The alarmone (p)ppGpp is part of the heat shock response of Bacillus subtilis

Here, B. subtilis was used as a model organism to investigate how cells respond and adapt to proteotoxic stress conditions. Our experiments suggested that the stringent response, caused by raised levels of the (p)ppGpp alarmone, plays a role during thermotolerance development and the heat shock response. Accordingly, our experiments revealed a rapid increase of cellular (p)ppGpp levels upon heat shock as well as salt- and oxidative stress. Strains lacking (p)ppGpp exhibited increased stress sensitivity, while raised (p)ppGpp levels conferred increased stress tolerance to heat- and oxidative stress. During thermotolerance development, stress response genes were highly up-regulated together with a concurrent transcriptional down-regulation of the rRNA, which was influenced by the second messenger (p)ppGpp and the transcription factor Spx. Remarkably, we observed that (p)ppGpp appeared to control the cellular translational capacity and that during heat stress the raised cellular levels of the alarmone were able to curb the rate of protein synthesis. Furthermore, (p)ppGpp controls the heat-induced expression of Hpf and thus the formation of translationally inactive 100S disomes. These results indicate that B. subtilis cells respond to heat-mediated protein unfolding and aggregation, not only by raising the cellular repair capacity, but also by decreasing translation involving (p)ppGpp mediated stringent response to concurrently reduce the protein load for the cellular protein quality control system. Author Summary Here we demonstrate that the bacterial stringent response, which is known to slow down translation upon sensing nutrient starvation, is also intricately involved in the stress response of B. subtilis cells. The second messengers (p)ppGpp act as pleiotropic regulators during the adaptation to heat stress. (p)ppGpp slows down translation and is also involved in the transcriptional down-regulation of the translation machinery, together with the transcriptional stress regulator Spx. The stress-induced elevation of cellular (p)ppGpp levels confers increased stress tolerance and facilitates an improved protein homeostasis by reducing the load on the protein quality control system.

175°C display a relative much higher alarmone level (Fig. 1B,C). The primed thermotolerant cells 176 appear to be able to somehow limit the alarmone synthesis, when exposed to the lethal heat 177 shock. 178 The synthesis of (p)ppGpp that occurs during activation of the SR is normally 179 accompanied by a fast reduction of cellular GTP levels in cells treated with serine 180 hydroxamate (SHX) or DL-norvaline (NV) [27] and also after exposure to salt or diamide 181 (Fig. S1A). Therefore, we were also interested in monitoring changes in GTP levels under 182 conditions of heat shock but interestingly we do not observe a reduction in GTP levels after 183 exposure to 50 °C (Fig. S1A). Notably, the GTP levels were at a comparable high level 184 (FigS1B) during temperature upshifts of 37/48 °C, 37/53 °C and 48/53 °C, however GTP 185 levels appeared a little lower for all temperature upshifts after 15 min incubation (Fig. S1B). 186 Taken together, we show that exposure to heat shock elicits a fast, but transient, 187 increase of the alarmones pGpp, ppGpp and pppGpp, while not immediately affecting the 188 GTP levels. Therefore, it seems that alarmone levels exhibit a graded response to stress, 189 which appears to correlate to the temperature levels and possibly the heat stress intensity the 190 cells are exposed to.
191 Rel is the main source for (p)ppGpp synthesis during stress response 192 Next, we aimed to identify the major source of (p)ppGpp during the heat stress response. To 193 this end, strains with mutations that disrupt the (p)ppGpp synthetase activity of the proteins 194 encoded by either sasA/ywaC and sasB/yjbM (sasA/Bstrain) or rel (rel E324V ; inactive 195 synthetase) were assayed for (p)ppGpp accumulation and GTP levels upon heat shock at 50 196°C for 2 min (Fig. 1E, S1C). As a control, (p)ppGpp accumulation was also measured in a 197 (p)ppGpp 0 strain bearing inactivating mutations in all three alarmone synthetase genes (sasA, 198 sasB and rel) (Fig 1E). In addition to monitoring (p)ppGpp accumulation directly, the 199 (p)ppGpp-dependent transcription of hpf was employed as an additional read-out for the 250 shock at 48 °C for 15 min before being exposed to the lethal heat shock at 53 °C (Fig. 2G).
251 Similarly, B. subtilis strains with single deletions in sasA or sasB phenocopied the wildtype 252 strain for thermotolerance development (Fig. S2C-D), as they did for heat shock resistance 253 ( Fig. 2A-B and Fig. S2E). By contrast, we observed that rel deletion resulted in strongly 254 increased thermoresistance, which was apparent from the high number of cells still able to 255 form colonies during the otherwise lethal heat shock ( Fig 2G). Consistently, we also observed 256 a strong reduction in protein aggregation during the 37/53 °C heat shock (Fig 2 I). While no 257 significant effect on thermotolerance development was observed in the (p)ppGpp 0 strain ( Fig.   258 2 H), the (p)ppGpp 0 strain exhibited more protein aggregation when exposed to 37/53 °C heat 259 shock (Fig 2 I).  (Fig. S4). However, we could not observe the strongly 280 increased thermoresistance as we observed before in the presence of raised (p)ppGpp levels 281 (Fig 2 F, G, Fig S3). In addition, higher decoyinine concentrations (1000 µg ml -1 ) even 282 abolished both thermoresistance and thermotolerance development ( Fig S4).

436
The observed influence of (p)ppGpp on translation suggests that the major impact of 437 (p)ppGpp appears not to be its effect on transcription (Fig. 4, 5), but the direct modulation of

446
In summary, these observations indicate that the intracellular (p)ppGpp second 447 messenger can immediately control translation during heat stress and is involved in the 448 protection of ribosomes from damage upon severe heat stress (Fig. 7).  (Fig. 7).
458 The activation of the stringent response during heat stress 459 The presented results clearly demonstrate a rapid accumulation of (p)ppGpp during heat and 460 other environmental stresses (Fig. 1). In addition, strains unable to synthesize (p)ppGpp are 461 rendered sensitive to high temperatures and accumulate more heat-induced protein aggregates 462 ( Fig. 2A-D

479
Our experiments demonstrate that Rel activation during heat-or oxidative stress can 480 be inhibited by chloramphenicol similarly as during amino acid starvation (Fig. 1F, S1E). alarmones, but no decrease of the GTP concentration, was observed (Fig. 1, S1). It is possible 501 that the transient pulse, its kinetic and the generated total amount of (p)ppGpp induced by the 502 raised temperature might not be sufficient to promote the strong reduction of cellular GTP 503 that is observed during amino acid starvation (Fig. S1) [27]. It should be noted that a strong 504 reduction of cellular GTP levels would most likely also interfere with the ability of B. subtilis 505 cells to grow at 50 °C with a growth rate comparable to that at 37 °C (Fig. S9 B). When 506 designing the RNA-seq experiment, we choose 48 °C as a simple heat shock condition for the 507 mutant strains since it resembled the thermotolerance protocol (Fig. 1A) and the condition of 508 previously published microarrays [14]. However, many phenotypes of Spx and (p)ppGpp 509 could be observed best upon a stronger, but non-lethal, heat shock at 50 °C [14]. In contrast, 510 while wildtype cells treated with 37/53 °C exhibit a strong increase of (p)ppGpp within the 511 first minutes of stress (Fig 1D), the examination of cellular physiology is confounded by the 512 rapid reduction of viability at this lethal condition (Fig. 2G) [5,14].

513
We reported previously that transcription of rRNA can be down-regulated by the 514 global regulator Spx. During heat stress however, down-regulation of rRNA was independent 515 of Spx, suggesting that the loss of Spx was compensated by additional regulators [14].
516 Strikingly, we now observed that (p)ppGpp also engages in this down-regulation of rRNA 517 during heat stress and that the concurrent activity of both Spx and (p)ppGpp is required to 518 reach the full strength of this effect (Fig. 5A). This functional relationship of Spx and the SR 519 is corroborated by the observation that the (p)ppGpp 0 Δspx mutant strain displays strongly 520 impaired growth at both 37 °C and 50 °C (Fig. 5B). In addition, the observation that a

576
Interestingly, accumulation of (p)ppGpp upon heat or oxidative stress and its 577 importance for stress resistance has also been reported in other Firmicutes and also 578 Proteobacteria that differ widely in terms of (p)ppGpp signaling [56-58,74,75].
579 Accumulation of (p)ppGpp was shown to protect cells from salt or osmotic stress [76,77].
580 Conversely, the lack of (p)ppGpp is known to renders cells sensitive to heat or oxidative