. Intriguingly, this method showed even more

387 clearly that EsrA, 10 min into phage infection, is depleted in LMW fractions, with its main

388 peak shifting to fractions 10-11 where 30S ribosomes sediment (Fig. 9E). A similar, albeit

389 less pronounced shift was observed for the PhrS ncRNA. By contrast, phage infection did not

390 affect the in-gradient distribution of 5S rRNA and 6S RNA, as predicted by the digital data.

391 While the biological meaning of the redistribution of EsrA remains to be understood, its

392 independent validation by northern blot adds confidence for the predicted infection-induced

393 shifts of other cellular transcripts.

394

395 Phage mRNAs are efficiently transferred from transcription to translation

396 At the early time point of ΦKZ infection used here, nearly all (366/376) annotated phage

397 genes (NC_004629.1, NCBI nucleotide) are expressed (Table S1, (30, 51)). For an initial

398 glimpse at the sedimentation profiles of phage transcripts, we probed two well-expressed

399 viral operons on northern blots; specifically, operons 54 and 88 (30), which encode metabolic

400 and structural proteins of the phage, respectively. Intriguingly, in both cases the strongest

401 signal obtained were from the pellet fraction, indicating that these mRNAs are heavily

402 engaged by the translation apparatus (Figs. 10 and S5F,G). In addition, signals within the

403 gradient were primarily seen in fractions 10-13, overlapping with the 30S ribosome peak.

404 Global inspection of the Grad-seq data reinforced the notion that phage mRNAs are

405 prioritized for translation. That is, in contrast with host mRNAs which were primarily

406 detected in fractions 3-8 (Fig. 6B), the phage mRNAs were clearly more abundant in fractions

407 containing 30S and 70S ribosomes (Fig. 10B,C). In addition, different from host tRNAs, the

408 phage-encoded tRNAs also appeared in the 30S-containing fractions (Figs. 6B, 10C, S5E,F),

409 which appears to be a type of association (74). Thus, two classes of ΦKZ transcripts indicate

410 the existence of mechanisms that ensure efficient synthesis of phage proteins.

411

412 Grad-seq guides the identification of phage-encoded ncRNAs

413 We reasoned that if phage mRNAs generally co-migrate with ribosomes, non-coding phage

414 transcripts might be revealed through different in-gradient distributions, as already evident

415 from the phage-encoded tRNAs. Indeed, manual inspection of Grad-seq profiles along the

416 280-kbp phage genome revealed two ncRNA candidates. The first example was a ~100-nt

417 RNA species from the intergenic space between phage genes PHIKZ298 and PHIKZ297,

418 predicted to sediment primarily in fractions 3-4 (Fig. 11A,B). Northern blot probing proved

419 this species to be abundant and also confirmed its predicted length. Interestingly, the probe

420 used here simultaneously detects a larger transcript(s) with an mRNA-like in-gradient

421 distribution. Combined with the lack of a mapped TSS downstream of the stop codon of

422 PHIKZ298 (51), we assume that the detected 100-nt RNA species is generated by 3'UTR

423 processing of the PHIKZ298 mRNA. According to in silico prediction, the ncRNA forms two

424 stem-loops, the second of which might constitute the Rho-independent transcriptional

425 terminator of the PHIKZ298 gene (Fig. 11C).

426 The other ncRNA candidate also accumulates in the LMW part of the gradient, yet

427 shows a broader distribution (fractions 4-10; Fig. 11A,D). This putative ncRNA comprises

428 the first 180-nts of the 5'UTR of phage gene p40. Similar to the above candidate, northern

429 blot probing confirmed both the proposed length of the candidate ncRNA itself and detected

430 larger transcripts with mRNA-like behaviour in heavier fractions. Note that for reasons

431 unknown, the signals of both these putative ncRNAs are shifted by one fraction towards the

432 top of the gradient when comparing the northern blot to the Grad-seq data. The 5'UTR-

433 derived ncRNA species also seems highly structured, showing two stem loops (Fig. 11E), the

434 second of which might promote premature termination before transcription reaches the start

435 codon of p40.

436 We carefully checked both UTR-derived RNA species for potential small ORFs,

437 including alternative start codons, but found none that would be preceded by a strong Shine-

438 Dalgarno sequence. We therefore consider both as ncRNAs and take them as illustrative

439 examples how the extra information provided by Grad-seq, as compared to simple RNA-seq,

440 may help to discover overlooked transcripts in phage genomes.

441

442 DISCUSSION

443 The primary objectives of this work have been: i) to provide the interested microbiologist

444 with previously unavailable Grad-seq profiles of cellular transcripts and proteins in the

445 model bacterium P. aeruginosa from a commonly-studied growth condition, ii) to assess the

446 quality of these high-throughput results by independent methods, iii) to illustrate how the

447 technique might be utilized in the future to study bacteriophage-host interactions, and iv) to

448 organize the data such that they can be accessed and interrogated by non-specialists. To

449 facilitate the latter, we have set up an online browser (Fig. 12), which is available

450 https://helmholtz-hiri.de/en/datasets/gradseqpao1/ and allows the user to cross-compare

451 the present Pseudomonas data with Grad-seq results for several other species (22, 28, 29). In

452 addition, the browser offers comparison with GradR (75), which is a recently developed

453 variation of Grad-seq trying to predict potential RNA association/dependency of cellular

454 proteins, and more specifically, unrecognized RBPs. Although those GradR data were

455 obtained with Salmonella, some of the many proteins affected by the RNase treatment used

456 in this method will also be conserved in P. aeruginosa.

457 Even though the present P. aeruginosa Grad-seq data covers only one growth

458 condition, we believe that it already offers a huge discovery space. Given the bacterium’s 21

459 impressive metabolic capabilities, the MS-profiles of all detected proteins should give new

460 insights into metabolic multi-enzyme complexes. Obviously, not all these complexes are

461 sufficiently stable to survive the long (over-night) ultra-centrifugation procedure during

462 which cellular complexes separate according to size and shape in a glycerol gradient. For

463 example, the PDH complex dissociated into stable smaller subcomplexes (Fig. 5B), which is

464 in keeping with recent size-exclusion experiments that revealed a salt-labile structure of the

465 macromolecular complex (38). Nonetheless, in addition to the examples highlighted in

466 Fig. 5B, there are many proteins that sedimented between the fractions of the small and large

467 ribosomal subunits. In other words, these proteins must be part of stable complexes in the

468 2-4 MDa range, an information that should provide useful leads for future functional analysis.

469 From an RNA biology perspective, the initial Grad-seq data provided here promises to

470 be a treasure trove. Known RBPs such as Hfq and RsmA/N sedimented in a much higher

471 molecular weight range than their individual size would predict. We have recently shown for

472 Hfq (and other RBPs) in Salmonella that this sedimentation behaviour is primarily due to RNA

473 interactions. Indeed, when transcripts are removed by RNase treatment of the lysate, these

474 proteins shift to LMW fractions (75). In the present P. aeruginosa data, the associations of the

475 Hfq and RsmA/N RBPs are also obvious by a clear co-migration with their major RNA ligands

476 (Fig. 7A,B). While these three RBPs have seen extensive work, our Grad-seq data strongly

477 indicate that there is yet another global RBP. That is, we have detected peptides of a candidate

478 ProQ RBP (protein PA2582), which is encoded in a chromosomal region close to exonuclease

479 repair gene uvrC and the regulatory protein PA2583 (Fig. S6). The predicted ProQ of

480 P. aeruginosa not only carries a full-length FinO domain, it also shows conservation of several

481 important residues for RNA binding by E. coli ProQ (76). The pseudomonal ProQ proteins are

482 small (~20 kDa), and similarly to Neisseria ProQ (21), lack the typical C-terminal or

483 N-terminal extensions of the central FinO domain (25), which makes them attractive

484 candidates for understanding the core recognition mode of RNA by the FinO domain.

485 The complex of 6S RNA and RNAP is an RNA-based regulation previously undescribed

486 in P. aeruginosa. The two partners show in an almost ideal form both, narrow peaks and

487 exceptionally high correlation in the gradient, primarily in fractions 5-6 (Fig. 2A). While

488 similarly high correlations have been shown before in both conventional and Grad-seq based

489 analyses of 6S RNA-RNAP (22, 28, 29), the new observation in the present P. aeruginosa Grad-

490 seq experiment are the 14-nt pRNAs. These pRNAs are a natural product of the 6S RNA

491 regulatory cycle in which 6S RNA captures RNAP by mimicking an open promoter complex

492 of transcribed DNA, and once bound, changes the affinity of RNAP for different σ factors, the

493 result of which is a global change in transcription (34). In order to dissociate from 6S RNA,

494 RNAP uses it as a template for transcription, yielding pRNAs. Thus far, these pRNAs have been

495 considered as an inevitable side product of 6S RNA release, possessing no independent

496 function and awaiting degradation. However, our detection of a pRNA peak spanning several

497 fractions (2-5) may now hint that these short transcripts go on to interact with other cellular

498 molecules, perhaps as part of serve a currently unrecognized independent function in the cell.

499 Next to Grad-seq analysis of naïve P. aeruginosa cells, we provide an RNA snapshot

500 with the corresponding experiment performed at an early time point of phage infection.

501 Phages by definition manipulate host cell metabolism in exquisitely diverse ways, as

502 reviewed recently (68). Specifically, Pseudomonas bacteriophage ΦKZ is known to rapidly

503 shift towards host-independent viral replication. This is achieved through transcription by

504 both, virion-delivered and virus-encoded RNAPs (30), a complete reorganization of the

505 metabolism (77), and modulation of the host RNA turnover machinery (78). However,

506 insights into RNA/protein interactions between phage and host remain scarce. Therefore, the

507 RNA data provided in the next section allow a first glimpse at how ΦKZ alters RNA processing

508 in the host, and hint at modulation of translation rates of some host mRNAs, and of phage

509 mRNAs in general. Importantly, active manipulation of protein synthesis has been reported

510 for coliphages T4 (79) and T7 (80-84), and is also implied by prediction of phage-encoded S1

511 proteins (85, 86).

512 On the host side, phage infection impacts the in-gradient distributions of both mRNAs

513 and ncRNAs. Many host mRNAs shift away from ribosomal fractions, and this effect is even

514 more pronounced for those transcripts with a particular strong ribosome association in the

515 first place. Primary examples include transcripts related to oxidative phosphorylation

516 (Fig. S7); a strong reduction of the synthesis of these proteins may eventually lead to a

517 collapse of cellular energy production. In general, given that active translation stabilizes

518 bacterial mRNAs, the predicted interruption of translation may offer one explanation for the

519 observed ~5-fold depletion of host transcripts 10 min after phage infection.

520 Yet, we also observe the reciprocal behaviour, i.e., host mRNAs that move into

521 ribosome-associated fractions. A particularly noteworthy case is the mRNA of protein

522 PA4441, which carries a predicted DUF1043 domain that is also found in cyclic-

523 oligonucleotide-based anti-phage signalling system (CBASS) effectors (87). Put simply, after

524 phage predation, the mRNA of a potential phage defence protein migrates to the 30S-related

525 fraction as the consequence of increased translational initiation. This may indicate the

526 existence of a specific mechanism to rapidly activate synthesis of host proteins with

527 protective functions.

528 In addition to mRNAs moving out of or into ribosomal fractions, Grad-seq predicts

529 interesting cases of ncRNAs that seem to associate with different or additional cellular

530 partners after lytic infection. These shifts for the ErsA and PhrS sRNAs were independently

531 validated on northern blots (Fig. 9E). Because both these ncRNAs are well-characterized,

532 they would serve as ideal models to understand the mechanisms behind the observed shift.

533 In the case of PhrS, it is worth mentioning that in P. aeruginosa strain PA14 this sRNA may

534 promote adaptive immunity against bacteriophages through suppression of Rho-dependent

535 termination of early CRISPR transcripts (88). This CRISPR leader is conserved in the PAO1

536 strain used here despite the lack of a CRISPR array, and we thus speculate that the observed

537 shift of PhrS RNA might be the result of enhanced PhrS-CRISPR leader interaction upon phage

538 infection.

539 Of well-understood molecular mechanisms whereby phages usurp bacteria, several

540 include active manipulation of host RNA decay (89, 90). The present Grad-seq data is of

541 insufficient depth to allow for a statistically sound global analysis of phage-induced alteration

542 of host RNA processing. However, during inspection of rRNA profiles, we observed that

543 biogenesis of 5S rRNA in phage-infected cells might be compromised, as suggested by a

544 smeary northern blot signal for 5S rRNA in several gradient fractions (Fig. S8A). Processing

545 of 5S rRNA from longer ribosomal transcripts crucially depends on the major

546 endoribonuclease RNase E, and ΦKZ expresses a protein called Dip that functionally

547 interferes with the RNase E-associated degradosome (78, 89, 91).

548 In further support of altered RNase E activity, we observe a differential accumulation

549 of 5' mRNA fragments of RNase E itself (Fig. S8B,C), which is well-established to cleave in the

550 5'UTR of its own mRNA to feedback-control its own protein levels (92-94). Upon ΦKZ

551 infection, a longer band that we attribute to cleavage at the rne-II motif (Rfam RF01756, (95))

552 disappears. Whether the altered processing eventually causes a decrease in RNase E protein

553 levels needs to be shown. Interestingly, this mechanism would be the opposite to what was

554 reported with the marine cyanobacterium Prochlorococcus MED4 in which phage infection

555 boosts RNase E synthesis by altered processing of its mRNA (96).

556 Finally, one of the most intriguing phage-related observation in our Grad-seq data is

557 the distribution of phage versus host mRNAs. Contrary to mRNAs for which reads where

558 detected in both LMW and ribosome-containing HMW fractions (Figs. 3B, S5E), phage

559 mRNAs sediment almost exclusively in the HMW part of the gradient as well as in the pellet

560 (Figs. 10C, S5F,G), the latter of which contains 70S monosomes and polysomes. On the face

561 of it, this would suggest that ΦKZ mRNAs are prioritized for translation, perhaps even at the

562 expense of making host proteins, as has been reported previously in other systems (79-86)

563 We would like to caution, however, that mRNA reads in LMW fractions represent a steady-

564 state picture in which we see host mRNAs undergoing degradation, whereas the newly

565 produced phage mRNAs, only 10 min after the phage has attacked, may still be fully protected

566 by ongoing translation. In addition, phage ΦKZ segregates its DNA from immunity nucleases

567 of P. aeruginosa by constructing a proteinaceous nucleus-like compartment (69), which

568 might provide further protection of phage mRNAs. While this new model does not question

569 generally that phage mRNAs are translated by cytoplasmic ribosomes, the influence of this

570 intracellular spatial segregation on phage mRNA stability is not clear. Future work with

571 higher time resolution will be needed to determine whether phage mRNAs are indeed

572 prioritized for translation, and if so, which phage or host factors are responsible.

573 Many of the above observations will benefit from a further mechanistic analysis of the

574 Grad-seq protein data of infected host cells, which is currently ongoing. In studying the

575 molecular mechanisms governing phage-bacteria interactions, it should be obvious that

576 Grad-seq offers unique opportunities to reveal RNA-protein interactions, including phage-

577 CRISPR-Cas complex interactions, usage and modifications of tRNAs or the discovery and

578 functional elucidation of phage ncRNAs, all of which have remained largely unstudied to date.

579 Indeed, the potential of differential sedimentation profiles in a single sample became

580 apparent as we were able to point to two previously unannotated ncRNAs in the phage

581 (Fig. 11), whereas TSS mapping in ΦKZ (51) failed to predict such candidates. To put this

582 into perspective, a mere 21 ncRNAs have been described in phages to date (97), hence

583 Grad-seq offers an orthogonal approach for identification of RNA processing sites and

584 ncRNAs.

585

586 MATERIAL AND METHODS

587 Cell preparation, lysis, and gradient sedimentation

588 Pseudomonas aeruginosa strain PAO1 (JVS-11761, Table S2, German Collection of

589 Microorganisms and Cell cultures GmbH: DSM 22644, BacDive ID: 12801, NCBI tax-ID:

590 208964) was grown in 400 mL LB media to OD600 0.3 at 37°C. Optionally, the cells were

591 infected with phage ΦKZ at a multiplicity of infection (MOI) of 15 to ensure immediate

592 infection of the entire culture. To ensure that infected cell culture was in the early infection

593 phase, the infected culture was mixed and incubated for 5 min at room-temperature without

594 shaking, followed by incubation at 37°C for 5 min with shaking, as described previously (30).

595 The culture was cooled rapidly down in ice and the cells were pelleted at 6,000 × g for 15 min

596 at 4°C, and washed three times with ice-cold TBS (20 mM Tris/HCl pH 7.4, 150 mM NaCl).

597 The gradient analysis was performed as published (22), with minor modifications.

598 Cells were resuspended in 0.5 ml lysis buffer (20 mM Tris/HCl pH 7.5, 150 mM KCl, 1 mM

599 MgCl2, 1 mM DTT, 1 mM PMSF, 0.2% (v/v) Triton X-100, 20 U/ml DNase I (Thermo Fisher

600 Scientific), 200 U/ml SUPERase-IN (Life Technologies)) and one volume of 0.1 mm glass

601 beads. Lysis was obtained by vortexing this mix for 30 s, followed by 15 s cooling on ice. This

602 process was repeated ten times and the lysate was cleared at 12,000 × g for 10 min at 4°C.

603 The supernatant was used for SDS-PAGE analysis and RNA preparation by TRIzol™ (Thermo

604 Fischer Scientific). Aliquots of 180 μl supernatant were layered on a linear 10-40% (w/v)

605 glycerol gradient and were sedimented in a SW40Ti rotor at 100,000 × g for 17 h at 4°C. The

606 gradient was fractionated in 590 μl fractions and the absorption was determined at 260 nm.

607 Protein samples for SDS-PAGE were prepared for each individual fraction and analysed as

608 previously described (22). A 0.5 ml volume of each fraction was used for RNA isolation by

609 addition of 1% SDS, and subsequently 600 μl acidic PCI (Carl Roth). Samples were vortexed

610 extensively and the phases separated by centrifugation. The upper aqueous layer was

611 extracted repeatedly with chloroform. The aqueous layer was selected and precipitated by

612 three volumes ice-cold ethanol with 0.3 M NaOAc (pH 6.5). GlycoBlue® (1 μl) (Thermo Fischer

613 Scientific) was added as tracer. After overnight storage at -20°C the precipitated RNA was

614 pelleted and washed with 70% ethanol, dried and dissolved in nuclease-free water for by

615 northern blot analysis, as previously described (98) with oligomers listed in Table S2.

616

617 28

618 RNA sequencing

619 RNA samples from the gradient factions of naïve or ΦKZ-infected bacteria were diluted 1:10

620 with nuclease-free water and 10 μl of the dilution were mixed with 10 μl of 1:100 diluted

621 ERCC RNA Spike-In Mix 1 (Thermo Fischer Scientific). This mix was used as an external RNA

622 control for read normalization and to determine the dynamic range in sequencing, as

623 suggested in best practice guidelines by the External RNA Controls Consortium (Baker et al.

624 2005). RNA-sequencing libraries were prepared by VERTIS Biotechnologie AG (Freisingen,

625 Germany) as previously described (29). In essence, RNA samples were fragmented by

626 ultrasound (four pulses of 30 s at 4°C) followed by 3' adapter ligation. The 3' adapter served

627 as primer, synthesis of the first-strand cDNA was performed using M-MLV reverse

628 transcriptase. After purification, 5' Illumina TruSeq sequencing adapter was ligated to the

629 end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10–20 ng/μl using

630 a high-fidelity DNA polymerase and purified with the Agencourt AMPure XP Kit (Beckman

631 Coulter Genomics). The cDNA samples were pooled based on the ratios according to the RNA

632 concentrations of the input samples, and a size range of 200–550 bp was eluted from a

633 preparative agarose gel. This size-selected cDNA pool was finally subjected to sequencing on

634 an Illumina NextSeq 500 system using 75-nt single-end read length.

635

636 Mass spectrometry

637 The nanoLC-MS/MS analysis was performed as previously described (29). Protein samples

638 diluted in 1.25× protein loading buffer were homogenized by ultrasound [five cycles of 30 s

639 on followed by 30 s off, high power at 4°C, Bioruptor Plus, Diagenode]. Insoluble material was

640 removed by pelleting at full speed. A 20 μl sample of the cleared protein was mixed with 10 μl

641 of UPS2 spike-in (Sigma-Aldrich) and diluted in 250 μl 1.25× protein loading buffer. The

642 samples were subsequently reduced in 50 mM DTT for 10 min at 70°C and alkylated with

643 120 mM iodoacetamide for 20 min at room temperature in the dark. The proteins were

644 precipitated in four volumes of acetone overnight at -20°C. Pellets were washed four times

645 with ice-cold acetone and dissolved in 50 μl 8 M urea, 100 mM ammonium bicarbonate.

646 Digestion of the proteins was performed by 0.25 μg Lys-C (Wako) for 2 h at 30°C, followed by

647 dilution to 2 M urea by addition of 3 volumes 100 mM ammonium bicarbonate, pH 8 and

648 overnight digestion with 0.25 μg trypsin at 37°C.

649 Peptides were purified through C-18 Stage Tips (99). Each Stage Tip was prepared

650 with three disks of C-18 Empore SPE Disks (3M) in a 200 μl pipette tip. Peptides were eluted

651 with 60% acetonitrile/0.3% formic acid, lyophilized in a laboratory freeze-dryer (Christ), and

652 stored at -20°C. Prior to nanoLC-MS/MS, the peptides were dissolved in 2%

653 acetonitrile/0.1% formic acid.

654

655 NanoLC-MS/MS analysis

656 NanoLC-MS/MS analysis was performed as previously described (29) with an Orbitrap

657 Fusion (Thermo Fisher Scientific) equipped with a PicoView Ion Source (New Objective) and

658 coupled to an EASY-nLC 1000 (Thermo Fisher Scientific). Peptides were loaded on capillary

659 columns (PicoFrit, 30 cm × 150 μm ID, New Objective) self-packed with ReproSil-Pur 120

660 C18-AQ, 1.9 μm (Dr. Maisch) and separated with a 140-min linear gradient from 3 to 40%

661 acetonitrile and 0.1% formic acid at a flow rate of 500 nl/min.

662 Both MS and MS/MS scans were acquired in the Orbitrap analyser with a resolution

663 of 60,000 for MS scans and 15,000 for MS/MS scans. HCD fragmentation with 35%

664 normalized collision energy was applied. A Top Speed data-dependent MS/MS method with

665 a fixed cycle time of 3 s was used. Dynamic exclusion was applied with a repeat count of 1

666 and an exclusion duration of 60 s. Precursors that were singly charged were excluded from

667 selection. Minimum signal threshold for precursor selection was set to 50,000. Predictive

668 AGC was used with a target value of 2 × 105 for MS scans and 5 × 104 for MS/MS scans. EASY-

669 IC was used for internal calibration.

670

671 Grad-seq RNA data analysis

672 Reads were mapped to the Pseudomonas and ΦKZ reference sequences (NC_002516 and

673 NC_004629, respectively) using the READemption align function (READemption version

674 0.4.3, (100)). About ~96% of reads were aligned, for details see the statistics files in the

675 READemption analysis folder. Coverage wig-files were generated with the coverage function

676 and read allocation to genomic features was quantified by the gene_quanti function (gff files

677 are supplied in the READemption analysis folder, Zenodo 4024332). Annotation of 3'/5'UTRs

678 was used from our parallel study and sRNAs from PseudoCAP (42), and manually annotated

679 transcripts were added. Sequencing coverages were visualized in the Integrative Genomics

680 Viewer (IGV, Broad Institute, (101)) based on uniquely mapped reads and normalized to the

681 total number of aligned reads.

682

683

684 Grad-seq MS data analysis

685 Raw MS data files were analysed with MaxQuant version 1.6.2.2 (102). Database search was

686 performed with Andromeda, which is integrated in the utilized version of MaxQuant. The

687 search was performed against the UniProt Pseudomonas aeruginosa UP000002438 (strain

688 PAO1), the ΦKZ proteome UP000002098 (37) and a database containing the proteins of the

689 UPS2 proteomic standard. In addition, a database containing common contaminants was

690 used. The search was performed with tryptic cleavage specificity with three allowed missed

691 cleavages. Protein identification was under control of the false-discovery rate (FDR, 1% on

692 protein and peptide level). In addition to MaxQuant default settings, the search was

693 additionally performed for the following variable modifications: Protein N-terminal

694 acetylation, glutamine to pyro-glutamic acid formation (N-term. glutamine) and oxidation

695 (methionine). Carbamidomethyl (cysteine) was set as fixed modification. For protein

696 quantitation, the iBAQ intensities were used (103). Proteins that could not be distinguished

697 by peptides were listed individually.

698

699 Normalization and dose-response curves

700 Relative protein abundance in gradient fractions were calculated by iBAQ, as described

701 previously (75). Relative protein abundance was estimated by correcting for alterations in

702 digestion and C18 purification in fractions by normalization to human albumin (Table S1,

703 norm. to spike-in, P02768ups|ALBU_HUMAN_UPS Serum albumin, chain 26-609, Fig. S1A-C)

704 that was spiked-in as part of the UPS2 standard (Sigma-Aldrich). We selected albumin for

705 normalization because of the highest number of recovered peptides (104). In bar-diagram

706 representation, the abundances for each individual protein were transformed into

707 distributions across the gradient by dividing the abundance in each fraction by the maximal

708 abundance across the gradient (Table S1, norm. to max.). For visualization purposes of

709 differential sedimentation representation, the y-axes of individual profiles were scaled to the

710 largest value in both represented gradients. For RNAs basically the same approach was used

711 with the ERCC-0130 spike in, as all spike-in RNAs behaved well correlated in sample-wise

712 quantification. Based on the corrected quantification values, we setup a dose-response curve

713 plotting the measured relative abundance vs. the input abundance (Fig. S1D-F). The linear

714 range of recovery was determined for proteins being log10iBAQ >7.5 and for log10RNA levels

715 >1.5. All proteins and RNAs that were recovered below the threshold were neglected

716 representing 614 proteins (Fig. S1B) and 2,182 RNAs (Fig. S1E).

717 Oligo probe labelling and northern blotting

718 Oligos were radioactively labelled at 5'-OH with [γ-32P]ATP by the T4-polynucleotide kinase,

719 as reported previously (75). Northern probing was performed as previously reported (98).

720

721 Data availability

722 MS data are accessible at the ProteomeXchange consortium (105) via the PRIDE partner

723 repository (106) with the dataset identifier PXD021359. Raw data after MaxQuant and

724 sequencing analysis are listed in Table S1 in the tab ‘RAW’. Sequencing raw FASTQ and

725 analysed WIG and TDF coverage files are accessible at Gene Expression Omnibus (GEO,

726 (107)) with the accession no. GSE157708. The code for the Gradseq browser is deposited at

727 Zenodo (DOI:10.5281/zenodo.3955585). The GradR browser is online accessible at

728 https://helmholtz-hiri.de/en/datasets/ radseq 1. READemption 0.4.5 is deposited at

729 Zenodo 1134354 (DOI:10.5281/zenodo.1134354).g pao The READemption analysis folder is

730 deposited at Zenodo 4024332 (DOI:10.5281/zenodo. 4024332).

731

732 ACKNOWLEDGEMENTS

733 We thank Barbara Plaschke for technical assistance, Stephanie Lamer, Andreas Schlosser,

734 Jens Hör, Lars Barquist, Konrad Förstner and Silvia di Giorgio for helpful discussions about

735 experiments and data analysis. We thank the Helmholtz Institute for RNA-based Infection

736 Research (HIRI) who supported this work with a seed grant through funds from the Bavarian

737 Ministry of Economic Affairs and Media, Energy and Technology (Grant allocation nos.

738 0703/68674/5/2017 and 0703/89374/3/2017). This work was also supported by a

739 Gottfried Wilhelm Leibniz award (DFG Vo875/18). L.W. holds a predoctoral scholarship from

740 FWO-fundamental research (11D8920N). This manuscript was supported by funding from

741 the European Research Council (ERC) under the European Union’s Horizon 2020 research

742 and innovation programme (Grant agreement No. 819800) awarded to R.L.

743 L.W., M.G., J.V. designed and performed experiments. M.G. analysed the Grad-seq data

744 and carried out bioinformatics analyses and generated figures for the manuscript. M.G. and

745 J.V. wrote the manuscript with input from all authors. J.V. supervised and guided the project

746 and interpreted Grad-seq data. All authors approved the final version.

747 We declare no competing interests.

34 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 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.

748 REFERENCES

749 1. Jernigan JA, Hatfield KM, Wolford H, Nelson RE, Olubajo B, Reddy SC, McCarthy N, Paul

750 P, McDonald LC, Kallen A, Fiore A, Craig M, Baggs J. 2020. Multidrug-resistant bacterial

751 infections in U.S. hospitalized patients, 2012–2017. N Engl J Med 382:1309-1319.

752 2. Mulani MS, Kamble EE, Kumkar SN, Tawre MS, Pardesi KR. 2019. Emerging strategies

753 to combat ESKAPE pathogens in the era of antimicrobial resistance: a review. Front

754 Microbiol 10:539.

755 3. Lamut A, Peterlin Masic L, Kikelj D, Tomasic T. 2019. Efflux pump inhibitors of

756 clinically relevant multidrug resistant bacteria. Med Res Rev 39:2460-2504.

757 4. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FSL,

758 Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-

759 Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K,

760 Spencer D, Wong GKS, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock REW, Lory S,

761 Olson MV. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an

762 opportunistic pathogen. Nature 406:959-964.

763 5. Grenga L, Little RH, Malone JG. 2017. Quick change: post-transcriptional regulation in

764 Pseudomonas. FEMS Microbiol Lett 364.

765 6. Holmqvist E, Vogel J. 2018. RNA-binding proteins in bacteria. Nat Rev Microbiol

766 16:601-615.

767 7. Sonnleitner E, Schuster M, Sorger-Domenigg T, Greenberg EP, Bläsi U. 2006. Hfq-

768 dependent alterations of the transcriptome profile and effects on quorum sensing in

769 Pseudomonas aeruginosa. Mol Microbiol 59:1542-1558.

770 8. Chihara K, Bischler T, Barquist L, Monzon VA, Noda N, Vogel J, Tsuneda S. 2019.

771 Conditional Hfq association with small noncoding RNAs in Pseudomonas aeruginosa

772 revealed through comparative UV cross-linking immunoprecipitation followed by

773 high-throughput sequencing. mSystems 4:e00590-19.

774 9. Kambara TK, Ramsey KM, Dove SL. 2018. Pervasive targeting of nascent transcripts

775 by Hfq. Cell Rep 23:1543-1552.

776 10. Zhang Y-F, Han K, Chandler CE, Tjaden B, Ernst RK, Lory S. 2017. Probing the sRNA

777 regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of

778 pathogenicity and antibiotic susceptibility. Mol Microbiol 106:919-937.

779 11. Han K, Tjaden B, Lory S. 2016. GRIL-seq provides a method for identifying direct

780 targets of bacterial small regulatory RNA by in vivo proximity ligation. Nat Microbiol

781 2:16239.

782 12. Pei XY, Dendooven T, Sonnleitner E, Chen S, Bläsi U, Luisi BF. 2019. Architectural

783 principles for Hfq/Crc-mediated regulation of gene expression. eLife 8:e43158.

784 13. Sonnleitner E, Wulf A, Campagne S, Pei X-Y, Wolfinger MT, Forlani G, Prindl K, Abdou

785 L, Resch A, Allain FH-T, Luisi BF, Urlaub H, Bläsi U. 2017. Interplay between the

786 catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational

787 regulation in Pseudomonas aeruginosa. Nucleic Acids Res 46:1470-1485.

788 14. Marden JN, Diaz MR, Walton WG, Gode CJ, Betts L, Urbanowski ML, Redinbo MR, Yahr

789 TL, Wolfgang MC. 2013. An unusual CsrA family member operates in series with RsmA

790 to amplify posttranscriptional responses in Pseudomonas aeruginosa. Proc Natl Acad

791 Sci U S A 110:15055-15060.

792 15. Morris Elizabeth R, Hall G, Li C, Heeb S, Kulkarni Rahul V, Lovelock L, Silistre H,

793 Messina M, Cámara M, Emsley J, Williams P, Searle Mark S. 2013. Structural

794 rearrangement in an RsmA/CsrA ortholog of Pseudomonas aeruginosa creates a

795 dimeric RNA-binding protein, RsmN. Structure 21:1659-1671.

796 16. Pessi G, Williams F, Hindle Z, Heurlier K, Holden MT, Cámara M, Haas D, Williams P.

797 2001. The global posttranscriptional regulator RsmA modulates production of

798 virulence determinants and N-acylhomoserine lactones in Pseudomonas aeruginosa. J

799 Bacteriol 183:6676-6683.

800 17. Chihara K, Barquist L, Takasugi K, Noda N, Tsuneda S. 2020. Global identification of

801 RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq. bioRxiv

802 doi:10.1101/2020.08.24.265819:2020.08.24.265819.

803 18. Romero M, Silistre H, Lovelock L, Wright VJ, Chan K-G, Hong K-W, Williams P, Cámara

804 M, Heeb S. 2018. Genome-wide mapping of the RNA targets of the Pseudomonas

805 aeruginosa riboregulatory protein RsmN. Nucleic Acids Res 46:6823-6840.

806 19. Gebhardt MJ, Kambara TK, Ramsey KM, Dove SL. 2020. Widespread targeting of

807 nascent transcripts by RsmA in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A

808 117:10520-10529.

809 20. Holmqvist E, Li L, Bischler T, Barquist L, Vogel J. 2018. Global maps of ProQ binding in

810 vivo reveal target recognition via RNA structure and stability control at mRNA 3' ends.

811 Mol Cell 70:971-982.

812 21. Bauriedl S, Gerovac M, Heidrich N, Bischler T, Barquist L, Vogel J, Schoen C. 2020. The

813 minimal meningococcal ProQ protein has an intrinsic capacity for structure-based

814 global RNA recognition. Nat Commun 11:2823.

815 22. Smirnov A, Forstner KU, Holmqvist E, Otto A, Gunster R, Becher D, Reinhardt R, Vogel

816 J. 2016. Grad-seq guides the discovery of ProQ as a major small RNA-binding protein.

817 Proc Natl Acad Sci U S A 113:11591-11596.

818 23. Melamed S, Adams PP, Zhang A, Zhang H, Storz G. 2020. RNA-RNA interactomes of

819 ProQ and Hfq reveal overlapping and competing roles. Mol Cell 77:411-425.

820 24. Westermann AJ, Venturini E, Sellin ME, Förstner KU, Hardt W-D, Vogel J. 2019. The

821 major RNA-binding protein ProQ impacts virulence gene expression in Salmonella

822 enterica serovar Typhimurium. mBio 10:e02504-18.

823 25. Olejniczak M, Storz G. 2017. ProQ/FinO-domain proteins: another ubiquitous family

824 of RNA matchmakers? Mol Microbiol 104:905-915.

825 26. Michaux C, Holmqvist E, Vasicek E, Sharan M, Barquist L, Westermann AJ, Gunn JS,

826 Vogel J. 2017. RNA target profiles direct the discovery of virulence functions for the

827 cold-shock proteins CspC and CspE. Proc Natl Acad Sci U S A 114:6824-6829.

828 27. Smith T, Villanueva E, Queiroz RML, Dawson CS, Elzek M, Urdaneta EC, Willis AE,

829 Beckmann BM, Krijgsveld J, Lilley KS. 2020. Organic phase separation opens up new

830 opportunities to interrogate the RNA-binding proteome. Curr Opin Chem Biol 54:70-

831 75.

832 28. Hör J, Di Giorgio S, Gerovac M, Venturini E, Förstner KU, Vogel J. 2020. Grad-seq shines

833 light on unrecognized RNA and protein complexes in the model bacterium Escherichia

834 coli. Nucleic Acids Res 48:9301–9319.

835 29. Hör J, Garriss G, Di Giorgio S, Hack L-M, Vanselow JT, Förstner KU, Schlosser A,

836 Henriques-Normark B, Vogel J. 2020. Grad-seq in a Gram-positive bacterium reveals

837 exonucleolytic sRNA activation in competence control. EMBO J 39:e103852.

838 30. Ceyssens P-J, Minakhin L, Van den Bossche A, Yakunina M, Klimuk E, Blasdel B, De

839 Smet J, Noben J-P, Bläsi U, Severinov K, Lavigne R. 2014. Development of giant

840 bacteriophage ϕKZ is independent of the host transcription apparatus. J Virol

841 88:10501-10510.

842 31. Kavaliauskas D, Nissen P, Knudsen CR. 2012. The busiest of all ribosomal assistants:

843 Elongation Factor Tu. Biochemistry 51:2642-2651.

844 32. Huter P, Müller C, Arenz S, Beckert B, Wilson DN. 2017. Structural basis for ribosome

845 rescue in bacteria. Trends Biochem Sci 42:669-680.

846 33. Vogel DW, Hartmann RK, Struck JC, Ulbrich N, Erdmann VA. 1987. The sequence of the

847 6S RNA gene of Pseudomonas aeruginosa. Nucleic Acids Res 15:4583-4591.

848 34. Wassarman KM, Storz G. 2000. 6S RNA regulates E. coli RNA polymerase activity. Cell

849 101:613-623.

850 35. Wassarman KM, Saecker RM. 2006. Synthesis-mediated release of a small RNA

851 inhibitor of RNA polymerase. Science 314:1601-1603.

852 36. Gildehaus N, Neusser T, Wurm R, Wagner R. 2007. Studies on the function of the

853 riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro

854 transcription and synthesis of RNA-directed de novo transcripts. Nucleic Acids Res

855 35:1885-1896.

856 37. The UniProt Consortium. 2018. UniProt: a worldwide hub of protein knowledge.

857 Nucleic Acids Res 47:D506-15.

858 38. Lee J, Oh S, Bhattacharya S, Zhang Y, Florens L, Washburn MP, Workman JL. 2020. The

859 plasticity of the pyruvate dehydrogenase complex confers a labile structure that is

860 associated with its catalytic activity. bioRxiv

861 doi:10.1101/2020.06.02.130369:2020.06.02.130369.

862 39. Joyce MA, Fraser ME, James MNG, Bridger WA, Wolodko WT. 2000. ADP-binding site

863 of Escherichia coli succinyl-CoA synthetase revealed by X-ray crystallography.

864 Biochemistry 39:17-25.

865 40. Saikawa N, Akiyama Y, Ito K. 2004. FtsH exists as an exceptionally large complex

866 containing HflKC in the plasma membrane of Escherichia coli. J Struct Biol 146:123-

867 129.

868 41. Dötsch A, Eckweiler D, Schniederjans M, Zimmermann A, Jensen V, Scharfe M, Geffers

869 R, Häussler S. 2012. The Pseudomonas aeruginosa transcriptome in planktonic

870 cultures and static biofilms using RNA sequencing. PloS One 7:e31092.

871 42. Winsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA, Brinkman Fiona SL. 2015. Enhanced

872 annotations and features for comparing thousands of Pseudomonas genomes in the

873 Pseudomonas genome database. Nucleic Acids Res 44:D646-53.

874 43. Hör J, Matera G, Vogel J, Gottesman S, Storz G. 2020. Trans-acting small RNAs and their

875 effects on gene expression in Escherichia coli and Salmonella enterica. EcoSal Plus 9.

876 44. Papenfort K, Förstner KU, Cong J-P, Sharma CM, Bassler BL. 2015. Differential RNA-

877 seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm

878 formation. Proc Natl Acad Sci U S A 112:E766-75.

879 45. Gómez-Lozano M, Marvig RL, Molin S, Long KS. 2012. Genome-wide identification of

880 novel small RNAs in Pseudomonas aeruginosa. Environ Microbiol 14:2006-2016.

881 46. Ferrara S, Brugnoli M, De Bonis A, Righetti F, Delvillani F, Dehò G, Horner D, Briani F,

882 Bertoni G. 2012. Comparative profiling of Pseudomonas aeruginosa strains reveals

883 differential expression of novel unique and conserved small RNAs. PLoS One

884 7:e36553.

885 47. Wagner EGH, Romby P. 2015. Chapter Three - Small RNAs in Bacteria and Archaea:

886 Who They Are, What They Do, and How They Do It. Adv Genet 90:133-208.

887 48. Chakravarty S, Massé E. 2019. RNA-dependent regulation of virulence in pathogenic

888 bacteria. Front Cell Infect Microbiol 9:337.

889 49. Miyakoshi M, Chao Y, Vogel J. 2015. Regulatory small RNAs from the 3′ regions of

890 bacterial mRNAs. Curr Opin Microbiol 24:132-139.

891 50. Adams PP, Storz G. 2020. Prevalence of small base-pairing RNAs derived from diverse

892 genomic loci. Biochim Biophys Acta Gene Regul Mech 1863:194524.

893 51. Wicke L, Ponath F, Coppens L, Gerovac M, Lavigne R, Vogel J. 2020. Introducing

894 differential RNA-seq mapping to track the early infection phase for Pseudomonas

895 phage ɸKZ. RNA Biol doi:10.1080/15476286.2020.1827785:1-12.

896 52. Wurtzel O, Yoder-Himes DR, Han K, Dandekar AA, Edelheit S, Greenberg EP, Sorek R,

897 Lory S. 2012. The single-nucleotide resolution transcriptome of Pseudomonas

898 aeruginosa grown in body temperature. PLoS Pathog 8:e1002945.

899 53. Gill EE, Chan LS, Winsor GL, Dobson N, Lo R, Ho Sui SJ, Dhillon BK, Taylor PK, Shrestha

900 R, Spencer C, Hancock REW, Unrau PJ, Brinkman FSL. 2018. High-throughput

901 detection of RNA processing in bacteria. BMC Genomics 19:223.

902 54. Chao Y, Vogel J. 2016. The 3′UTR-derived small RNA provides the regulatory

903 noncoding arm of the inner membrane stress response. Mol Cell 61:352-363.

904 55. Thomason MK, Voichek M, Dar D, Addis V, Fitzgerald D, Gottesman S, Sorek R,

905 Greenberg EP. 2019. A rhlI 5′ UTR-derived sRNA regulates RhlR-dependent quorum

906 sensing in Pseudomonas aeruginosa. mBio 10:e02253-19.

907 56. Wright PR, Georg J, Mann M, Sorescu DA, Richter AS, Lott S, Kleinkauf R, Hess WR,

908 Backofen R. 2014. CopraRNA and IntaRNA: predicting small RNA targets, networks

909 and interaction domains. Nucleic Acids Res 42:W119-23.

910 57. Sonnleitner E, Gonzalez N, Sorger-Domenigg T, Heeb S, Richter AS, Backofen R,

911 Williams P, Hüttenhofer A, Haas D, Bläsi U. 2011. The small RNA PhrS stimulates

912 synthesis of the Pseudomonas aeruginosa quinolone signal. Mol Microbiol 80:868-885.

913 58. Sonnleitner E, Bläsi U. 2014. Regulation of Hfq by the RNA CrcZ in Pseudomonas

914 aeruginosa carbon catabolite repression. PLoS Genet 10:e1004440.

915 59. Brencic A, Lory S. 2009. Determination of the regulon and identification of novel

916 mRNA targets of Pseudomonas aeruginosa RsmA. Mol Microbiol 72:612-632.

917 60. Duss O, Michel E, Yulikov M, Schubert M, Jeschke G, Allain FHT. 2014. Structural basis

918 of the non-coding RNA RsmZ acting as a protein sponge. Nature 509:588-592.

919 61. El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, Qureshi M, Richardson

920 LJ, Salazar GA, Smart A, Sonnhammer EL L, Hirsh L, Paladin L, Piovesan D, Tosatto SC E,

921 Finn RD. 2018. The Pfam protein families database in 2019. Nucleic Acids Res

922 47:D427-32.

923 62. Djapgne L, Panja S, Brewer LK, Gans JH, Kane MA, Woodson SA, Oglesby-Sherrouse AG.

924 2018. The Pseudomonas aeruginosa PrrF1 and PrrF2 small regulatory RNAs promote

925 2-alkyl-4-quinolone production through redundant regulation of the antR mRNA. J

926 Bacteriol 200:e00704-17.

927 63. Sonnleitner E, Sorger-Domenigg T, Madej MJ, Findeiss S, Hackermüller J, Hüttenhofer

928 A, Stadler PF, Bläsi U, Moll I. 2008. Detection of small RNAs in Pseudomonas aeruginosa

929 by RNomics and structure-based bioinformatic tools. Microbiology (Reading)

930 154:3175-3187.

931 64. Gómez Lozano M. 2013. Genome-wide identification of novel small RNAs in

932 Pseudomonas aeruginosa. Novo Nordisk Foundation Center for Biosustainability,

933 Technical University of Denmark.

934 65. Weaver J, Mohammad F, Buskirk AR, Storz G. 2019. Identifying small proteins by

935 ribosome profiling with stalled initiation complexes. mBio 10:e02819-18.

936 66. Grady SL, Malfatti SA, Gunasekera TS, Dalley BK, Lyman MG, Striebich RC, Mayhew MB,

937 Zhou CL, Ruiz ON, Dugan LC. 2017. A comprehensive multi-omics approach uncovers

938 adaptations for growth and survival of Pseudomonas aeruginosa on n-alkanes. BMC

939 Genomics 18:334-334.

940 67. Mesyanzhinov VV, Robben J, Grymonprez B, Kostyuchenko VA, Bourkaltseva MV,

941 Sykilinda NN, Krylov VN, Volckaert G. 2002. The genome of bacteriophage ϕKZ of

942 Pseudomonas aeruginosa. J Mol Biol 317:1-19.

943 68. De Smet J, Hendrix H, Blasdel BG, Danis-Wlodarczyk K, Lavigne R. 2017. Pseudomonas

944 predators: understanding and exploiting phage–host interactions. Nat Rev Microbiol

945 15:517-530.

946 69. Mendoza SD, Nieweglowska ES, Govindarajan S, Leon LM, Berry JD, Tiwari A,

947 Chaikeeratisak V, Pogliano J, Agard DA, Bondy-Denomy J. 2020. A bacteriophage

948 nucleus-like compartment shields DNA from CRISPR nucleases. Nature 577:244-248.

949 70. Förster-Fromme K, Höschle B, Mack C, Bott M, Armbruster W, Jendrossek D. 2006.

950 Identification of genes and proteins necessary for catabolism of acyclic terpenes and

951 leucine/isovalerate in Pseudomonas aeruginosa. Appl Environ Microbiol 72:4819-

952 4828. 44

953 71. Falcone M, Ferrara S, Rossi E, Johansen HK, Molin S, Bertoni G. 2018. The small RNA

954 ErsA of Pseudomonas aeruginosa contributes to biofilm development and motility

955 through post-transcriptional modulation of AmrZ. Front Microbiol 9:238.

956 72. Ferrara S, Carloni S, Fulco R, Falcone M, Macchi R, Bertoni G. 2015. Post-

957 transcriptional regulation of the virulence-associated enzyme AlgC by the σ22-

958 dependent small RNA ErsA of Pseudomonas aeruginosa. Environ Microbiol 17:199-

959 214.

960 73. Sonnleitner E, Pusic P, Wolfinger MT, Bläsi U. 2020. Distinctive regulation of

961 carbapenem susceptibility in Pseudomonas aeruginosa by Hfq. Front Microbiol 11.

962 74. Khade P, Joseph S. 2010. Functional interactions by transfer RNAs in the ribosome.

963 FEBS Lett 584:420-426.

964 75. Gerovac M, El Mouali Y, Kuper J, Kisker C, Barquist L, Vogel J. 2020. Global discovery

965 of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new

966 FinO domain protein. RNA doi:10.1261/rna.076992.120:1448-1463.

967 76. Pandey S, Gravel CM, Stockert OM, Wang CD, Hegner CL, LeBlanc H, Berry KE. 2020.

968 Genetic identification of the functional surface for RNA binding by Escherichia coli

969 ProQ. Nucleic Acids Res 48:4507-4520.

970 77. De Smet J, Zimmermann M, Kogadeeva M, Ceyssens P-J, Vermaelen W, Blasdel B, Bin

971 Jang H, Sauer U, Lavigne R. 2016. High coverage metabolomics analysis reveals phage-

972 specific alterations to Pseudomonas aeruginosa physiology during infection. ISME J

973 10:1823-1835.

974 78. Van den Bossche A, Hardwick SW, Ceyssens P-J, Hendrix H, Voet M, Dendooven T,

975 Bandyra KJ, De Maeyer M, Aertsen A, Noben J-P, Luisi BF, Lavigne R. 2016. Structural

976 elucidation of a novel mechanism for the bacteriophage-based inhibition of the RNA

977 degradosome. eLife 5:e16413.

978 79. Uzan M, Miller ES. 2010. Post-transcriptional control by bacteriophage T4: mRNA

979 decay and inhibition of translation initiation. Virol J 7:360-360.

980 80. Olins PO, Devine CS, Rangwala SH, Kavka KS. 1988. The T7 phage gene 10 leader RNA,

981 a ribosome-binding site that dramatically enhances the expression of foreign genes in

982 Escherichia coli. Gene 73:227-235.

983 81. Kalousek S, Schrot G, Lubitz W, Bläsi U. 1994. Expression of the Alcaligenes eutrophus

984 phbA gene in Escherichia coli using a positive selection vector based on phage Lambda

985 lysis genes. J Biotechnol 33:15-19.

986 82. Robertson ES, Nicholson AW. 1992. Phosphorylation of Escherichia coli translation

987 initiation factors by the bacteriophage T7 protein kinase. Biochemistry 31:4822-4827.

988 83. Robertson ES, Aggison LA, Nicholson AW. 1994. Phosphorylation of elongation factor

989 G and ribosomal protein S6 in bacteriophage T7-infected Escherichia coli. Mol

990 Microbiol 11:1045-1057.

991 84. Herrlich P, Rahmsdorf HJ, Pai SH, Schweigher M. 1974. Translational control induced

992 by bacteriophage T7. Proc Natl Acad Sci U S A 71:1088-1092.

993 85. Mizuno CM, Guyomar C, Roux S, Lavigne R, Rodriguez-Valera F, Sullivan MB, Gillet R,

994 Forterre P, Krupovic M. 2019. Numerous cultivated and uncultivated viruses encode

995 ribosomal proteins. Nat Commun 10:752.

996 86. Al-Shayeb B, Sachdeva R, Chen L-X, Ward F, Munk P, Devoto A, Castelle CJ, Olm MR,

997 Bouma-Gregson K, Amano Y, He C, Méheust R, Brooks B, Thomas A, Lavy A, Matheus-

998 Carnevali P, Sun C, Goltsman DSA, Borton MA, Sharrar A, Jaffe AL, Nelson TC, Kantor

999 R, Keren R, Lane KR, Farag IF, Lei S, Finstad K, Amundson R, Anantharaman K, Zhou J,

1000 Probst AJ, Power ME, Tringe SG, Li W-J, Wrighton K, Harrison S, Morowitz M, Relman

1001 DA, Doudna JA, Lehours A-C, Warren L, Cate JHD, Santini JM, Banfield JF. 2020. Clades

1002 of huge phages from across Earth’s ecosystems. Nature 578:425-31.

1003 87. Millman A, Melamed S, Amitai G, Sorek R. 2020. Diversity and classification of cyclic-

1004 oligonucleotide-based anti-phage signalling systems. Nat Microbiol

1005 doi:10.1038/s41564-020-0777-y:1608-1615.

1006 88. Lin P, Pu Q, Wu Q, Zhou C, Wang B, Schettler J, Wang Z, Qin S, Gao P, Li R, Li G, Cheng Z,

1007 Lan L, Jiang J, Wu M. 2019. High-throughput screen reveals sRNAs regulating crRNA

1008 biogenesis by targeting CRISPR leader to repress Rho termination. Nature Commun

1009 10:3728.

1010 89. Dendooven T, Lavigne R. 2019. Dip-a-Dee-Doo-Dah: Bacteriophage-mediated

1011 rescoring of a harmoniously orchestrated RNA metabolism. Annu Rev Virol 6:199-

1012 213.

1013 90. Okamoto K, Sugino Y, Nomura M. 1962. Synthesis and turnover of phage messenger

1014 RNA in E. coli infected with bacteriophage T4 in the presence of chloromycetin. J Mol

1015 Biol 5:527-534.

1016 91. Dendooven T, Van den Bossche A, Hendrix H, Ceyssens P-J, Voet M, Bandyra KJ, De

1017 Maeyer M, Aertsen A, Noben J-P, Hardwick SW, Luisi BF, Lavigne R. 2017. Viral

1018 interference of the bacterial RNA metabolism machinery. RNA Biol 14:6-10.

1019 92. Jain C, Belasco JG. 1995. Autoregulation of RNase E synthesis in Escherichia coli.

1020 Nucleic Acids Symp Ser:85-88.

1021 93. Diwa A, Bricker AL, Jain C, Belasco JG. 2000. An evolutionarily conserved RNA stem–

1022 loop functions as a sensor that directs feedback regulation of RNase E gene expression.

1023 Genes Dev 14:1249-1260.

1024 94. Schuck A, Diwa A, Belasco JG. 2009. RNase E autoregulates its synthesis in Escherichia

1025 coli by binding directly to a stem-loop in the rne 5′ untranslated region. Mol Microbiol

1026 72:470-478.

1027 95. Weinberg Z, Wang JX, Bogue J, Yang J, Corbino K, Moy RH, Breaker RR. 2010.

1028 Comparative genomics reveals 104 candidate structured RNAs from bacteria, archaea,

1029 and their metagenomes. Genome Biol 11:R31.

1030 96. Stazic D, Pekarski I, Kopf M, Lindell D, Steglich C. 2016. A novel strategy for

1031 exploitation of host RNase E activity by a marine cyanophage. Genetics 203:1149-59.

1032 97. Bloch S, Lewandowska N, Węgrzyn G, Nejman-Faleńczyk B. 2020. Bacteriophages as

1033 sources of small non-coding RNA molecules. Plasmid

1034 doi:https://doi.org/10.1016/j.plasmid.2020.102527:102527.

1035 98. Sharma CM, Papenfort K, Pernitzsch SR, Mollenkopf H-J, Hinton JCD, Vogel J. 2011.

1036 Pervasive post-transcriptional control of genes involved in amino acid metabolism by

1037 the Hfq-dependent GcvB small RNA. Mol Microbiol 81:1144-1165.

1038 99. Rappsilber J, Ishihama Y, Mann M. 2003. Stop and go extraction tips for matrix-

1039 assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample

1040 pretreatment in proteomics. Anal Chem 75:663-670.

1041 100. Förstner KU, Vogel J, Sharma CM. 2014. READemption—a tool for the computational

1042 analysis of deep-sequencing–based transcriptome data. Bioinformatics 30:3421-

1043 3423.

1044 101. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov

1045 JP. 2011. Integrative genomics viewer. Nat Biotechnol 29:24-26.

1046 102. Cox J, Mann M. 2008. MaxQuant enables high peptide identification rates,

1047 individualized p.p.b.-range mass accuracies and proteome-wide protein

1048 quantification. Nat Biotechnol 26:1367-1372.

1049 103. Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M.

1050 2011. Global quantification of mammalian gene expression control. Nature 473:337-

1051 342.

1052 104. Lott SC, Schäfer RA, Mann M, Backofen R, Hess WR, Voß B, Georg J. 2018. GLASSgo –

1053 Automated and reliable detection of sRNA homologs from a single input sequence.

1054 Front Genetics 9.

1055 105. Deutsch EW, Csordas A, Sun Z, Jarnuczak A, Perez-Riverol Y, Ternent T, Campbell DS,

1056 Bernal-Llinares M, Okuda S, Kawano S, Moritz RL, Carver JJ, Wang M, Ishihama Y,

1057 Bandeira N, Hermjakob H, Vizcaíno JA. 2016. The ProteomeXchange consortium in

1058 2017: supporting the cultural change in proteomics public data deposition. Nucleic

1059 Acids Res 45:D1100-6.

1060 106. Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ,

1061 Inuganti A, Griss J, Mayer G, Eisenacher M, Pérez E, Uszkoreit J, Pfeuffer J, Sachsenberg

1062 T, Yılmaz Ş, Tiwary S, Cox J, Audain E, Walzer M, Jarnuczak AF, Ternent T, Brazma A,

1063 Vizcaíno JA. 2018. The PRIDE database and related tools and resources in 2019:

1064 improving support for quantification data. Nucleic Acids Res 47:D442-50.

1065 107. Edgar R, Domrachev M, Lash AE. 2002. Gene Expression Omnibus: NCBI gene

1066 expression and hybridization array data repository. Nucleic Acids Res 30:207-210.

1067 108. Zuker M. 2003. Mfold web server for nucleic acid folding and hybridization prediction.

1068 Nucleic Acids Res 31:3406-3415.

1069

1070

1071 FIGURES

1072 Figure 1. Grad-seq in Pseudomonas. (A) Cellular lysate is analysed in a gradient and

1073 fractionated. High-throughput analysis by mass-spectrometry (LC-MS/MS) and RNA-

1074 sequencing reveals sedimentation profiles. (B) Complexes are resolved in a 10-40% glycerol

1075 gradient by size and density. The 260 nm-absorption profile of Pseudomonas lysate indicates

1076 LMW fractions at the top, 30S small, 50S large ribosomal subunits at fractions 9 and 15,

1077 respectively, and an elevated absorption in the pellet at HMW fractions. The profile is shifted

1078 by about one fraction compared to E. coli and S. enterica. The RNAP sediments between the

1079 top and 30S subunit. (C) The apparent proteome resembles the positions in (B). EF-Tu is a

1080 transient translation factor and sedimented in the top, RNAP subunits RpoB/C and GroEL

1081 sediment before 30S fractions. (D) tRNAs sedimented in top fractions, ribosomal RNAs

1082 resemble well the distribution in (B,C), 6S RNA sedimentation correlated with RNAP in (C).

1083 (E) tmRNA sedimented in the pellet and at RNAP fractions, 6S RNA sedimentation correlated

1084 with RNAP fractions and 5S rRNA with 50S ribosomal subunit fractions. (F) High throughput

1085 determined sedimentation profiles matched well with in gel and previously published results

1086 in other species.

1087 Figure 2. Grad-seq allows for mapping of pRNAs. (A) RNAP subunits, 6S RNA and pRNAs

1088 co-sedimented in the gradient. pRNAs were mapped by differential sedimentation.

1089 Importantly, pRNAs sedimented at a much larger position in fraction three indicating

1090 presence in a complex. (B) The precise position of pRNAs transcription initiation in the 6S

1091 initiation bubble was mapped based on differential read coverage from (A). The structure of

1092 6S RNA was mapped based on the Rfam annotation RF00013.

1093 Figure 3. Grad-seq pipeline and all detected sedimentation profiles. (A) Proteins

1094 sedimented mainly in the first couple of fractions. Defined clusters appeared in fraction

1095 below 10. Ribosomal proteins sedimented around fractions 10 and 16. (B) Transcripts

1096 (merged CDS and UTRs) sedimented mainly in RNAP and ribosomal fractions. tRNAs did not

1097 enter the gradient and ribosomal RNAs allocated to HMW fractions.

1098 Figure 4. MS detection of complexes and RNPs. (A) 2,182 proteins were recovered above

1099 the threshold of total iBAQ > 7.5 that represents about 40% of the total proteome. Membrane

1100 proteins (yellow) were depleted in the analysed soluble fraction. (B) 21 fraction

1101 sedimentation profiles were reduced in dimensionality to two principal components (PCs)

1102 resembling sedimentation complexity and coefficient. In the central part RNAP and ATP-

1103 synthase complexes cluster in proximity with major RBPs Hfq, ProQ, and RsmA/N.

1104 Figure 5. Large complexes co-sediment in higher-molecular weight fractions.

1105 (A) Sedimentation profiles of proteins that peaked in fractions 8-20 were clustered and

1106 labelled by protein name. Dashed circles indicate 30S and 50S ribosomal protein clusters.

1107 (B) Sedimentation profiles of metabolic complexes are listed with indicated calculated

1108 masses. The three dehydrogenase complexes sedimented between fractions 6-12. The TCA

1109 cycle complexes succinyl-CoA synthase and aspartate carbamoyltransferase sedimented in

1110 fractions 3 and 5, respectively. The chaperone GroEL sedimented in fraction 5 and the

1111 associated subunit GroES was dissociated and allocated to fraction 2. The protease complex

1112 FtsH/HflKC was detected in fraction 11. The relative abundance is ranging from 0-1 and is

1113 colour coded in black for proteins and in blue for RNA.

1114 Figure 6. Sedimentation of RNAs. (A) 73.6% of P. aeruginosa PAO1 transcriptome was

1115 detected. mRNAs represented about 9% and ncRNAs about 0.4% sequenced reads. (B) RNAs 52

1116 were clustered based on sedimentation position. ncRNAs accumulated together with UTRs in

1117 cluster 2. (C) Comparison of relative positions of CDS and corresponding UTRs revealed

1118 differential sedimentation that attributed to processing and rise of ncRNAs fragments. (D)

1119 The 3'UTR region of PA2758 was probed by northern for a 50-nt fragment, position was

1120 determined previously based on read-coverage in the annotated UTR. (E) An alignment of

1121 the 3'UTR region of PA2758 revealed a conserved motif in the read-covered part (red arrow).

1122 RNA Co-fold (https://e-rna.org/cofold/) resembled a two stemmed structure that is

1123 indicated in the dot-bracket annotation.

1124 Figure 7. RBPs co-sedimented with targeted RNAs. (A) Hfq associated RNAs co-

1125 sedimented in a correlated fashion (>0.7) with Hfq mainly in fractions 3-8. (B) The same

1126 holds true for Rsm RNAs and the corresponding RBPs RsmA and RsmN. (C) Other putative

1127 RBPs are the cold-shock protein CspA and three putative Csps that sedimented in the first

1128 fractions as also previously reported. The FinO-domain protein ProQ sedimented

1129 interestingly in between fractions 4 and 11. (D) Clustering of annotated ncRNAs by

1130 sedimentation position revealed clusters that could be bound by novel RBPs. Interestingly, 5

1131 ncRNAs sedimented in 30S subunit fractions, e.g. OsiS.

1132 Figure 8. Host RNome is altered at early infection. (A) ΦKZ conquering of Pseudomonas

1133 is strongly linked to modulation of RNA homeostasis. Viral RNAPs are utilized and produced

1134 to transcribe phase genes. Additional phage tRNAs are utilized in translation, and the RNA

1135 degradation is blocked through the Dip protein. (B) Host CDS and ncRNA levels were

1136 significantly reduced by about 5-fold in a non-parametric paired t-test. The threshold for

1137 statistical significance was a two-tailed P value of <0.05 (***p < 0.001; ****p < 0.0001). Prism

1138 9 was used for statistical analyses. Lines connect individual RNA levels. Outliers that

1139 increased levels upon ΦKZ infection are labelled. (C) Individual phage CDS are two

1140 magnitudes more abundant than host RNAs, whereas phage tRNAs remain underrepresented

1141 10 min post infection.

1142 Figure 9. ΦKZ infection shifts transcripts and ncRNAs. (A) Transcripts that sediment in

1143 HMW fractions are more shifted towards LMW fractions. (change in level <3-fold).

1144 (B) Annotated ncRNAs were shifted in direction of HMW fractions. (C) Sedimentation

1145 profiles of host CDS that shift strongly towards H/LMW fractions from (A). (D) Sedimentation

1146 profiles of phage-infection affected host ncRNAs, of which ErsA represents an important

1147 regulator in biofilm formation and is shifted to HMW fractions. (E) The ErsA shift in

1148 sedimentation was validated by northern probing against the end of stem-loop I in ErsA and

1149 accumulated strongly in the pellet fraction. A less pronounced shift was observed for PhrS

1150 that was strongly depleted upon infection.

1151 Figure 10. Phage RNAs associate strongly with ribosomes. (A,B) Expression and

1152 sedimentation of early infection operons 54 and 88 was northern probed and revealed a

1153 strongly ribosome associated profile. Interestingly, smaller fragments sedimented

1154 exclusively before fraction six. (C) In general, phage transcripts sedimented substantially at

1155 ribosomal fractions indicating an efficient transfer to translation or stabilization at the

1156 ribosome. Phage tRNAs were also strongly utilized in translation or stabilized at ribosomal

1157 subunits judged by sedimentation in ribosomal fractions.

1158 Figure 11. Grad-seq guides the discovery of phage-related ncRNA candidates. (A) Loci

1159 of operons 132 and 50 including candidate ncRNAs. (B) In the 3'UTR of PHIKZ298, we

1160 observed sedimentation exclusively around fraction four that was confirmed by probing a

1161 ~100-nt fragment that M-folded (108) into a two stemmed structure (C). (D) Read-coverage 54

1162 profiles in the 5'UTR of p40 revealed an LMW weighted sedimentation profile different from

1163 phage CDS transcripts (Fig. 10C). A ~180-nt fragment was detected in RNAP associated

1164 fractions and at 30S subunit fractions. A longer fragment (~880-nt) was substantially shifted

1165 to fraction 12. (E) The 5'UTR-p40 fragment M-folded into a two stemmed structure. Northern

1166 probing was conducted against the nucleotides indicated in orange.

1167 Figure 12. Differential Grad-seq browser. Protein and RNA sedimentation profiles can be

1168 searched for non-infected and infected experiments. Selected entries are displayed as bar-

1169 chart and can be added in a display list that is plotted as bar-chart, lines, or heatmap. The

1170 browser is accessible at https://helmholtz-hiri.de/en/datasets/gradseqpao1/

1171

1172 SUPPLEMENTARY MATERIAL

1173 Figure S1: Analysis and normalization of Grad-seq. (A) Dose-response curve for the MS

1174 analysis, plotted as relative concentration in UPS2 spike-in vs. recovered MS counts. The

1175 linear dynamic range of detection is valid down to log10 total iBAQ of 7.5. (B) 2,182 proteins

1176 were selected above the threshold from (A). 614 proteins were discarded. (C) Normalization

1177 of protein levels by human albumin (UPS2) spike-in. (D) Dose-response curve for the RNA-

1178 seq analysis based on the ERCC spike-in. The linear dynamic range of detection was valid

1179 down to log10 sum of RNA reads of 1.5. (E) 8,297 annotations were detected above the

1180 threshold of >100 reads/gradient, 2,971 annotations were excluded. (F) Normalization of

1181 RNA levels by the ERCC-130 spike-in.

1182 Figure S2. Sedimentation profiles of complexes that peak in fractions 8-20. (A) About

1183 30 uncharacterized proteins sedimented in HMW fractions and are named here by UniProt

1184 identifier. (B) Selected abundant protein bands in the Grad-seq experiment were selected for

1185 identification. (C) Protein entries were filtered by selected fraction where the protein was

1186 mostly abundant in (B) and a range of molecular weight based on the molecular size marker.

1187 The resulting table was ranked by abundance resulting in candidates. The abundance (iBAQ)

1188 was evaluated in contrast to the second-best hit. (D) Digital sedimentation profiles were

1189 correlated with the sedimentation profiles in (B).

1190 Figure S3. CopraRNA analysis of RNA-RNA interaction of the PA2758-3'UTR fragment.

1191 (A) Predicted interaction region around start codons were determined being in the first stem

1192 loop that may represent the seed sequence. (B) An example is the intergenic region between

1193 napB and napC where the fragment could pair with the Shine-Dalgarno-sequence (SDseq)

1194 and modulate translation initiation. (C) Predicted interaction regions around stop codons.

1195 (D) A second example covers an interaction in the 5'UTR of the universal stress protein A

1196 family protein uspK.

1197 Figure S4. ncRNA OsiS may harbour a hidden small open reading frame that may drive

1198 sedimentation in 30S fractions. TEX treatment indicates a TEX unaffected 5'-end

1199 downstream of the annotated TSS (51). Sequencing of RNA fragments recovered from

1200 monosomes (Ribo-seq, (66), re-analysed with HRIBO (https://github.com/RickGelhausen

1201 /HRIBO)) supports the possibility of a short translated fragment starting at a putative start

1202 codon that is out-of-frame with ptrB. In addition, an inverted repeat in-between may serve

1203 as a Rho-independent terminator because no stop-codon is reached in the reading frame.

1204 Figure S5. Gradient fractionation of ΦKZ-infected Pseudomonas at early infection time

1205 point. (A) The absorption profile and apparent proteome is not affected upon infection.

1206 (B) The global picture of the RNome is consistent with Fig. 1D and not altered upon infection. 56

1207 (C) Specific RNAs were probed by northern blotting and resulted in consistent sedimentation

1208 behaviour with non-infected cells. (D) Phage transcripts represented about 3% of all reads.

1209 Host CDS transcripts remained present at 7% - relative representation. (E) In non-infected

1210 cells, the sedimentation profile of CDS, UTRs, and ncRNAs was strongly balanced on the side

1211 of RNAP fractions and marginally on ribosomal fractions. tRNAs sedimented exclusively in

1212 LMW fractions. (F) In infected cells, the sedimentation profiles of the host transcripts were

1213 only marginally affected and shifted towards LMW fractions. (G) Phage transcripts

1214 sedimented strongly in ribosomal fractions.

1215 Figure S6. ProQ in Pseudomonas harbours important residues of a FinO-domain.

1216 (A) Genomic locus of ProQ in PAO1. (B) Conserved residues in a FinO-domain (PF04352) are

1217 represented in red. Important residues in ProQ for RNA-binding (76) are indicated with

1218 corresponding residues in ProQ from E. coli. (C) ProQ proteins in Pseudomonas represent the

1219 condensed representatives in the diverse class of FinO-domain proteins that can have C- or

1220 N-terminally associated domains.

1221 Figure S7. Transcripts for oxidative phosphorylation are shifted most strongly

1222 towards LMW fractions. (A) Overrepresentation analysis for biological processes revealed

1223 that 5'UTR transcripts that are shifted by >0.5 rel. fractions are enriched for transcription

1224 factors. 3'UTR transcripts that are shifted by more than 1 rel. fraction towards the top are

1225 enriched for factors of aerobic respiration and proton transmembrane transport.

1226 (B) 49 transcripts encoding oxidative phosphorylation factors are significantly more shifted

1227 towards the top compared to transcripts coding for metabolic factors. At the same time the

1228 relative position in the reference condition was for these transcripts significantly higher and

1229 matches the trend in (A). (C) Averaged sedimentation profile of the oxidative

1230 phosphorylation pathway pae00190 CDS, and selected examples coding for ATP synthase,

1231 NADH-oxidoreductase, and cytochrome C oxidase.

1232 Figure S8. 5S rRNA indicates processing effects and its major RNase E is altered in the

1233 5'UTR. (A) 5S rRNA sedimentation was not affected upon infection, but it become smeary in

1234 northern probing that may indicate processing defects that could lead to diminished reads in

1235 sequencing. (B) The read-coverage of the 5’UTR of rne was altered upon infection. (C) The

1236 5'UTR appeared in two fragments of which the larger one was allocated to RNAP fractions

1237 and strongly diminished upon ΦKZ infection.

1238 Table S1. Grad-MS & -seq data (xls file)

1239 Table S2. STAR methods (xls file)

bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 1 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P S A fractionation D - 16/23S 600 - rRNA gradient +/- analysis by 300 - ΦKZ phage size & density LC-MS/MS RNA-seq lysate complex - 6S RNA preparation separation protein RNA gradient profiles gradient profiles 100 - - 5S rRNA

- tRNA

6% Urea-PAGE, EtBr B P. aeruginosa S. enterica E. coli pellet

E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P S

331 - tmRNA 190 - 6S RNA A260 (a.u.) 147 - top RNAP 30S 50S 70S 5S rRNA 6% Urea-PAGE, phosphogram

LMW -10-40% glycerol gradient - HMW F RNAP30S 50S 70S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P ribosomal subunits C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P S RpsB - S2 250 - - RpoB/C 130 - 16S RNA 100 - 70 - 55 - - GroEL color-coding - EF-Tu RpsB - S2 16S RNA 35 - RplB - L2 23S RNA 25 - 5S RNA ribosomal proteins signal recognition particle (SRP) Ffh 4.5S RNA 15 - 10 - 0 max. abundance per gradient SDS-PAGE, Coomassie protein RNA

Figure 1. Grad-seq in Pseudomonas. (A) Cellular lysate is analysed in a gradient and fractionated. High-throughput analysis by mass-spectrometry (LC-MS/MS) and RNA-sequencing reveals sedimentation profiles. (B) Complexes are resolved in a 10-40% glycerol gradient by size and density. The 260 nm-absorption profile of Pseudomonas lysate indicates LMW fractions at the top, 30S small, 50S large ribosomal subunits at fractions 9 and 15, respectively, and an elevated absorption in the pellet at HMW fractions. The profile is shifted by about one fraction compared to E. coli and S. enterica. The RNAP sediments between the top and 30S subunit. (C) The apparent proteome resembles the positions in (B). EF-Tu is a transient translation factor and sedimented in the top, RNAP subunits RpoB/C and GroEL sediment before 30S fractions. (D) tRNAs sedimented in top fractions, ribosomal RNAs resemble well the distribution in (B,C), 6S RNA sedimentation correlated with RNAP in (C). (E) tmRNA sedimented in the pellet and at RNAP fractions, 6S RNA sedimentation correlated with RNAP fractions and 5S rRNA with 50S ribosomal subunit fractions. (F) High throughput determined sedimentation profiles matched well with in gel and previously published results in other species. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 2 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P RNA-polymerase (RNAP) RpoA RpoB RpoC RpoZ RpoD RpoN 6S RNA pRNAs

ssrS

pRNAs

5,884,320-

0 max. abundance per gradient protein RNA

B pRNA (14 nt.) GGAC CAG U C G G C U A UGC AC AA CGG A C GU UG U A A A A A A U U 5´ U C U C G C C G GG GU A G U C C C U GGU GU G U G GC G U CC U G A G CC GGAGG U U G C UUG G A C C GC UCCGC A C GGGGA C C A C A G GCGC GGGC CA GGUU CCGCC A A C G CA U C U G G GC CGGA AG 3´ G A U A G A U A C AA C A C C A U U A A U C GC C C U U motif based on RF00013 UC AA GU initiation bubble

Figure 2. Grad-seq allows for mapping of pRNAs. (A) RNAP subunits, 6S RNA and pRNAs co-sedimented in the gradient. pRNAs were mapped by differential sedimentation. Importantly, pRNAs sedimented at a much larger position in fraction three indicating presence in a complex. (B) The precise position of pRNAs transcription initiation in the 6S initiation bubble was mapped based on differential read coverage from (A). The structure of 6S RNA was mapped based on the Rfam annotation RF00013. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 3 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P

- braE, xylS, tadB, nirB

~ membrane assoc.

~ ox. phosphorylation

~ histidine metabolism & hypothetical protein

mRNAs

YjiK, PagL, bacterioferritin, RimP, aσ-factor FliA, DNA primase, WspC

50S 30S ribosomal prot. and maturation factors, HflC/K, ~ OsiS, RhlE, membrane pant ncRNAs assoc. proteins ncRNA

polymerase, transcript. factors, - rRNA/ cold-shock-prot., ffs/tmRNA ribosomal prot., - tRNA ATP synthase, ProQ 0, normalized to max. n = 2,182 0, normalized to max. n = 5,084

Figure 3. Grad-seq pipeline and all detected sedimentation profiles. (A) Proteins sedimented mainly in the first couple of fractions. Defined clusters appeared in fraction below 10. Ribosomal proteins sedimented around fractions 10 and 16. (B) Transcripts (merged CDS and UTRs) sedimented mainly in RNAP and ribosomal fractions. tRNAs did not enter the gradient and ribosomal RNAs allocated to HMW fractions. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 4 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A MS detected (2,182) Pseudomonas proteome not detected (3,462) metal-/nucleotide-bd. RNA-bd. membrane other I I I I I I DNA-bd. meta- uncharact- bolism erized MS detected proteome

log10 totla iBAQ > 7.5

50S

tRNA-ligases

aspartate carbaomyltransferase RsmA ProQ ATP synthase branched-chain alpha-keto acid dehydrogenase complex 30S Cytochrome c oxidase Hfq FtsH/HflKC GroEL/ES ATP-synthase oxoglutarat dehydrogenase complex RNAP RsmN pyruvate dehydrogenase complex polymerase subunits ribosomal proteins RBPs sRNA assoc. RBPs and tRNA-ligases Succinyl-CoA synthetase PC2 ~ profile complexity

central fractions HMW fractions

PC1 ~ sedimentation coefficient

Figure 4. MS detection of complexes and RNPs. (A) 2,182 proteins were recovered above the threshold of total iBAQ > 7.5 that represents about 40% of the total proteome. Membrane proteins (yellow) were depleted in the analysed soluble fraction. (B) 21 fraction sedimentation profiles were reduced in dimensionality to two principal components (PCs) resembling sedimentation complexity and coefficient. In the central part RNAP and ATP-synthase complexes cluster in proximity with major RBPs Hfq, ProQ, and RsmA/N. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 5 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P pyruvate dehydrogenase complex MW of complex E1-AceE ~ 4.6 MDa MutM E2-AceF E3-LpdG peaking fraction oxoglutarat dehydrogenase complex RibH MvaT 7.5 - 10 CcoQ2 10 - 12.5 E1-SucA 12.5 - 15 E2-SucB RbfA RlmL YajC 15 - 17.5 PssA 17.5 - 20 RimP branched-chain α-keto acid dehydrogenase complex PbpG E1-BkdA1 DnaG E1-BkdA2 FtsH FliA E2-BkdB RNase E E3-LpdV PepA RpsS BfrB HflK AlgH succinyl-CoA synthetase NrdJb SucC OprD RluE ~ 140 kDa DeaD OprHRsfS SucD HflC SucA PcnB HflX WaaF KptA aspartate carbamoyltransferase SucB RsmA FecA RpsT PyrC´ ~ 300 kDa RNase R Der 50S PyrB subunit PhaF 30S RhlE GroEL/ES subunit RluC PoxBInfC GroEL ~ 840 kDa RluB GroES AceF AtuDRsgA circle size ~ log iBAQ FtsH/HflKC holoenzyme 10 FtsH

PC2 ~ profile complexity HflC ~ 1,000 kDa HflK PC1 ~ sedimentation coefficient

Figure 5. Large complexes co-sediment in higher-molecular weight fractions. (A) Sedimentation profiles of proteins that peaked in fractions 8-20 were clustered and labelled by protein name. Dashed circles indicate 30S and 50S ribosomal protein clusters. (B) Sedimentation profiles of metabolic complexes are listed with indicated calculated masses. The three dehydrogenase complexes sedimented between fractions 6-12. The TCA cycle complexes succinyl-CoA synthase and aspartate carbamoyltransferase sedimented in fractions 3 and 5, respectively. The chaperone GroEL sedimented in fraction 5 and the associated subunit GroES was dissociated and allocated to fraction 2. The protease complex FtsH/HflKC was detected in fraction 11. The relative abundance is ranging from 0-1 and is colour coded in black for proteins and in blue for RNA. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 6 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A E

strain strand PAO1 + PA2758 PA2759 PA2760 UCBPP-PA14 - PA14_28420 PA14_28410 PA7 - PSPA7_2479 PSPA7_2477 PAK-Cain - PAKAF_02329 PAKAF_02328 LESB58 - PLES_23201 PLES_23191 5′UTR (1,818, ~1%) ncRNA (365, ~0.5%) LysR transcriptional hypothetical outer membrane CDS (4,643, ~7.3%) tRNA (62, ~6.5%) regulator ? protein protein 3′UTR (1,410, ~0.5%) r-/tmRNA (14, ~84%) I 3,118,296 ncRNA0222/SPA0115 (500 Bp) pant259

PAO1 UV

B cluster n 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P characteristics + 0-3,055 planktonic 1 13 tRNAs - 2 122 ncRNAs, UTRs + 0-1,869 3 8,058 mRNAs biofilm 4 101 mRNAs - 5 4 16S rRNA 6 13 5/23S rRNA CLIP-seq Hfq (Chihara et al. 2020)

C correlated sedimentation between CDS and UTRs? PA2578 3'CDS... stop 3'UTR... PAO1 2192 5′- 5′UTR CDS 3′UTR UCBPP-PA14 LESB58 PAK-assembly no yes 19BR UTR PABCH10 PAK-Cain CDS PA7 5' ncRNA0222/SPA0115 prediction... PAO1 2192 12 UTRs UCBPP-PA14 in LMW LESB58 PAK-assembly 11 19BR PABCH10 10 PA4972 PAK-Cain PA7 PA0450 9 PAO1 2192 8 UCBPP-PA14 LESB58 PAK-assembly 7 19BR rhlI/RhlS PABCH10 6 PAK-Cain rel. pos. CDS PA3825 PA7 PA2758 5 Grad-seq / CLIP-seq covered ((((((...... )))))).(((((((( 4 sdaB PA4643 PAO1 2192 3 UCBPP-PA14 2 4 6 8 10 12 LESB58 PAK-assembly rel. pos. UTR 19BR PABCH10 PAK-Cain PA7

D PA2758 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P hyperconserved region JVO-18951 CDS (...(..(...... )..)..))))))))).. 3′UTR PAO1 2192 501 - 404 - UCBPP-PA14 331 - LESB58 PAK-assembly 242 - 19BR PABCH10 190 - PAK-Cain PA7 147 - JVO-18951

111/110 -

67 - PA2758-3′UTR -

JVO-18951

Figure 6. Sedimentation of RNAs. (A) 73.6% of P. aeruginosa PAO1 transcriptome was detected. mRNAs represented about 9% and ncRNAs about 0.4% sequenced reads. (B) RNAs were clustered based on sedimentation position. ncRNAs accumulated together with UTRs in cluster 2. (C) Comparison of relative positions of CDS and corresponding UTRs revealed differential sedimentation that attributed to processing and rise of ncRNAs fragments. (D) The 3'UTR region of PA2758 was probed by northern for a 50-nt fragment, position was determined previously based on read-coverage in the annotated UTR. (E) An alignment of the 3'UTR region of PA2758 revealed a conserved motif in the read-covered part (red arrow). RNA Co-fold (https://e- rna.org/cofold/) resembled a two stemmed structure that is indicated in the dot-bracket annotation. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 7 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A C Hfq assoc. RNAs other putative RBPs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P correl. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P Hfq CspA PhrS 0.83 Q9I4Z8 probable Q9I4H8 Q9I662 cold-shock-proteins 190 - ProQ Crc CrcZ 0.73 404 -

cluster 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P B CsrA/RsmA/RsmN assoc. RNAs n examples 1 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P correlation 2 7 RsmY/Z/W RsmA/CsrA RsmA/N 3 9 PhrD, ReaL RsmN/CsrA´ 4 6 AmiL 5 15 4.5S RNA, ErsA RsmY 0.79/0.98 6 13 RsmV, P14, pRNAs RsmZ 0.78/0.97 7 1 RNase P RsmV 0.82/0.99 8 2 CrcZ, ssrS RsmW 0.80/0.95 9 299 PhrX/S, RhlS, PrrF1/H 10 5 OsiS OsiS

Figure 7. RBPs co-sedimented with targeted RNAs. (A) Hfq associated RNAs co-sedimented in a correlated fashion (>0.7) with Hfq mainly in fractions 3-8. (B) The same holds true for Rsm RNAs and the corresponding RBPs RsmA and RsmN. (C) Other putative RBPs are the cold-shock protein CspA and three putative Csps that sedimented in the first fractions as also previously reported. The FinO-domain protein ProQ sedimented interestingly in between fractions 4 and 11. (D) Clustering of annotated ncRNAs by sedimentation position revealed clusters that could be bound by novel RBPs. Interestingly, 5 ncRNAs sedimented in 30S subunit fractions, e.g. OsiS. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 8 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A host genome ΦKZ viral RNAP viral subunits nucleus-like early infection phase structure

tRNAs mRNAs Pseudomonas aeruginosa Dip transcription RNA polymerase (RNAP) phage translation proteins ribosome degradation DNA degradosome RNA degraded RNA

B CDS ncRNA C CDS tRNA - +ΦKZ - +ΦKZ PAO1 ΦKZ PAO1 ΦKZ 7 7

6 CrcZ 6

5 5

4 PA5181.1 4 reads + Φ KZ reads/transcript

PrrH 10

10 RnpB

3 log 3 log

2 (4,643) (29) 2

Figure 8. Host RNome is altered at early infection. (A) ΦKZ conquering of Pseudomonas is strongly linked to modulation of RNA homeostasis. Viral RNAPs are utilized and produced to transcribe phase genes. Additional phage tRNAs are utilized in translation, and the RNA degradation is blocked through the Dip protein. (B) Host CDS and ncRNA levels were significantly reduced by about 5-fold in a non-parametric paired t-test. The threshold for statistical significance was a two-tailed P value of <0.05 (***p < 0.001; ****p < 0.0001). Prism 9 was used for statistical analyses. Lines connect individual RNA levels. Outliers that increased levels upon ΦKZ infection are labelled. (C) Individual phage CDS are two magnitudes more abundant than host RNAs, whereas phage tRNAs remain underrepresented 10 min post infection. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 9 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P CDS 12 PA2746a

pyrG +ΦKZ 10 pant401 PA4441 8 liuR

PA4441 6 ncRNA pant512 tRNA ErsA CDS pant253 liuR

rel. position in reference 4 PA2746a

y = 6.94 - 0.91x tRNALeu 2 Pearson‘s r = -0.46 R² = 0.21 pyrG -5 -4 -3 -2 -1 0 1 2 3 Δrel. position

tRNALeu B tRNA (62) ncRNAs (29)

CDS (4,597)

-5 -4 -3 -2 -1 0 1 2 3 Δrel. position

E PA5491 ErsA PA5494 D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P I cc4 PA5492 polA thrB 6,186,525 ncRNA ErsA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P

110 - ErsA +ΦKZ 5'

pant512 110 - +ΦKZ

JVO-18950 PhrS (~23x) 190 - pant253 190 - +ΦKZ JVO-16164

190 - 6S RNA 190 - +ΦKZ pant401 JVO-15874 147 - 5S rRNA (~8x) 147 - +ΦKZ 6% Urea-PAGE, phosphogram JVO-15848

Figure 9. ΦKZ infection shifts transcripts and ncRNAs. (A) Transcripts that sediment in HMW fractions are more shifted towards LMW fractions. (change in level <3-fold). (B) Annotated ncRNAs were shifted in direction of HMW fractions. (C) Sedimentation profiles of host CDS that shift strongly towards H/LMW fractions from (A). (D) Sedimentation profiles of phage- infection affected host ncRNAs, of which ErsA represents an important regulator in biofilm formation and is shifted to HMW fractions. (E) The ErsA shift in sedimentation was validated by northern probing against the end of stem-loop I in ErsA and accumulated strongly in the pellet fraction. A less pronounced shift was observed for PhrS that was strongly depleted upon infection. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 10 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A C probe operon 54 PHIKZ125 I 126,122 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P operon 88 host CDS PHIKZ185 p56 PHIKZ186 I 199,270

ΦKZ CDS B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P operon

- 54 ~500 - host tRNA 331 -

JVO-16261 ΦKZ tRNA

~500 - - 88 50% transparent bars 242 - indicate standard deviation

6% Urea-PAGE, phosphogram JVO-16029

Figure 10. Phage RNAs associate strongly with ribosomes. (A,B) Expression and sedimentation of early infection operons 54 and 88 was northern probed and revealed a strongly ribosome associated profile. Interestingly, smaller fragments sedimented exclusively before fraction six. (C) In general, phage transcripts sedimented substantially at ribosomal fractions indicating an efficient transfer to translation or stabilization at the ribosome. Phage tRNAs were also strongly utilized in translation or stabilized at ribosomal subunits judged by sedimentation in ribosomal fractions. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 11 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

operon A 131 132 133 C 3′UTR_PHIKZ298 tRNAPro tRNAAsp tRNAMet A A 70 Asn A U A C tRNA 297 298 299 p87 301 302 U A G U I A A Asn Leu A U 269,557 ? tRNA p86 tRNA 20 U A C G U A A U 30 G U U A G U G U U A operon 49 50 51 G U A U 60 G A 113 115 p37 116 p40 p41p42 p43 G C C G A U I U A 80 114 p36 p38 p39 ? A U C G 117,450 10 U A G A A U U A C G A A U U 40 U 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P U C C G A U A U B U A A C A U 50 A U PHIKZ U G C C A C U A 5´ 3´ -298

270,649 - ? E 5′UTR_p40 C G A A G 60 881 - A U C G U A A U probed part U A 50 U A A C A G C G U G 70 A U A 110 - A U A U C G A A JVO-18830 40 A U A U U A A C 80 150 A U U A U G U A U C D U G C G G C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P U G A U C G U A U A 30 U A A A U G A G U A ? A A 140 G C C G 90 A U 160 U A G G G U A A U U A p40 A U C C 114,529 - U A U U p39 U C 20 U A A C A U U A G U 130 G C U U 170 U A A U C G 100 C G 881 - U G C G U A C GA 180 A U C G U C C C G C U U A A U 10 A U U G G C G A G C C G U 190 - U 120 G C 190 A U C G U A U G G C G C U A 110 A U 110 - A U G C U G A G U 5´ 3´ JVO-18831

Figure 11. Grad-seq guides the discovery of phage-related ncRNA candidates. (A) Loci of operons 132 and 50 including candidate ncRNAs. (B) In the 3'UTR of PHIKZ298, we observed sedimentation exclusively around fraction four that was confirmed by probing a ~100-nt fragment that M-folded (102) into a two stemmed structure (C). (D) Read-coverage profiles in the 5'UTR of p40 revealed an LMW weighted sedimentation profile different from phage CDS transcripts (Fig. 10C). A ~180-nt fragment was detected in RNAP associated fractions and at 30S subunit fractions. A longer fragment (~880-nt) was substantially shifted to fraction 12. (E) The 5'UTR-p40 fragment M-folded into a two stemmed structure. Northern probing was conducted against the nucleotides indicated in orange. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure 12 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

select protein/RNA database

search field

select entry from search table

view selected entry from search table

add entry to display table

display table

change representation bars/lines/heat map

displayed entries as bar chart

displayed entries as overlay line plot

displayed entries in a heat map

Figure 12. Differential Grad-seq browser. Protein and RNA sedimentation profiles can be searched for non-infected and infected experiments. Selected entries are displayed as bar-chart and can be added in a display list that is plotted as bar-chart, lines, or heatmap. The browser is accessible at https://helmholtz-hiri.de/en/datasets/gradseqpao1/ bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure S1 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A 12 D reference PAO1 human albumin 6 infected 11

3 total iBAQ 10 8 log 2 sum of RNA reads in gradient sum of RNA

7 10

Log 1

0 0 1 2 3 4 5 -2 -1 0 1 2 3 4 5 log RNA level in ERCC spike-in log10 protein level in UPS2 spike-in 10

B E 600 3k MS detected (2,182) RNA-seq detected (8,297) log iBAQ >7.5 sum of reads/gradient > 100 500 10 2.5k not detected (614) not detected (2,971) 400 2k

300 1.5k

200 counts 1k counts

100 0.5k

0 0 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8

log10 total iBAQ log10 reference reads

C F reference reference infected 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P bfrB (bacterioferritin) RplB 992,710,000 - 12,000 - raw MS intensities raw RNA seq reads

- 7,311,200,000 human albumin ERCC-130 spike-in 79,411 - spike-in

1 - 1 - corrected and corrected and norm. to max. norm. to max.

Figure S1. Analysis and normalization of Grad-seq. (A) Dose-response curve for the MS analysis, plotted as relative concentration in UPS2 spike-in vs. recovered MS counts. The linear dynamic range of detection is valid down to log10 total iBAQ of 7.5. (B) 2,182 proteins were selected above the threshold from (A). 614 proteins were discarded. (C) Normalization of protein levels by human albumin (UPS2) spike-in. (D) Dose-response curve for the RNA-seq analysis based on the ERCC spike-in. The linear dynamic range of detection was valid down to log10 sum of RNA reads of 1.5. (E) 8,297 annotations were detected above the threshold of >100 reads/gradient, 2,971 annotations were excluded. (F) Normalization of RNA levels by the ERCC-130 spike- in. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyrightQ9HTZ4 holder for this preprint Q9HUG2 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.pcnB Figure S2 der phaF pbpG ribH mutM Q9HTY1 mvaT rlmL Q9I4H8 Q9HVU8 rimP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P rbfA A yajC Q9I756 Q9I4Z8 AlgH NrdJa DnaG PepA FliA FtsH Q9I6B1 NrdJb PssA Q9HWA9 Q9HUC7 FecA Q9HYR5 RpsK AtuD RpsN Q9I057 RpsR RpmI RpsT RpmJ RsmA RpmH SucB Q9I2Q3 SucA RplT RpsE RplM RpsM RplR RpsB RpmD RpsG RlmE RpsP RpmB RpsC RplB RpsF RplQ RpsA RplW RpsJ RplP RpsH KptA Rnr RplX RpsI RplD RpsQ RplI RpsO RpmE Q9I4M5 RplO PoxB RpmC RpsL RplU RpsD RplA RpsU RpmE2 RsgA Q9I4B0 Q9HYU6 RplC InfC RplK DeaD RpmG HflX RplL Q9I083 RpmF Rne Q9HVT3 Q9I4Q6 Q9HZL4 Q9HXR4 RplJ CcoQ2 RpmA RpsS RplE Q9HXI3 RplV OprD RplS OprH RplN HflK RplF HflC Q9HZG2 Q9I456 RplY Q9HUG4 Q9HTZ4 RhlE Q9HUG2 RluC PcnB Q9HUN6 Der P95453 PhaF RluB PbpG Q9HYE0 RibH Q9I6D9 MutM AceF Q9HTY1 BfrB MvaT WaaF RlmL Q9HUG7 Q9I4H8 Q9I6W8 Q9HVU8 Q9HUD1 RimP RsfS RbfA Q9I0A5 YajC RluE Q9I4Z8 nrdJa pepA ftsH nrdJb B Q9HWA9 C fecA RNAP30S 50S 70S rpsK rpsN rpsR rpsT rsmA sucB sucA rpsE rpsM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P S rpsB fract. norm. rpsG 250 - rpsP filter MW filter MW proteins Log10iBAQ rpsC 130 - rpsF rpsA 100 - rpsJ 6 130-250 150.8 RNAP β 2.7E9 70 - rpsH rnr 154.4 RNAP β' 2.1E9 55 - rpsI rpsQ 140.6 PurL 1.7E8 rpsO Q9I4M5 3 80-120 93.6 aconitase B 5.3E9 poxB 35 - rpsL 81.6 isocitrat dehydr. 3.2E9 rpsD rpsU 107.1 RN.-diphos. red. 2.5E9 25 - rsgA Q9HYU6 8 55-65 57.1 GroEL 1.9E10 ribosomalinfC deaD 61.9 RPS1 1.4E9 proteinshflX Q9I083 10 60-75 rne 61.9 RP S1 6.4E9 Q9I4Q6 70.1 RhlE 2.3E8 15 - Q9HXR4 ccoQ2 1 40-55 10 - rpsS 43.4 EF-Tu 4.5E10 Q9HXI3 SDS-PAGE, Coomassie oprD 43.5 enoyl-ACP red. 4.3E9 oprH hflK hflC Q9I456 Q9HUG4 rhlE rluC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 1 2 3Q9HUN6 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P D P95453 rluB Q9HYE0 EF-Tu Q9I6D9 aconitase B RNAP β aceF bfrB waaF Q9HUG7 Q9I6W8 Q9HUD1 rsfS enoyl-ACP-red. Q9I0A5 isocitrat dehydr. RNAP β' rluE

GroEL RN.-diphos. red. PurL

RP S1 RhlE bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure S2 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure S2. Sedimentation profiles of complexes that peak in fractions 8-20. (A) About 30 uncharacterized proteins sedimented in HMW fractions and are named here by UniProt identifier. (B) Selected abundant protein bands in the Grad-seq experiment were selected for identification. (C) Protein entries were filtered by selected fraction where the protein was mostly abundant in (A) and a range of molecular weight based on the molecular size marker. The resulting table was ranked by abundance resulting in candidates. The abundance (iBAQ) was evaluated in contrast to the second-best hit. (D) Digital sedimentation profiles were correlated with the sedimentation profiles in (B). bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure S3 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A C Predicted interaction regions sRNA in PA2578 3'UTR Predicted interaction regions sRNA in PA2578 3'UTR

0.89 0.88

0.7 0.69

0.51 0.5

0.32 0.31

0.13 0.13

0 0 pa1172_napC pa2708_N/A pa2863_lipH pa1173_napB pa0566_N/A pa1423_bdlA pa1554_ccoN1 pa5447_wbpZ pa2866_mttC pa4938_purA pa4667_N/A pa2862_lipA pa4073_N/A pa3455_N/A pa3324_N/A pa3323_N/A pa3454_N/A pa3578_N/A pa2544_N/A pa3319_plcN pa1256_N/A pa2832_tpm pa1265_N/A pa3308_N/A pa2510_catR pa1257_N/A pa0360_N/A pa0418_N/A pa3309_N/A pa2759_N/A ((((((...... )))))).(((((((((...(..(...... )..)..))))))))).. ((((((...... )))))).(((((((((...(..(...... )..)..))))))))).. GCGGCCATCCTCCGTTTGCGGCCGTAGAGCTCCGGACACTTCATGCTAGGGGGGCCGGAGCTCTT GCGGCCATCCTCCGTTTGCGGCCGTAGAGCTCCGGACACTTCATGCTAGGGGGGCCGGAGCTCTT nt 1 nt 65 1 65 Predicted interaction regions mRNAs Predicted interaction regions mRNAs

0.13 0.09

0.1 0.07

0.08 0.05

0.05 0.03

0.02 0.01

−200 −100 start 100 200 100 stop 100 codon codon

B D PA3309 nitrate reductase operon PAO1 PA1171 PAO1 PA3308 universal stress napEFDABC probable RNAP assoc. prot. protein A family PA3310 transglycosylase napA napF phoPoprH (rapA) protein (uspK) sulfatase

I I 1,270,972 napC napB napEnapD 3,707,090

start SDseq codon 272 301 92 115 | | | | PA1172 5'-CCAAU G CCU A AUGAA-3' PA3309 5'-AUC...GCGAA C A GCGGC...GCC-3' GCGGC CGCAAAC GAGGG GGCCGC GGCCGC AACGG G AUGGCCGC :|||| ||||||| ||||: |||||| |||||| ||||| | |||||||| UGCCG GCGUUUG CUCCU CCGGCG CCGGCG UUGCC C UACCGGCG PA2578 3'UTR 3'-CGAGA C A -5' PA2578 3'UTR 3'-UUC...AGAUG U U C -5' | | | | 26 1 24 1 -15.5 kcal/mol (CopraRNA) -15.7 kcal/mol (CopraRNA)

Figure S3. CopraRNA analysis of RNA-RNA interaction of the PA2758-3'UTR fragment. (A) Predicted interaction region around start codons were determined being in the first stem loop that may represent the seed sequence. (B) An example is the intergenic region between napB and napC where the fragment could pair with the Shine-Dalgarno-sequence (SDseq) and modulate translation initiation. (C) Predicted interaction regions around stop codons. (D) A second example covers an interaction in the 5'UTR of the universal stress protein A family protein uspK. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure S4 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Ribo-seq 0-221 (monosome) RNA-seq

Grady et al. 2017 0-886 no TEX

+TEX

prtN prtR (essential) ptrB PA0613

I 672,777 pant67/ type III OsiS screction system 0-751,000 no TEX

+TEX Wicke et al. 2020

TSS, OsiS 5'... PAO1 UCBPP-PA14 PA7 LESB58 monosome read coverage (Grady et al. 2017) 5'-OH-transcript ... PAO1 UCBPP-PA14 PA7 LESB58 monosome read coverage (Grady et al. 2017) putative start (out-of-frame) inverted repeat/ Rho-independent terminator (((((( PAO1 UCBPP-PA14 PA7 LESB58 Shine-Dalgarno putative start (in-frame) ((((.(.....).)))))))))) PAO1 UCBPP-PA14 PA7 LESB58

PAO1 UCBPP-PA14 PA7 LESB58

PAO1 UCBPP-PA14 PA7 LESB58 putative start (in-frame) ...OsiS 3' PAO1 UCBPP-PA14 PA7 LESB58 Shine-Dalgarno start ptrB CDS... PAO1 UCBPP-PA14 PA7 LESB58

PAO1 UCBPP-PA14 PA7 LESB58

Figure S4. ncRNA OsiS may harbour a hidden small open reading frame that may drive sedimentation in 30S fractions. TEX treatment indicates a TEX unaffected 5'-end downstream of the annotated TSS (Wicke et al., 2020). Sequencing of RNA fragments recovered from monosomes (Ribo-seq, (Grady et al., 2017), re-analysed with HRIBO (https://github.com/RickGelhausen/HRIBO)) supports the possibility of a short translated fragment starting at a putative start codon that is out-of-frame with ptrB. In addition, an inverted repeat in-between may serve as a Rho-independent terminator because no stop-codon is reached in the reading frame. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure S5 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A +ΦKZ Pa lysate D pellet ɸKZ 70S CDS A260 (a.u.) top 30S 50S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P S 5′UTR (1,818, ~1.3%) tRNA (62, ~11%) CDS (4,643, ~6.7%) r-/tmRNA (14, ~76%) - RpoB/C 3′UTR (1,410, ~0.7%) ɸKZ CDS (359, ~3%) ncRNA (365, ~0.8%) ɸKZ tRNA (7, ~5E-05%) 70 - - GroEL E -ΦKZ - EF-Tu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 5′UTR

ribosomal proteins CDS

SDS-PAGE, Coomassie 3′UTR

B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P S ncRNA 600 - 16/23S rRNA

300 - tRNA

6S RNA

100 - 5S rRNA F +ΦKZ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 5′UTR tRNA

6% Urea-PAGE, EtBr CDS

C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P S

190 - PhrS 3′UTR 404 - CrcZ

331 - tmRNA ncRNA 190 - 6S RNA 6% Urea-PAGE, phosphogram

tRNA bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint PHIKZ_p74 PHIKZ174 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. PHIKZ113 PHIKZ_p54 PHIKZ018 Figure S5 PHIKZ224 PHIKZ041 PHIKZ_p69 PHIKZ055 PHIKZ050 PHIKZ071 PHIKZ209 PHIKZ232 PHIKZ207 PHIKZ_p64 PHIKZ136 PHIKZ138 PHIKZ_p80 PHIKZ208 PHIKZ_p71 PHIKZ279 PHIKZ278 PHIKZ305 PHIKZ246 PHIKZ051 G PHIKZ201 PHIKZ_p09 PHIKZ184 PHIKZ142 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P PHIKZ049 PHIKZ178 PHIKZ_t01 PHIKZ035 PHIKZ_t07 PHIKZ_p29 PHIKZ_t03 PHIKZ271 PHIKZ_t04 PHIKZ149 PHIKZ_t02 PHIKZ286 PHIKZ_p06 PHIKZ233 PHIKZ_p34 PHIKZ_p61 PHIKZ199 PHIKZ027 PHIKZ_p81 PHIKZ077 PHIKZ072 PHIKZ236 PHIKZ132 PHIKZ_p85 5UTR_p40 PHIKZ237 PHIKZ111_rev PHIKZ273 PHIKZ_p21 PHIKZ177 3UTR_PHIKZ298 PHIKZ124 PHIKZ_p40 PHIKZ235 PHIKZ_p39 PHIKZ070 PHIKZ_p04 PHIKZ_p22 PHIKZ126 PHIKZ093 PHIKZ158 PHIKZ105 PHIKZ153 PHIKZ_p02 PHIKZ144 PHIKZ_p87 PHIKZ202 PHIKZ010 PHIKZ003 PHIKZ110 PHIKZ241 PHIKZ_p35 PHIKZ_p24 PHIKZ185 PHIKZ114 PHIKZ006 PHIKZ060 PHIKZ_p31 PHIKZ111 PHIKZ108 PHIKZ022 PHIKZ076 PHIKZ099 PHIKZ_p49 PHIKZ299 PHIKZ084 3UTR_p39 PHIKZ133 PHIKZ119 PHIKZ_p13 PHIKZ_p43 PHIKZ033 PHIKZ200 PHIKZ082 PHIKZ155 PHIKZ043 PHIKZ023 PHIKZ066 PHIKZ056 PHIKZ242 PHIKZ183 PHIKZ137 PHIKZ157 PHIKZ_p42 PHIKZ_p08 PHIKZ016 PHIKZ198 PHIKZ014 PHIKZ120 PHIKZ287 PHIKZ122 PHIKZ181 PHIKZ067 PHIKZ188 PHIKZ145 PHIKZ047 PHIKZ134 PHIKZ162 PHIKZ080 PHIKZ168 PHIKZ140 PHIKZ106 PHIKZ028 PHIKZ015 PHIKZ094 PHIKZ194 PHIKZ090 PHIKZ249 PHIKZ030 PHIKZ020 PHIKZ146 PHIKZ_p19 PHIKZ054 PHIKZ218 PHIKZ123 PHIKZ057 PHIKZ069 PHIKZ253 PHIKZ214 PHIKZ_p73 PHIKZ129 PHIKZ216 PHIKZ026 PHIKZ063 PHIKZ029 PHIKZ250 PHIKZ229 PHIKZ_p75 PHIKZ_p46 PHIKZ059 PHIKZ025 PHIKZ058 PHIKZ_p58 PHIKZ004 PHIKZ166 PHIKZ_p18 PHIKZ179 PHIKZ_p76 PHIKZ298 PHIKZ187 PHIKZ204 PHIKZ251 PHIKZ131 PHIKZ171 PHIKZ_p23 PHIKZ115 PHIKZ296 PHIKZ012 PHIKZ139 PHIKZ295 PHIKZ165 PHIKZ186 PHIKZ121 PHIKZ109 PHIKZ011 PHIKZ260 PHIKZ205 PHIKZ104 PHIKZ_p63 PHIKZ002 PHIKZ228 PHIKZ256 PHIKZ001 PHIKZ021 PHIKZ064 PHIKZ226 PHIKZ252 PHIKZ048 PHIKZ087 PHIKZ096 PHIKZ088 PHIKZ176 PHIKZ092 PHIKZ034 PHIKZ089 PHIKZ274 PHIKZ180 PHIKZ095 PHIKZ085 PHIKZ267 PHIKZ100 PHIKZ190 PHIKZ141 PHIKZ191 PHIKZ182 PHIKZ046 PHIKZ_p62 PHIKZ135 PHIKZ_p16 PHIKZ_p10 PHIKZ143 PHIKZ_p27 3UTR_PHIKZ_p04 PHIKZ203 PHIKZ_t05 PHIKZ276 PHIKZ_t06 PHIKZ_p20 PHIKZ_p55 PHIKZ_p60 PHIKZ017 PHIKZ245 PHIKZ270 PHIKZ_p44 PHIKZ_p78 PHIKZ128 PHIKZ281 PHIKZ101 PHIKZ_p66 PHIKZ297 PHIKZ197 PHIKZ_p05 PHIKZ238 PHIKZ_p91 PHIKZ222 PHIKZ_p67 PHIKZ215 PHIKZ_p33 PHIKZ038 PHIKZ053 PHIKZ243 PHIKZ231 PHIKZ_p59 PHIKZ_p15 PHIKZ_p68 PHIKZ_p32 PHIKZ244 PHIKZ007 PHIKZ234 PHIKZ_p53 PHIKZ239 PHIKZ167 PHIKZ151 PHIKZ302 PHIKZ221 PHIKZ_p79 PHIKZ283 PHIKZ_p14 PHIKZ259 PHIKZ045 PHIKZ078 PHIKZ254 PHIKZ268 PHIKZ_p82 PHIKZ266 PHIKZ005 PHIKZ269 PHIKZ_p56 PHIKZ193 PHIKZ_p77 PHIKZ227 PHIKZ261 PHIKZ288 PHIKZ293 PHIKZ195 PHIKZ264 PHIKZ112 PHIKZ_p36 PHIKZ294 PHIKZ_p37 PHIKZ277 PHIKZ_p38 PHIKZ210 PHIKZ116 PHIKZ_p70 PHIKZ262 PHIKZ_p17 PHIKZ_p86 PHIKZ291 PHIKZ107 PHIKZ019 PHIKZ_p25 PHIKZ_p65 PHIKZ301 PHIKZ285 PHIKZ_p88 PHIKZ206 PHIKZ_p89 PHIKZ275 PHIKZ_p90 PHIKZ_p11 PHIKZ013 PHIKZ212 PHIKZ008 PHIKZ152 PHIKZ103 PHIKZ211 PHIKZ172 PHIKZ_p03 PHIKZ_p01 PHIKZ196 PHIKZ257 PHIKZ289 PHIKZ290 PHIKZ_p57 PHIKZ248 PHIKZ223 PHIKZ263 PHIKZ068 PHIKZ170 PHIKZ031 PHIKZ_p83 PHIKZ265 PHIKZ247 PHIKZ061 PHIKZ062 PHIKZ284 PHIKZ258 PHIKZ272 PHIKZ_p84 PHIKZ_p74 PHIKZ304 PHIKZ174 PHIKZ125 PHIKZ113 PHIKZ_p26 PHIKZ_p54 PHIKZ_p07 PHIKZ018 PHIKZ_p28 PHIKZ224 PHIKZ303 PHIKZ041 PHIKZ040 PHIKZ_p69 PHIKZ280 PHIKZ055 PHIKZ240 PHIKZ050 PHIKZ037 PHIKZ071 PHIKZ075 PHIKZ209 PHIKZ074 PHIKZ232 PHIKZ219 PHIKZ207 PHIKZ036 PHIKZ_p64 PHIKZ150 PHIKZ136 PHIKZ_p41 PHIKZ138 PHIKZ189 PHIKZ_p80 PHIKZ220 PHIKZ208 PHIKZ_p72 PHIKZ_p71 PHIKZ148 PHIKZ279 PHIKZ147 PHIKZ278 PHIKZ039 PHIKZ305 PHIKZ081 PHIKZ246 PHIKZ175 PHIKZ051 PHIKZ_p30 PHIKZ201 PHIKZ097 PHIKZ_p09 PHIKZ163 PHIKZ184 PHIKZ_p12 PHIKZ142 PHIKZ098 PHIKZ049 PHIKZ160 PHIKZ178 PHIKZ130 PHIKZ035 PHIKZ_p45 PHIKZ_p29 PHIKZ271 PHIKZ149 PHIKZ286 0,PHIKZ233 normalized to max. PHIKZ_p61 PHIKZ027 PHIKZ077 PHIKZ236 PHIKZ_p85 PHIKZ237 PHIKZ273 PHIKZ177 PHIKZ124 PHIKZ235 PHIKZ070 PHIKZ_p22 PHIKZ093 PHIKZ105 PHIKZ_p02 PHIKZ_p87 PHIKZ010 Figure S5. Gradient fractionation of ΦKZ-infected PseudomonasPHIKZ110 at early infection time point. (A) The absorption profile PHIKZ_p35 PHIKZ185 PHIKZ006 and apparent proteome is not affected upon infection. (B) PHIKZ_p31The global picture of the RNome is consistent with Fig. 1D and not PHIKZ108 PHIKZ076 altered upon infection. Specific RNAs were probed by PHIKZ_p49northern blotting and resulted in consistent sedimentation behaviour (C) PHIKZ084 PHIKZ133 PHIKZ_p13 with non-infected cells. (D) Phage transcripts represented aboutPHIKZ033 3% of all reads. Host CDS transcripts remained present at 7% - PHIKZ082 PHIKZ043 PHIKZ066 relative representation. (E) In non-infected cells, the sedimentationPHIKZ242 profile of CDS, UTRs, and ncRNAs was strongly balanced on PHIKZ137 PHIKZ_p42 PHIKZ016 the side of RNAP fractions and marginally on ribosomal fractions.PHIKZ014 tRNAs sedimented exclusively in LMW fractions. (F) In infected PHIKZ287 PHIKZ181 cells, the sedimentation profiles of the host transcripts were onlyPHIKZ188 marginally affected and shifted towards LMW fractions. Phage PHIKZ047 (G) PHIKZ162 PHIKZ168 transcripts sedimented strongly in ribosomal fractions. PHIKZ106 PHIKZ015 PHIKZ194 PHIKZ249 PHIKZ020 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure S6 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

proQ histidine kinase PA2580 PA2582 PA2583 pgsA uvrC

I 2,916,158 PA2581 PA2581.1 PA2583.1

B FinO-domain (PF04352) putida F1 MGFEQLAELRDRLRAEKGQVKAESAKPPKRKSPPQ---AKPREMDPAVAAIWPLQKHFPL 57 PA7 MGFEQLAELRDRLRAQAEQAKPAKSAQSKAKASPGRAAKKREPVDPEVEAIWRLQRHFPL 60 LESB58 MGFEQLAELRDRLRAQAAQAKPAQTKSS------AGRAKKREAVEPGVEAIWRLQRHFPL 54 PAO1 MGFEQLAELRDRLRAQAAQAKPAQTKSS------AGRAKKREAVEPGVEAIWRLQRHFPL 54 UCBPP-PA14 MGFEQLAELRDRLRAQAAQAKPAQTKSS------AGRAKKREAVEPGVEAIWRLQRHFPL 54 conservation ***************: *.* .: * . ::* * *** **:****

important residues K35 R80 in ProQ from E. coli G37 Y70 D82 (Pandey et al. 2020) putida AFPVNPAPKVPLKEGIFKDAEQHLELLGLTREQLKLGISTWCRGARYWASMVENAPRLDL 117 PA7 AFPKNPAPKVPLKQGILEDAQQHLESLGITAEQLKQAIATWCQGNRYWSCMVEDAPRLDL 120 LESB58 AFPKSPAAKVPLKQGILQDAQQHLELLGITAEQLKQAIATWCQGSRYWSCMVEDAPRLDL 114 PAO1 AFPKSPAAKVPLKQGILQDAQQHLELLGITAEQLKQAIATWCQGSRYWSCMVEDAPRLDL 114 UCBPP-PA14 AFPKSPAAKVPLKQGILQDAQQHLELLGITAEQLKQAIATWCQGSRYWSCMVEDAPRLDL 114 conservation *** .** *****:**::**:**** **:* **** .*:***:* ***:.***:******

G85 putida NGQAAGTVTAAQALHAKQQAARQRSQDRRNRAKSKAQAQAQAPAPAAD-TATVQSTD--- 173 PA7 QGQVAGKVTAEQAVYAKRQASRRQRDQMREKRAKRARAATESAADKLESDA-----DAQP 175 LESB58 QGQVAGKVTAEQAVYARRQASRRQREQMREKRAKRAQADSEAAAATEAPTPEASATEASP 174 PAO1 QGQVAGKVTAEQAVYARRQASRRQREQMREKRAKRAQAGGEAPAATEAPTPEAPATEASP 174 UCBPP-PA14 QGQVAGKVTAEQAVYARRQASRRQREQMREKRAKRAQAGSEAPAATEAPTPEAPATEASP 174 conservation :**.**.*** **::*::**:*:: :: *:: .:*:* :: * :

putida ----- 173 PA7 DSSAF 180 LESB58 EAN-- 177 PAO1 EAN-- 177 UCBPP-PA14 EAN-- 177

C FinO-domain (PF04352) 0.45 - Pseudomonas (n=275) rel. frquency 0 - 0.45 - all (n=7,530) rel. frquency 0 - 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 MW (kDa)

Figure S6. ProQ in Pseudomonas harbours important residues of a FinO-domain. (A) Genomic locus of ProQ in PAO1. (B) Conserved residues in a FinO-domain (PF04352) are represented in red. Important residues in ProQ for RNA-binding (76) are indicated with corresponding residues in ProQ from E. coli. (C) ProQ proteins in Pseudomonas represent the condensed representatives in the diverse class of FinO-domain proteins that can have C- or N-terminally associated domains. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure S7 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A GO-biological process overrepresentation

CDS Δrel. position < -1 (n=834)

aerobic respiration

proton transmembrane transport carboxylic acid metabolic process organonitrogen compound metabolic process 0 1 2 3 4 5 fold-enrichment

B all (n=4,643)

metabolism (n=908)

oxidative phosphorylation (KEGG: pae00190, n=49)

-5 -4 -3 -2 -1 0 1 2 3 Δrel. position

all (n=4,643)

metabolism (n=908)

oxidative phosphorylation (KEGG: pae00190, n=49)

2 3 4 5 6 7 8 9 10 11 12 13 14 15 rel. position

C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P pae00190, CDS ATP synthase subunit γ

+infection +infection

NADH-quinone oxido- cytochrome C reductase subunit C oxidase subunit I

+infection +infection

Figure S7. Transcripts for oxidative phosphorylation are shifted most strongly towards LMW fractions. (A) Overrepresentation analysis for biological processes revealed that 5'UTR transcripts that are shifted by >0.5 rel. fractions are enriched for transcription factors. 3'UTR transcripts that are shifted by more than 1 rel. fraction towards the top are enriched for factors of aerobic respiration and proton transmembrane transport. (B) 49 transcripts encoding oxidative phosphorylation factors are significantly more shifted towards the top compared to transcripts coding for metabolic factors. At the same time the relative position in the reference condition was for these transcripts significantly higher and matches the trend in (A). (C) Averaged sedimentation profile of the oxidative phosphorylation pathway pae00190 CDS, and selected examples coding for ATP synthase, NADH-oxidoreductase, and cytochrome C oxidase. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.06.403469; this version posted December 6, 2020. The copyright holder for this preprint Figure S8 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 1 2 5S rRNA in reference 3 4 5 6 7 147 - 8 9 -ΦKZ 10 11 infected 12 13 14 15 16 17 147 - 18 19 20 6% Urea-PAGE, phosphogram JVO-15848 P

1 2 3 4 5 C 6 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 P 8 9 rne ~3x +ΦKZ 10 11 12 13 14 15 16 67 - 17 18 JVO-18826 19 20 infected P rne-II ? (RF01756) 5´ rne I I 67 - 3,332,368 -880

6% Urea-PAGE, phosphogram JVO-18826 probe

Figure S8. 5S rRNA indicates processing effects and its major RNase E is altered in in the 5'UTR. (A) 5S rRNA sedimentation was not affected upon infection, but it become smeary in northern probing that may indicate processing defects that could lead to diminished reads in sequencing. (B) The read-coverage of the 5’UTR of rne was altered upon infection. (C) The 5'UTR appeared in two fragments of which the larger one was allocated to RNAP fractions and strongly diminished upon ΦKZ infection.