The (p)ppGpp-binding GTPase Era promotes rRNA

Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified theStaphylococcus aureusRA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, although the function of Era in ribosome assembly is unclear. Era is highly conserved throughout the bacterial kingdom and is essential in many species. Here we show that Era is not essential inS. aureusbut is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures, virulence and 16S rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSA, with RelSA positively impacting the GTPase activity of Era. We propose that Era acts to direct enzymes involved in rRNA maturation and ribosome subunit assembly to their site of action, an activity that is regulated by components of the stringent response.The bacterial ribosome is an essential cellular component and as such is the target for a number of currently used antimicrobials. Correct assembly of this complex macromolecule requires a number of accessory enzymes, the functions of which are poorly characterised. Here we examine the function of Era, a GTPase enzyme involved in 30S ribosomal subunit biogenesis in the important human pathogenS. aureus. We uncover that Era is not an essential enzyme inS. aureus, as it is in many other species, but is important for correct ribosome assembly. In a bid to uncover the cellular function of this enzyme, we identify a number of protein interaction partners with roles in ribosomal RNA maturation and degradation, supporting the idea that Era acts as an intermediary protein facilitating ribosomal biogenesis. We also uncover a link between Era and the (p)ppGpp synthetase RelSA, revealing an additional level of control of rRNA levels by the stringent response. With this study we elaborate on the functions of GTPases in ribosomal assembly, processes that are controlled at multiple points by the stringent response.


Introduction
Era is not an essential protein in S. aureus, it is important for optimal growth and 161 ribosome maturation. 162 163

Uncovering Era protein interaction dynamics in a native background 164
In S. aureus, Era is encoded in an operon with five other genes, one of which encodes 165 YbeY (Fig 1a). YbeY is an endoribonuclease implicated in the maturation of the 3' 166 terminus of 16S rRNA in E. coli, as well as a quality control checkpoint protein that 167 together with RNase R is involved in eliminating defective 70S ribosomes [19,28]. 168 Era interacts with YbeY in E. coli [18], although these two genes are not encoded in 169 the same operon in that organism (Fig 1a). To determine whether Era from S. aureus 170 interacts with YbeY, and/or any other proteins encoded in the S. aureus era operon, 171 we first used a bacterial two-hybrid approach, heterologously expressing Era in 172 combination with YbeZ, YbeY, DgkA, Cdd or RecO in E. coli. Using this approach, 173 we observed that Era potentially interacts with YbeY and YbeZ (Fig 2a). 174 In order to look at these interactions natively in an S. aureus background, we 175 adapted a split-luciferase system recently developed to analyse protein-protein 176 interactions in Clostridium difficile for use in S. aureus (Fig 2b) [29]. The era gene, in 177 combination with itself and each of the operon genes, were cloned into the split-178 luciferase plasmid pAP118 and introduced into S. aureus strain LAC*. Induction of 179 protein expression with Atet revealed a strong positive interaction for Era with itself 180 (Fig 2c), indicating that Era can form dimers. Dimerisation of Era has previously been 181 observed while solving the crystal structure of the E. coli protein [30], and so this 182 interaction confirms the functionality of the split-luciferase system in S. aureus. In 183 addition, a significant interaction was also observed for Era and YbeY (2 x above the 184 control), although not for YbeZ (1.40 x above the control) (Fig 2c). In agreement with 185 the bacterial two-hybrid results, no interactions occurred between Era and DgkA,Cdd 186 or RecO. A decrease in luminescence below the Era control pAP256-era SB -LB occurred 187 when co-expressing Era with DgkA and RecO. The reason for this is unclear but could 188 be due to toxicity upon over-expression of a second copy of these genes from a multi-189 copy plasmid. To confirm the validity of the Era-YbeY interaction in vitro, we 190 performed pulldown assays using glutathione beads coupled to either GST or  tagged Era and incubated them with His-tagged YbeY (Fig 2d). Here we observed 192 low-level cross reactivity of YbeY with the gluthatione beads that could not be 193 reduced despite increased washes. However, His-YbeY was pulled down significantly 194 more in the presence of Era (Fig 2d). Altogether the bacterial two-hybrid, split 195 luciferase and affinity pulldown assays confirm an interaction between Era  interaction between Era and CshA, full-length cshA was cloned into both pUT18 and 209 pUT18C. Upon co-transformation, interactions occurred between CshA-T18 and both 210 T25-Era and Era-T25 (Fig 3a). 211 To assess this interaction in vitro, we performed pulldown assays using 212 glutathione beads coupled to either GST or GST-tagged CshA and incubated them 213 with His-tagged Era, revealing that while His-Era did not interact with the control 214 GST protein, it was pulled down in the presence of CshA (Fig 3b). Finally, to analyse 215 and confirm this interaction in the context of the native host, we used the split 216 luciferase assay (Fig 2b). Assaying for luminescence revealed a 4-fold increase in 217 luciferase activity upon co-expression of Era and CshA (Fig 3c), confirming the 218 identification of a novel Era interaction partner. 219 220

The N-terminal region of CshA is crucial for interacting with Era 221
CshA is a DEAD-box RNA helicase with an N-terminal helicase core containing two 222 RecA-like domains and a disordered C-terminal (CT) region involved in RNA binding 223 (Fig 3d)  assays reveal that the G domain is sufficient for this interaction (Fig 3f). 237 238

Interactions between Era and CshA do not affect enzymatic activity 239
CshA is an RNA helicase, while Era has GTPase activity. To investigate whether the 240 interaction between these two proteins affects the enzymatic activity of either one, we 241 first performed helicase assays. A double stranded RNA oligo was incubated with each 242 protein singly or in combination. While CshA was able to unwind the dsRNA, the 243 addition of Era had no effect on its activity ( Fig S2a). In addition, the GTPase activity 244 of Era was unaltered in the presence of either CshA or YbeY (Fig S2b and S2c), 245 indicating that while these proteins do interact, this binding has little effect on their 246 enzymatic functions. As previously proposed, we suggest that these interactions fit 247 with a role for Era as a guide for rRNA/ribosome maturation enzymes to their showed significant growth defects at 37°C (Fig 1b and S3a), the growth of both was 257 severely compromised at 25°C (Fig 4a and S3b). This defect was enhanced in a double 258 Δera cshA mutant, which failed to grow at 25°C even after 48 h (Fig 4a and S3b),

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highlighting that these two proteins are essential for bacteria to cope with growth at 260 suboptimal temperatures. 261 Era is an RA-GTPase that, along with a second RA-GTPase RsgA, binds to the 262 While the wildtype bacteria were fully virulent, both the era and cshA mutant strains 272 displayed reduced killing, with the combined double mutant being completely 273 avirulent (Fig 4b). 274 To understand why Era and CshA are so important for growth at cold 275 temperatures and for virulence, we sought to assess the impact of deleting these genes 276 on the cellular rRNA. We observed that RNA extracted from both the single Δera and 277 double Δera cshA strains grown at 37°C had a processing or degradation intermediate 278 migrating slightly below the 16S band (Fig 4c). At 25°C we also observed a defect for 279 the ΔcshA mutant strain and multiple additional bands were evident in the Δera cshA 280 double mutant (Fig 4c), which could represent either processing intermediates from 281 the 16S, or degradation intermediates from both the 23S and the 16S. A build-up of 282 degradation products are expected in the cshA mutant due to its role in the RNA 283 degradosome. However, to determine whether processing defects could be occurring 284 13 in these mutant strains when grown at low temperatures, we performed qRT-PCR with 285 probes that either amplified internally to the 16S rRNA sequence, or either flanked the 286 5' or 3' processing junctions required to convert 17S into mature 16S rRNA (Fig 4d). 287 While the levels of 16S rRNA were not significantly different between wildtype and 288 mutants, large increases in unprocessed 5' and 3' rRNA were evident in the Δera strain 289 (Fig 4d). As Era is known to interact with the 30S ribosomal subunit, this could 290 potentially be due to mis-localisation of endonuclease YbeY in the absence of Era. It is 291 therefore, possible that Era acts as an intermediary protein on the 30S ribosomal 292 subunit, enabling proteins such as the endonuclease YbeY, and the RNA-degradosome 293 helicase CshA to access the 17S rRNA, allowing for processing (maturation or 294 degradation as appropriate) of rRNA under normal and stress conditions, such as 295 growth at cold temperatures. 296 297

Rel SA interacts with both Era and CshA 298
Our previous work demonstrated that the GTPase activity of Era is inhibited by the 299 stringent response alarmone ppGpp [22]. To examine whether ppGpp also interacts 300 with CshA we performed DRaCALA assays, revealing that this nucleotide does not 301 interact with CshA ( Fig S5a), nor does the affinity of ppGpp for Era alter in the 302 presence of CshA (K d of 3.1 ± 0.4 µM in the absence versus 3.2 ± 0.3 µM in the 303 presence of CshA) (Fig S5b). 304 To investigate whether the stringent response has any additional points of 305 interaction with either Era and CshA, we examined whether one of the (p)ppGpp 306 synthetases might directly interact with either protein via bacterial two-hybrid. This 307 revealed that Rel SA , but not RelP or RelQ, interacts with CshA and that Rel SA interacts 308 with Era (Fig 5a). To confirm this interaction, we first used affinity pulldown assays, 309 showing that although no significant interaction occurred with Era, CshA and Rel SA 310 interacted quite strongly (Fig 5b). Following this, we again used the split luciferase 311 assay, which confirmed an interaction between Rel SA and CshA (Fig 5c) or Era (Fig  312   5d). It was interesting to note that the CshA-Rel SA interaction only occurred in the 313 We wished to ascertain whether the interactions between Rel SA and Era impact the 319 activity of either enzyme. Recombinantly purified full-length Rel SA is known to be in 320 the synthetase-off/hydrolase-on conformation in vitro [37]. Using purified Rel SA , we 321 determined that Era and CshA have no effect on the pppGpp hydrolase activity of this 322 enzyme ( Fig S5c). However, upon examination of the GTPase activity of Era, we 323 observed significantly increased activity in the presence of Rel SA (Fig 6a). 324 Through Rel SA , the stringent response appears to interact with components of the 325 ribosome and 16S rRNA processing machinery. To analyse the importance of the 326 stringent response for rRNA processing, we grew the wildtype S. aureus LAC* at 327 to the 16S rRNA sequence, or either flanked the 5' or 3' processing junctions of the 332 17S rRNA. For the probe internal to the 16S rRNA we observed a significant decrease 333 in 16S rRNA in the presence of mupirocin (Fig 6b). This is expected, as it is known 334 that rRNA levels decrease upon activation of the stringent response. However, when 335 we compare the amount of unprocessed 5' or 3' sequences relative to the level of 16S 336 in each strain, we observed a significant increase in the amount of unprocessed leader 337 17S rRNA upon induction of (p)ppGpp (Fig 6c: p = 0.0031). We have previously 338 shown that ppGpp interacts with Era and that mature ribosome formation is inhibited proteins encoded in the era operon in S. aureus with Era (Fig 2). As in E. coli, YbeY 386 from S. aureus interacts with Era. YbeZ also interacted by bacterial two-hybrid but not 387 significantly using the quantitative luciferase assay (Fig 2). YbeZ has been shown to 388 interact with YbeY in E. coli [18]. This protein has an ATPase domain and an RNA 389 binding motif, and so is likely to also bind to the 17S rRNA and may form a complex 390 with Era and YbeY to aid in processing the 17S rRNA to the mature 16S form. 391 Here we use interaction studies to show that Era interacts with a protein 392 involved in survival at suboptimal temperatures, the DEAD-box RNA helicase CshA 393 that Era and CshA interact in vitro and when expressed heterologously in E. coli (Fig  402   3a and 3b). We additionally used an adapted split luciferase assay to show this 403 interaction natively in S. aureus (Fig 3c). Using this assay has also allowed us to 404 determine that the CT disordered region of CshA, which is important for binding to 405 mRNA and the degradosome [32], is dispensable for interacting with Era (Fig 3). 406 Using cshA and era deletion strains we show that both single mutants are 407 important for growth at cold temperatures, with a double mutant unable to survive at 408 25°C (Fig 4). By examining the rRNA of these strains we observe that the era mutant 409 strain has increased 17S unprocessed rRNA intermediates (Fig 4d). Additionally, a 410 double Dera cshA mutant has multiple processing and degradation defects (Fig 4c). It 411 has been proposed that Era may act to guide processing enzymes to their site of action 412 [18]. In keeping with this, we have observed that these proteins have little effect on the 413 enzymatic activity of each other. Therefore, we propose a model whereby Era acts as 414 an intermediary protein, allowing proteins such as the endonuclease YbeY and the 415 RNA helicase CshA, and potentially others, access to their substrate rRNA. It has been 416 suggested that CshA helps RNase components of the degradosome access cleavage 417 sites that may be otherwise inaccessible [45], and so it is tempting to speculate that Era 418 is responsible for recruiting CshA to target misfolded ribosomal subunits for 419 degradation under stress conditions. 420 interaction between the (p)ppGpp synthetase Rel SA and both CshA and Era (Fig 5), 426 and show that Rel SA has a positive effect on the GTPase activity of Era (Fig 6a). 427 Additionally, we observed that the activation of the stringent response results in 428 increased processing defects in a wildtype strain grown at 25°C (Fig 6c). RA-GTPases 429 are reported to have increased association to the ribosome in the GTP-bound form. 430 Hydrolysis of GTP then acts as a signal to promote dissociation, presumably following 431 a maturation event [49,50]. The increased GTPase activity of Era in the presence of 432 Rel SA could promote premature dissociation of Era from the ribosome, leading to 433 processing defects (Fig 6c) and contributing to the stalled growth phenotype 434 synonymous with the stringent response. The precise dynamics of processing-enzyme 435 association under stress conditions requires further in-depth research but is beyond the 436 scope of this study. For instance, under what growth conditions is Rel SA binding to Era 437 and CshA promoted and how does this affect the localisation of processing proteins to 438 the ribosome? 439 Taken together, we demonstrate a cellular function for Era as a protein 440 important for coordinating 30S ribosomal biogenesis in a stringent response-dependent 441 manner. It is possible that defects in ribosomal assembly lead to altered protein 442 translation, which may be the reason for the plethora of other phenotypes associated 443 with depletion of Era. As Era can complement a defect in RsgA (Fig S4a, [36 Synthesis of (p)ppGpp and differential radial capillary action of ligand assay 554 (DRaCALA). The synthesis of (p)ppGpp and DRaCALA binding assays were 555 performed as described previously [22]. Precipitated proteins were pelleted by centrifugation at 17,000 x g for 10 min. 594 Reaction products were then visualized by spotting 1 µl on PEI-cellulose thin layer 595 chromatography (TLC) plates (Merck Millipore) and separated using 1 M KH 2 PO 4 , 596 pH 3.6 buffer. The radioactive spots were visualised using an LA 7000 Typhoon 597 PhosphorImager and images quantified using ImageJ.