Role of VapBC4 toxin-antitoxin system ofSulfolobus acidocaldariusin heat stress adaptation

15 Toxin-antitoxin (TA) systems are important for stress adaptation in prokaryotes, including 16 persistence, antibiotic resistance, pathogenicity, and biofilm formation. Toxins can cause cell 17 death, reversible growth stasis, and direct inhibition of crucial cel lular processes through 18 various mechanisms, while antitoxins neutralize the effects of toxins. In bacteria, these 19 systems have been studied in detail, whereas their function in archaea remains elusive. 20 During heat stress, the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibited an 21 increase in the expression of several bicistronic type II vapBC TA systems, with the highest 22 expression observed in the vapBC4 system. In the current study, we performed a 23 comprehensive biochemical characterization of the VapBC4 TA system, establishing it as a 24 bonafide type II toxin -antitoxin system. The VapC4 toxin is shown to have high -temperature 25 catalyzed RNase activity specific for mRNA and rRNA, while the VapB4 antitoxin inhibits 26 the toxic activity of VapC4 by interacting with it. VapC4 toxin expression led to heat-induced 27 persister-like cell formation, allowing the cell to cope with the stress. Furthermore, th is study 28 explored the impact of vapBC4 deletion on biofilm formation, whereby deletion of vapC4 led 29 to increased biofilm formation, suggesting its role in regulating biofilm formation. Thus, 30 during heat stress, the liberated VapC4 toxin in cells could potentially signal a preference for 31 persister cell formation over biofilm growth. Thus, our findings shed light on the diverse 32 roles of the VapC4 toxin in inhibiting translation, inducing persister cell formation, and 33 regulating biofilm formation in S. acidocaldarius , enhancing our understanding of TA 34 systems in archaea. 35 36 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint IMPORTANCE 37 This research enhances our knowledge of Toxin-antitoxin (TA) systems in archaea, 38 specifically in the thermoacidophilic archaeon Sulfolobus acidocaldarius . TA systems are 39 widespread in both bacterial and archaeal genomes, indicating their evolutionary importance. 40 However, their exact functions in archaeal cellular physiology are still not well understood. 41 This study sheds light on the complex roles of TA systems and their critical involvement in 42 archaeal stress adaptation, including persistence and biofilm formation. By focusing on S. 43 acidocaldarius, which lives in habitats with fluctuating temperatures that can reach up to 44 90℃, the study reveals the unique challenges and survival mechanisms of this organism. The 45 detailed biochemical analysis of the VapBC4 TA system, and its crucial role during heat 46 stress, provides insights into how extremophiles can survive in harsh conditions. The findings 47 of this study show the various functions of the VapC4 toxin, including inhibiting translation, 48 inducing persister-like cell formation, and regulating biofilm formation. This knowledge 49 improves our understanding of TA systems in thermoacidophiles and has broader 50 implications for understanding how microorganisms adapt to extreme environments. 51 52

53 Toxin-antitoxin, heat-stress, biofilms, persisters, archaea, Sulfolobus acidocaldarius 54 55 56 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint

57 In recent years, the investigation of toxin -antitoxin (TA) systems has gained attention due to 58 their essential roles in regulating bacterial growth, aiding survival under stressful conditions, 59 and their potential implications in bacterial virulence and antibiotic resistance [ 1, 2]. TA 60 systems consist of a pair of closely linked genes encoding a stable toxin and a labile 61 antitoxin, wherein the antitoxin neutralizes the toxin's effects, maintaining cellular 62 equilibrium [ 3]. While extensively studied in bacteria, the presence and functional 63 significance of these systems in archaea remain relatively unexplored. Virulence- associated 64 proteins (Vaps) constitute a distinctive subset within the type-II toxin-antitoxin (TA) systems. 65 They exhibit a genetic arrangement with two linked genes, where a proteolytically unstable 66 antitoxin (VapB) is commonly encoded first, followed by a stable ribonucleolytic toxin 67 (VapC). Initially associated with virulence plasmids in human pathogens, like Salmonella 68 dublin, these proteins have gained recognition as a prominent variant of type -II toxins, 69 particularly prevalent among archaea [4, 5]. The significance of the presence of a large 70 number of type II vapBC TA systems in archaea, which have not shown any signs of 71 virulence, is yet to be determined. The functional scope of VapC toxins in bacteria 72 encompasses the degradation of diverse RNA molecules like mRNA, rRNA, and tRNA [ 6-73 11]. However, interestingly, tRNAs have emerged as the primary RNA targets for most 74 VapCs [12]. 75 Sulfolobus acidocaldarius , a thermoacidophilic archaeon, thrives in a challenging 76 environment of hot mud pools and solfataric springs where temperatures range from 75℃ to 77 80℃ and the pH levels are highly acidic (pH 2 -3) [13]. The temperature of these habitats is 78 constantly fluctuating owing to the geothermal activities of the earth’s crust, with the 79 potential to reach as high as 90℃, presenting a serious threat to the resident organisms. 80 Surviving under such extreme conditions requires utilizing intricate molecular strategies, 81 rendering S. acidocaldarius a model organism for investigating unique survival mechanisms 82 [14]. Recent genomic analyses have identified multiple TA systems in the S. acidocaldarius 83 genome, with the majority being type II TA systems [ 15, 16]. These type II TA systems 84 comprise a protein toxin targeting specific cellular processes, counteracted by a 85 corresponding antitoxin protein. Furthermore, within the phylum of thermoacidophilic 86 thermoproteota the vapBC system stands out as the most encountered type II TA system [ 16]. 87 Previous studies by Cooper et al. demonstrated that with the increase in the optimal growth 88 temperature of archaea, there is a concurrent increase in the count of vapBC TA genes within 89 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint the genome of archaea [ 17]. This correlation implies that the vapBC TA system could enable 90 archaea to adapt to higher temperatures. Also, disruption of the vapBC6 operon from S. 91 solfataricus through targeted gene inactivation resulted in two recessive phenotypes 92 associated with fitness: heightened sensitivity to high temperatures and a heat -dependent 93 decrease in the growth rate [18]. 94 The VapBC TA systems play critical roles in regulating cellular processes, stress responses, 95 and survival strategies. In bacteria, VapBC systems have been extensively studied for their 96 involvement in bacterial persistence, formation of persister cells, and stress adaptation [ 19, 97 20]. These systems influence growth arrest, dormancy, and antibiotic tolerance, which are 98 crucial for bacterial survival in challenging environments [ 21, 22]. The VapBC family 99 constitutes the predominant group of toxin -antitoxin loci in extreme thermoacidophiles, 100 implying their vital significance in the physiology of these organisms. For instance, S. 101 tokodaii has 25 vapBC toxin-antitoxin loci, while S. solfataricus has 22 [ 23]. Various vapBC 102 pairs within S. solfataricus demonstrated differential transcriptional patterns during heat 103 shock [17]. In vitro experiments involving a toxin responsive to heat shock, VapC6 from S. 104 solfataricus, revealed its ability to target mRNA specifically. This study demonstrated VapC6 105 mediated targeting of transcripts coding for its cognate antitoxin vapB6, a transcriptional 106 regulator (tetR), and an oligo/dipeptide transport permease (dppB-1) [18]. Strikingly, deleting 107 the gene encoding VapC6 from the S. solfataricus genome rendered the archaeon susceptible 108 to heat shock, indicating its pivotal involvement in response to thermal stress [ 18]. Under 109 uranium stress conditions, another thermoacidophile, Metallosphaera prunae, employs VapC 110 toxins in post-transcriptional regulation, inducing a cellular dormant state [ 24]. This strategic 111 adaptation counteracts the damage by toxic metals [ 24]. Moreover, previous investigations 112 successfully identified 18 unique type II vapBC TA systems in Sulfolobus acidocaldarius , 113 each harboring a PIN domain within its toxin protein [ 15]. An interesting observation from 114 our findings was the distinct upregulation observed for at least eight specific vapBC pairs out 115 of the sixteen encoded vapBC systems under various stressors, including heat stress , 116 oxidative stress, and nutrient limitations [15]. This suggested their involvement in facilitating 117 adaptive responses to individual stress types, a phenomenon termed cross -stress adaptation 118 [15]. 119 In the current study, we conducted a detailed biochemical and genetic analysis of the 120 saci_1812/saci_1813 Toxin-antitoxin pair which is upregulated under heat stress in S. 121 acidocaldarius [15]. In a recent study, Lewis et al. designated this TA pair as vapBC4 [25]. In 122 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint a separate study, we simultaneously denoted the identical pair as vapBC5, purely based on the 123 detection of the PIN -domain in the toxin counterpart of type II vapBC TA loci [ 15]. To 124 maintain uniformity in the nomenclature used in the field, we uphold the identi ty of this TA 125 pair as vapBC4 in the present study. Our investigation demonstrated that the vapBC4 TA 126 system is a bicistronic type II toxin -antitoxin. The toxin component of this system (VapC4 ) 127 exhibited a temperature-dependent ribonucleolytic activity targeted towards both mRNA and 128 rRNA molecules in vitro. This enzymatic activity is believed to hinder the translation process, 129 leading to cellular persistence efficiently. Subsequently, a deletion mutant of vapC4 toxin 130 resulted in decreased viability of the cells in response to heat stress at 85℃ in concordance 131 with the observed maximal upregulation of the vapBC4 operon in response to heat stress. 132 This mutant also showed an increased tendency to form biofilm, suggesting a role of VapC4 133 toxin in biofilm formation, as was already the case for VapC14 (Saci_2183) [25]. 134 Together, the present study demonstrated, the significance of the archaeal VapC4 toxin 135 component within the type II TA system in promoting cellular survival under heat stress by 136 inhibiting translation, allowing the cells to enter a persister -like state as a coping mechanism 137 for stress. 138 2. MATERIALS AND METHODS 139 Phylogenetic analysis 140 The SpeciesTreeAlignment.fa file generated by OrthoFinder was used as input for inference 141 of the best amino acid substitution model and for subsequent phylogenetic inference by 142 maximum likelihood [26]. Once we obtained the phylogenomic tree, we began searching for 143 homologs of Saci_1812 (VapB4) and Saci_1813 (VapC4). For this, we used blastp with the 144 parameters recommended by the developer [ 27]. Finally, the possible homologous proteins 145 obtained were aligned with the MAFFT tool to identify regions at the amino acid sequence 146 level that could be conserved [28]. The result of the phylogenomic analysis and the search for 147 homologs was visualized through the iTOL software [29]. 148 Strains and growth conditions 149 S. acidocaldarius MW2000 (WT), MW1363 (Δ vapC4) and MW1365 (Δ vapBC4) and 150 complementation strains were grown aerobically in Brock medium at pH 3, enriched with 151 0.1% N-Z-amine, 0.2% sucrose, and for strains not carrying a plasmid,10 µg/ml uracil, in a 152 75℃ incubator shaker (Thermo Scientific MaxQ 6000). Growth was tracked by assessing the 153 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint optical density at 600 nm (OD 600) using a UV-vis spectrophotometer (Dynamica HALO XB -154 10). 155 156 Generation of vapC4, vapBC4 knock-out strains 157 The plasmids pSV A6520 and pSV A6567 were generated by cloning 500 bp of the upstream 158 and downstream regions of the target genes into plasmid pSV A431 (Table S3). The plasmids 159 were methylated by transforming them into E. coli ER1821 cells harboring the additional 160 plasmid pM.EsaBC4I (New England Biolabs, Frankfurt am Main, Germany) to evade 161 restriction by the SuaI restriction system. 162 For generation of competent cells, S. acidocaldariu s strains were grown in Brock medium 163 enriched with 0.1% N-Z-amine, 0.2% dextrine, and 10 µg/ml uracil. When the optical density 164 (OD600) reached 0.5 –0.7, a calculated amount of the cell culture was transferred to fresh 165 medium and harvested the following day at an OD 600 of 0.2 –0.3. The culture was cooled on 166 ice, centrifuged for 20 min at 4000 × g at 4°C, and washed three times with 30 ml and once 167 with 1 ml of 20 mM ice -cold sucrose. Cells were resuspended in 20 mM sucrose to a 168 theoretical OD of 20, aliquoted into 50 μl and stored at −80°C. 169 Purified methylated plasmids were then used for transformation into competent MW2000 170 cells as described previously [ 30]. Colony PCR was performed to detect knock -out mutants 171 using primers binding outside of the flanking regions. The PCR products of positive colonies 172 were sent to sequencing analysis to confirm in frame deletion and no additional mutations. 173 Complementation assays 174 For complementation assays, we complemented ΔvapC4 with vapC4 by electroporating 175 plasmid pSV A6524 (vapBC4 operon along with its upstream 150 bps promoter cloned into 176 the pSV Aara-FX-Stop backbone with a premature stop codon in the vapB4 gene) into ΔvapC4 177 strain. The colonies obtained after electroporation were selected on first selection plates 178 lacking uracil. Similarly, the ΔvapBC4+vapBC4 complementation was generated by 179 electroporating pSV A6525 plasmid ( vapBC4 operon along with its upstream 150 bps 180 promoter cloned into the pSV Aara-FX-Stop backbone) into ΔvapBC4 strain. Since we could 181 not obtain the ΔvapB4 strain, we complemented the vapC4 gene into the ΔvapBC4 strain, 182 generating the ΔvapBC4+vapC4 strain. This was performed by electroporating pSV A6524 183 plasmid into ΔvapBC4 strain followed by uracil selection. 184 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint Construction of overexpression vectors for VapB4 antitoxin and VapC4 toxin proteins 185 For heterologous expression of the VapB4 antitoxin and VapC4 toxin, both with a C- terminal 186 histidine tag, the saci_1812 (vapB4) and saci_1813 (vapC4) genes were PCR-amplified from 187 gDNA of S. acidocaldarius DSM639. The PCR amplified product was digested with dual 188 restriction enzymes (NcoI and XhoI) and was subsequently ligated into pET28a (Kanamycin -189 resistant), yielding plasmid pAG153 and pAG154 for VapC4 and VapB4 respectively. The 190 PCR product of amplified vapB4 was additionally ligated into the multiple cloning site 2 191 (MCS2) of pETDuetI (Ampicillin -resistant), which harbors a C -terminal StrepII tag, 192 constructing plasmid pAG155. For conducting expression analysis, the respective vectors 193 were introduced into E. coli BL21 (DE3) cells containing the RIL Camr plasmid (Stratagene). 194 A comprehensive list of all the strains, primers, and plasmids generated is listed in 195 Supplementary information (Table S1-S3) 196 Toxicity test 197 To conduct streak toxicity tests, cultures of E. coli BL21(DE3) cells carrying pAG153 and 198 pAG154 grown overnight were streaked onto M9 minimal agar plates supplemented with 199 kanamycin. Also, E. coli cells harboring both the pAG153 and pAG155 plasmids were 200 streaked onto minimal agar plates containing a combination of both kanamycin and 201 ampicillin. These plates were prepared with and without the addition of 0.5 mM IPTG and 202 were incubated overnight at 37℃. 203 E. coli growth curve analysis and cell viability assay 204 Overnight cultures of E. coli BL21(DE3) cells carrying the empty pET28a vector, pAG153, 205 and pAG154 were diluted in 100 ml of fresh M9 minimal media. This media was 206 supplemented with kanamycin (50 µg/ml) and 0.5mM IPTG, and the dilution was adjusted to 207 initiate the cultures at an initial OD 600 of 0.01. Similarly, for E. coli BL21(DE3) with a 208 combination of the empty pET28a and pETDuet1 vectors, as well as a combination of 209 pAG153 and pAG155, the overnight cultures were diluted in 100 ml of fresh M9 minimal 210 media. This media was supplemented with kanamycin (50 µg/ml), ampicillin (100 µg/ml), 211 and 0.5 mM IPTG. The cultures were then incubated at 37 °C. OD 600 measurements were 212 taken at 30 mins intervals for up to 4 hours using a UV -vis spectrophotometer (Dynamica 213 HALO XB -10). Simultaneously, aliquots of cells were collected at each time interval for 214 assessing cell viability using the MTT (3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 215 bromide) assay. For the cell viability assessment, 10 µl of a 5 mg/ml MTT solution was added 216 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint to 100 µl of cells and incubated at 37 °C for 20 mins. After incubation, the cells were 217 centrifuged at highest speed in a benchtop centrifuge (Tarsons Spinwin MC03) for 1 min, and 218 the supernatant was discarded. The resulting cell pellet was reconstituted in 1 ml of DMSO. 219 Subsequently, 200 µl of the resulting purple solution was transferred to a 96 -well microtiter 220 plate, and the released purple color was measured spectroscopically at 550 nm using a Plate 221 reader (Thermo Scientific Multiskan GO). The intensity of the purple color signifies the 222 amount of viable cells present. 223 Recombinant protein expression in E. coli 224 A 10 ml volume of an overnight grown culture of E. coli BL21(DE3)-RIL cells, containing 225 the respective pAG153 and pAG154, was used to inoculate 1 L of Luria –Bertani medium 226 supplemented with kanamycin (50 µg/ml). The cells were cultivated at 37°C until reaching an 227 OD600 of 0.6. For expressing the VapC4 toxin, 150 µM IPTG (isopropyl ß -D-228 thiogalactopyranoside) was added, the incubation temperature was reduced to 16°C, and 229 growth continued overnight to minimize inclusion body formation. For the VapB4 antitoxin 230 expression, cells were induced with 500 µM IPTG and incubated at 37°C for 3 -4 hours. 231 Subsequently, the cells were collected through centrifugation (using rotor SA -300; SORV AL 232 RC6+ Thermo-Scientific), reconstituted in lysis buffer (50 mM Tris pH 8.0, 150 mM KCl, 233 and 10% glycerol) supplemented with the complete EDTA -free protease inhibitor cocktail (1 234 tablet/50 ml of lysate; Roche), frozen in liquid nitrogen, and preserved at -80 °C. 235 Recombinant protein purification from E.coli 236 Before purification, frozen cell pellets were thawed on ice and added to the lysis buffer. To 237 the lysis buffer, 1 mM lysozyme was added separately. After 30 minutes of incubation on ice, 238 cell lysis was achieved through sonication using Soniprep 150 (DJB Labcare, UK). The 239 resulting cell debris was eliminated by centrifugation at 15000 rpm for 30 minutes (using 240 rotor SA-300; SORV AL RC6+ Thermo-Scientific). The supernatant was then subjected to a 241 Ni2+-NTA affinity column (Qiagen) to isolate histidine -tagged proteins. The immobilized 242 proteins underwent a stepwise wash with lysis buffer containing 10 mM and 20 mM 243 imidazole. Depending on the specific protein, the fraction of bound proteins was then 244 released in a lysis buffer containing 100 -300 mM of imidazole. The eluted fraction was 245 assessed by running a reducing SDS -PAGE. Subsequently, the target protein fraction was 246 subjected to overnight dialysis in a lysis buffer (50 mM Tris pH 8.0, 150 mM KCl, and 10% 247 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint glycerol). All solutions used were nuclease -free or treated with DEPC to ensure RNase free 248 protein purification. 249 250 251 Purification of single stranded 7S SRP RNA, 23S rRNA, TetR mRNA, tRNA Met and 16S 252 rRNA 253 The 7S SRP-RNA, 23S rRNA, TetR mRNA, Met-tRNA, and the 16S-rRNA were synthesized 254 using in vitro transcription protocol (Thermo -Scientific). The respective genes were cloned 255 under the T7 promoter of the pGEM3z vector, and transcription was carried out by T7 RNA 256 polymerase using NTPs. After completion, the reaction mixture was purified by a column 257 using RNA Purification Kit (Qiagen). A Nanodrop (Thermo Scientific, USA) quantified the 258 purified RNA product. 259 260 RNase Assay 261 Increasing concentrations of purified VapC4 toxin (Saci 1813) were incubated at 37 ℃ and 262 60 ℃ for 30 mins with approximately 5 00 ng of purified total RNA from Sulfolobus 263 acidocaldarius DSM639 in a buffer containing 50 mM sodium phosphate (pH -7.0),150mM 264 KCl and 5mM MgCl 2. For the VapB4 neutralization assay, VapB4 antitoxin and VapC4 toxin 265 were pre-incubated together at 75 ̊ C for 10 mins in a 2:1 ratio, respectively, before adding 266 total RNA substrate, after which the reaction was held at 60 ̊ C for 30 mins. For determining 267 the optimal pH for VapC4, 500 ng of total RNA from Sulfolobus acidocaldarius DSM639 was 268 incubated with 2µM of purified VapC4 protein in different buffers: 50 mM Tris (pH-8.0), 150 269 mM KCl, and 5mM MgCl 2; 50 mM sodium phosphate (pH -6.0), 150 mM KCl, and 5 mM 270 MgCl2; 50 mM citrate buffer (pH -5.0), 150 mM KCl, and 5 mM MgCl 2; and 50 mM citrate 271 buffer (pH-3.0), 150 mM KCl, and 5 mM MgCl 2 at 60℃ for 30 minutes. Since the pH of a 272 solution is dependent on temperature, the pH of the buffer at 60℃ was monitored using pH 273 paper. In the case of in -vitro transcribed RNA substrates, 2µM of purified VapC4 was 274 incubated with 300ng of in-vitro transcribed RNA substrates in the same buffer and incubated 275 at 60℃ for various time intervals: 10 minutes, 20 minutes, 30 minutes, and 60 minutes. The 276 total reaction volume was 10 µl. Each reaction was stopped by adding 2µl of 6x loading 277 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint buffer and separated on 1% Tris -acetate-EDTA agarose gels with 1ug/ml ethidium bromide. 278 All solutions used were nuclease-free or treated with DEPC. 279 280 281 282 Co-purification assay 283 E. coli cells were co -transformed with toxin with His tag cloned into pET28a and antitoxin 284 with StrepII tag cloned into the pETDuet1 vector. The co -transformed cells were selected 285 onto LB agar plates containing the antibiotic kanamycin and ampicillin. Overnight primary 286 culture at 37℃ was given with the colony obtained on the plates with both selection markers . 287 100 ml of secondary culture was given the following day. After the OD 600 reached 0.6, 288 0.5mM IPTG induction was given for 3 hours. The cells were then harvested by 289 centrifugation followed by lysis with a sonicator. After sonication, the cell debris was 290 removed by centrifugation at 13000 rpm for 30 mins at 4℃. The cell lysates obtained after 291 centrifugation were subjected to a Ni2+-NTA affinity column (Qiagen). The immobilized 292 proteins underwent a stepwise wash with lysis buffer containing 10 mM and 20 mM 293 imidazole. The fraction of bound proteins was then released in a lysis buffer containing 100-294 300 mM of imidazole. The eluted fraction was assessed by running a reducing SDS -PAGE 295 followed by Western blot analysis utilizing anti-His and anti-StrepII antibodies. 296 Fluorescent labeling of VapC4 and VapB4 297 Purified VapB4 and VapC4 were labeled with Tetramethyrhodamine (TRITC): Alexa-532 298 and Fluorescein isothiocyanate (FITC): Alexa-488 fluorescent probes, respectively, following 299 the manufacturer's instructions (Sigma-Aldrich, USA). In brief, VapC4 (50 µM) and VapB4 300 (75 µM) in 50 mM Na-Phosphate Buffer supplemented with 100 mM Sodium bicarbonate 301 (pH 9.0) were combined with FITC or TRITC fluorescent probes, respectively. FITC and 302 TRITC stock solutions were prepared in anhydrous DMSO at 600 µg/ml concentrations. For 303 labeling the protein samples, 50 µl of the FITC or TRITC fluorescent probe was taken from 304 the stock solution and gradually introduced into 1 ml of the protein. The protein sample was 305 gently and continuously stirred for 2 hours at room temperature. A Bio-Gel A column (Bio-306 Rad) was utilized to segregate the labeled protein from the unbound probe. The collected 307 fraction containing the labeled VapB4 or VapC4 was dialyzed against a phosphate buffer (50 308 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint mM, pH 7.2) at room temperature for 24 hours. The evaluation of the protein labeled with 309 FITC or TRITC involved measuring the absorbance of the protein at 280 nm and the 310 corresponding fluorescence probes at 495 nm (for FITC) or 542 nm (for TRITC). The extent 311 of FITC or TRITC labeling on the protein was determined based on the F/P ratio, which was 312 approximately 0.5 for both probes, confirming optimal protein labeling. Lastly, the labeled 313 proteins were verified by running an SDS-PAGE and scanning using a Typhoon scanner (GE 314 Healthcare, Danderyd, Sweden). 315 Fluorescence resonance energy transfer (FRET) 316 The fluorescence resonance energy transfer (FRET) method was employed to ascertain the 317 interaction between VapC4 toxin and VapB4 antitoxin proteins. FITC -labeled VapC4 (100 318 nM), serving as the donor, was titrated with TRITC -labeled VapB4 (25-2000 nM) acting as 319 acceptor. A no FRET control set was also set where FITC -labeled VapC4 (100 nM), serving 320 as the donor, was titrated with only buffer. The reaction mixture was excited at 495 nm, and 321 the fluorescence emission spectra were taken from 505 -625 nm at 60°C using a JASCO f -322 8500 spectrophotometer (JASCO International Co., Ltd., Japan). Subsequently, the donor and 323 acceptor emission spectra were deconvoluted using Igor pro 6 (WaveMetrics, Portland, 324 Oregon, USA). The emission intensity of FITC at 520 nm was individually measured during 325 various titrations and used to compute the dissociation rate constant (Kd) using the 326 subsequent equation: 327 𝐹0 − 𝐹𝑡 𝐹0 = 𝐵 × [𝑉𝑎𝑝𝐵4] Kd + [𝑉𝑎𝑝𝐵4] Where Ft represents fluorescence intensity at 520 nm for various concentrations of VapB4 , 328 and F0 is the fluorescent intensity at [VapB4] = 0. Kd denotes the dissociation rate constant, 329 and B represents the maximum intensity reached at the saturation of binding. 330 S. acidocaldarius growth curve analysis 331 Exponentially growing cultures of S. acidocaldarius MW2000 (WT), ΔvapC4, and ΔvapBC4 332 were diluted into 50 ml of Brock medium at pH 3, supplemented with 0.1% N-Z-amine, 0.2% 333 sucrose, and 10 µg/ml uracil. The dilution was adjusted to an initial OD 600 of 0.01. The 334 cultures were then incubated in a 75℃ incubator shaker (Thermo Scientific MaxQ 6000). The 335 growth progression of the organisms was checked at four -hour intervals by measuring the 336 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint optical density at 600 nm (OD 600) using a UV-vis spectrophotometer (Dynamica HALO XB -337 10). The experiment was performed in three biological replicates. 338 To assess growth under heat stress, cell cultures in the exponential growth phase were diluted 339 to an OD 600 of 0.5. Subsequently, 1 ml of the diluted cells was dispensed into 2 ml 340 microcentrifuge tubes and subjected to heat stress at 85°C in a shaking dry heat block. 341 Growth progression was tracked by recording the OD 600 at hourly intervals. As a control, the 342 diluted cell cultures were maintained at 75°C in a shaking dry heat block. The experiment 343 was performed in three biological replicates. 344 Cell viability assay for S. acidocaldarius 345 To evaluate the ability of the generated Sulfolobus strains to survive under heat stress, an 346 MTT assay was conducted at various time points during heat stress exposure. All knock -out 347 and complementation strains were cultured until the mid -exponential phase (OD 600 0.5). 348 Subsequently, 2 ml of culture from each strain was transferred to microcentrifuge tubes. 349 These tubes were then exposed to heat stress by incubating at 85°C in a Thermo Shaker 350 incubator (BenchTop Lab Systems BT -MTH-100) for different durations (15, 30, 45, and 60 351 mins), with strains maintained at 75℃ serving as controls. The heat stress temperature was 352 determined following a previous study by Baes et al. [ 31]. After incubation, 100 µl of the 353 cultures were mixed with 10 µl of MTT reagent (5 mg/ml) and incubated for 1 hour at 75°C 354 in an incubator (Thermo Scientific MaxQ 6000). Following centrifugation at 13,000 rpm 355 using a benchtop centrifuge (Tarsons Spinwin MC03) for 1 min, the supernatant was 356 discarded, and the pellets were dissolved in 1 ml DMSO. Subsequently, 200 µl of the 357 resulting purple solution was transferred to a 96 -well microtiter plate, and the released purple 358 color was measured spectroscopically at 550 nm using a Plate reader (Thermo Scientific 359 Multiskan GO). The intensity of the purple color signifies the amount of viable cells present. 360 To calculate the percentage survivability based on the MTT assay absorbance at 550 nm, the 361 following formula was used: 362 Percentage Survivability = 100 × (Absorbance of Sample at 550 nm) (Absorbance of Control at 550 nm) 363 Where the "Absorbance of Sample" refers to the absorbance measured for each time point 364 after heat stress, and the "Absorbance of Control" is the absorbance of the corresponding 365 control set (not subjected to heat stress). The experiment was performed in three biological 366 replicates. 367 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint Biofilm formation 368 All knock -out and complementation strains were grown to mid -exponential phase (OD 600 369 0.5). Subsequently, cells were inoculated into 24 -well microtiter plates filled with Brock 370 medium at pH 3, enriched with 0.1% N-Z-amine, 0.2% sucrose, and 10 µg/ml uracil (for WT, 371 ΔvapC4, and Δ vapBC4), or only 0.1% N -Z-amine and 0.2% sucrose (for the 372 complementation strains). The cells were inoculated into 24 well plates in such a way such 373 that their initial OD600 was 0.01. For the heat stress-induced biofilm formation, the cells were 374 initially subjected to heat stress at 85℃ for different durations (15 mins, 30 mins, 45 mins, 375 and 60 mins). After each heat stress period, the cells were collected and subjected to an MTT 376 assay to quantify viable cell counts. Following the assay, an equal number of heat -stressed 377 viable cells, determined from the MTT results, were inoculated into each well of the 24 -well 378 plates. The 24 -well plate was sealed tightly with a gas -permeable sealing membrane 379 (Breathe-Easy, Diversified Biotech, Boston, MA, USA) and incubated at 75 °C in an 380 incubator (Thermo Scientific MaxQ 6000) for 48 hours within a humidity chamber to avoid 381 evaporation. The initial OD 600 was adjusted to 0.01, following the procedure outlined by 382 Koerdt et al. [32]. After 48 -hour incubation, the 24 -well microtiter plates were cooled, and 383 200 µl of vegetative cells from each sample were transferred to a new 96 -well microtiter 384 plate. The OD 600 of the supernatant containing the vegetative cells was determined using a 385 96-well plate reader (Thermo Scientific MultiSkan GO). The remaining supernatants were 386 carefully discarded without disturbing the biofilm adhered to the 96 -well microtiter plate . 387 Subsequently, 200 µl of a 0.5% w/v crystal violet (CV) solution was added to each well and 388 incubated for 10 minutes at room temperature. Once again, the CV supernatant was removed, 389 and the biofilm adhered to the well plate was washed three times with Brock medium at pH 5 390 until residual crystal violet was eliminated. One ml of 30% acetic acid was added to each 391 well to release the crystal violet absorbed by the biofilm -forming cells, and absorbance was 392 measured at 570 nm. The OD 570/OD600 correlation index was employed to assess the 393 efficiency of biofilm formation. The experiment was conducted in biological replicates. 394 Statistical Analysis 395 GraphPad Prism 6 was utilized for statistical analyses. The values in the figures represent the 396 mean of three replicates ± the standard error of the mean. We used two-way ANOVA and the 397 Bonferroni post-test for the statistical data comparison. In all analyses, a significance level of 398 p < 0.05 was considered. 399 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint 400 3. RESULTS 401 The vapB4 and vapC4 genes are co-transcribed in S. acidocaldarius 402 The Sulfolobus acidocaldarius DSM 639 genome contains a potential vapBC4 operon 403 (saci_1812 and saci_1813, depicted in Figure 1a). Subsequent evaluation of the toxin gene 404 sequence encoded a VapC-like member of the PIN domain superfamily of ribonucleases. The 405 identified antitoxin gene encoded a CopG family transcriptional regulator, possessing an 406 RHH-like domain usually observed in VapB antitoxins. Notably, sequence analyses showed 407 an overlap for the translational start codon of vapC4 with the translational stop codon of 408 vapB4, strongly implying translational coupling ( Figure 1a ). To confirm the existence of 409 these two genes as a bicistronic operon, reverse transcriptase (RT) -PCR experiments were 410 conducted. The primers selected targeted a segment that spanned from the end of the 411 antitoxin gene to the start of the toxin gene. As shown (Figure 1b), these genes are indeed 412 expressed as a single transcriptional unit. Since most of the TA systems are inherited through 413 horizontal gene transfer, we sought to assess the conservation of the VapBC4 TA system 414 beyond Sulfolobus species. We searched for homologous genes in a set of 78 archaeal 415 genomes (Supplementary Information Table S4). We identified 25 pairs of toxin -antitoxin 416 genes in various lineages, including the superphylum Asgardarchaeota, DPANN, TACK, and 417 the phylum Euryarchaeota. The specific organisms harboring these potential homologs are 418 detailed in Supplementary Information ( Table S5 and Figures S 1-S2). None of the listed 419 VapBCs have been functionally characterized to date. This distribution suggests that the 420 VapBC4 TA system, or its homologs, are not confined to a specific taxonomic group but are 421 instead widely distributed among archaeal taxa, suggesting their significance in archaeal 422 biology, and highlighting the need for further investigation into their roles and evolutionary 423 dynamics. 424 425 Heterologous expression of VapC4 leads to cell growth inhibition in E. coli. 426 E. coli cells harboring different constructs, including the empty pET28a vector, pAG153 427 (VapC4 toxin) and pAG154 (vapB4 antitoxin), and the combination of both pAG153 and 428 pAG155 (vapB4 antitoxin), were streaked onto M9 minimal media agar plates supplemented 429 with the appropriate antibiotics for selection and 0.5 mM of IPTG to induce gene expression. 430 Cells expressing the VapC4 toxin exhibited growth inhibition in comparison to cells 431 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint expressing the VapB4 antitoxin or carrying the empty vector ( Figure 2a, upper row ). 432 Conversely, the co -expression of the VapB4 antitoxin and the VapC4 toxin rescued the cell 433 growth ( Figure 2a, lower row ). A growth curve analysis of bacterial cells expressing the 434 different genes was performed to further investigate this impaired growth phenomenon upon 435 toxin expression and the alleviation of toxin activity upon antitoxin expression. The VapC4 436 toxin-expressing cells showed significant growth defects compared to those carrying empty 437 pET28a and VapB4 antitoxin expressing plasmid ( Figure 2b ). The growth was partially 438 rescued when the VapB4 antitoxin was co-expressed along with the VapC4 toxin (Figure 2b). 439 A similar observation was noted in bacteria when VapC toxins from various sources, 440 including a plant pathogen Acidovorax citrulli, a deep-sea marine bacterium Streptomyces sp. 441 SCSIO02999, and a human pathogen non -typeable Haemophilus influenzae, were expressed 442 heterologously in E. coli cells [ 33-35]. It is particularly surprising to observe that the toxin 443 derived from a thermoacidophilic archaeon can effectively induce bacteriostasis in the 444 mesophilic E. coli cells. 445 446 VapC4 toxin expression in E. coli leads to persister cells 447 Previous reports in bacteria have indicated that the expression of toxins can induce alterations 448 in cellular metabolism, potentially triggering the emergence of the persister phase or leading 449 to cell death [36]. The cells' fate depends on the specific nature of stress and the type of toxin 450 in play. To understand what is happening to the E. coli cells that result in the growth 451 impairment caused by the Saci VapC4 toxin expression, we conducted a MTT assay to assess 452 cell viability. We aimed to investigate the fate of E. coli cells upon the induction of the VapC4 453 toxin. In the cell viability test, the absorbance at 550 nm displayed a gradual increase for cells 454 containing the empty pET28a vector (control), showing no influence on their growth. In 455 contrast, following 1 hour of induction, a reduction in absorbance (OD 550) became apparent, 456 signifying that upon VapC4 overexpression, a portion of the cells dies ( Figure 2c). After 2 457 hours, cell growth was gradually restor ed and remained stable. This pattern indicates that the 458 overexpression of VapC4 generates a bacteriostatic effect where the cells are viable but 459 incapable of dividing. This phenomenon was also observed when Leptospira VapC and 460 Mycobacteria VapC46 were expressed in E. coli [10, 37]. 461 The RNase activity of VapC4 from S. acidocaldarius is catalyzed by high temperatures 462 and is effectively counteracted by the inhibitory action of VapB4 463 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint The purification of the VapC4 toxin protein from E. coli presented a significant challenge due 464 to the observed inhibition of cell growth upon protein overexpression. In an attempt to 465 overcome this hurdle, VapC4 expression was gradually induced at a very low temperature of 466 16℃ and different IPTG concentrations were tested. Surprisingly, at an IPTG concentration 467 of 0.1 mM, the cells could express the toxic protein without causing any detrimental impact 468 on their growth (as depicted in Figure 3a ). This successful outcome was corroborated by 469 SDS-PAGE analysis, which indicated the presence of VapC4 protein with a migration pattern 470 aligning with that of the 16 kDa marker (as illustrated in Figure 3b). Conversely, the VapB 471 antitoxin was produced in E. coli growing at 37℃ at and induced with 0.5 mM IPTG for 3 -4 472 hours. Subsequent SDS -PAGE analysis confirmed the presence of VapB4 protein whose 473 migration aligned with that of the 9 kDa marker (as represented in Figure 3c). 474 The VapC proteins are known for their ribonucleolytic activity due to the presence of the 475 characteristic PIN domain in their structure [ 38]. When the purified VapC4 toxin was 476 incubated with total RNA extracted from S. acidocaldarius at two different temperatures, 477 37℃ and 60℃, it was observed that VapC4 was able to degrade the RNA at both 478 temperatures. Complete degradation of RNA was observed with 1.3 µM of VapC4 at 60℃ 479 compared to that at 37℃, aligning with the fact that S. acidocaldarius is a thermophilic 480 organism. ( Figure 4 a-b). A similar observation was made with the S. solfataricus VapC6 481 toxin, demonstrating more efficient cleavage of total RNA at 80°C in contrast to temperatures 482 of 37°C and 60°C [ 18]. As the vapC4 toxin gene was identified within a bicistronic operon 483 alongside the vapB4 antitoxin gene, a characteristic feature of type II Toxin -antitoxin 484 systems, we proceeded to investigate the inhibitory function of the cognate VapB4 antitoxin 485 on the VapC4 toxin. In this case, purified VapB4 antitoxin was incubated with the VapC4 486 toxin at two different temperatures, 37℃ and 60℃. At 60℃ the RNase activity of VapC4 was 487 substantially inhibited compared to the activity observed at 37℃ (as illustrated in Figure 4c). 488 This observation validates the protective role of the VapB4 antitoxin against VapC's action on 489 substrates. Additionally, it confirms that only the VapC4 toxin is responsible for RNase 490 activity, ruling out the involvement of any other potential RNase from E. coli. 491 An RNase assay was conducted to determine the optimal pH conditions under which the 492 VapC4 toxin exhibits its highest efficacy. The VapC4 protein was subjected to incubation with 493 total RNA from S. acidocaldarius at different pH values, all maintained at a temperature of 494 60℃. As expected, VapC4 demonstrated enhanced RNA hydrolysis efficiency across the pH 495 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint range of 6 to 8 ( Figure 4 d). This observation aligns with expectations, considering the 496 internal pH of S. acidocaldarius is around 6.5 [39]. 497 VapC4 toxin can hydrolyze mRNA and rRNA in vitro 498 A prior study proposed that VapC1 and VapC2 derived from non -typeable Haemophilus 499 influenzae (NTHi) can inhibit translation by cleaving initiator tRNA fMet, thereby inducing the 500 formation of dormant cells [ 40]. However, VapC6 from S. solfataricus has been shown to 501 cleave dppB-1, tetR, and vapB6 mRNA [18]. Given that VapC exhibits varying RNA substrate 502 specificity depending on the organism, and with the confirmed RNAse activity of the VapC4 503 toxin, we wanted to identify its specific substrate. We synthesized different class es of RNA 504 substrates from S. acidocaldarius and evaluated the VapC4 RNase activity on these 505 transcribed RNA molecules to determine the substrate specificity ( Figure 5a). To this end, 506 the purified VapC4 toxin was incubated with various in -vitro transcribed RNAs –7S SRP 507 RNA, tRNA Met, 16S rRNA, 23S rRNA, and TetR mRNA - at 60℃. Notably, the VapC4 508 cleaved the rRNA and mRNA, while it did not hydrolyse the 7S SRP RNA and tRNA Met 509 (Figure 5b-f). Interestingly, while SRP RNA and tRNAMet remained unaffected, the complete 510 degradation of 23S rRNA and TetR mRNA required 10 mins, whereas 16S rRNA took 20 511 mins for complete breakdown ( Figure 5 b-f). Hence, the VapC4 enzyme from S. 512 acidocaldarius can degrade mRNA and rRNA in vitro . It can be speculated that the 513 significance of mRNA hydrolysis lies in the direct inhibition of translation, while its rRNA 514 hydrolysis indirectly inhibits translation. As published previously, the cleavage of 16S rRNA 515 causes a separation of the anti -Shine-Dalgarno sequence from the ribosome [ 41]. 516 Consequently, the ribosome's ability to scan the initiation codon of the mRNA is impaired, 517 leading to a subsequent hindrance in protein translation. On the other hand, the cleavage of 518 the 23S rRNA triggers the disassembly of the 50S and 30S ribosomal subunits, thereby 519 compromising the entire translation apparatus [42]. This suggested that VapC4 might serve as 520 a dual substrate-specific RNase, targeting both mRNA and rRNA for degradation, ultimately 521 leading to the inhibition of translation. Previous studies on Metallosphaera VapCs 522 demonstrated that VapC4 specifically recognized a GAAG consensus motif, whereas VapC7 523 and VapC8 targeted the degenerate consensus motifs (A/U)AG(G)A and (U/G)AAU, 524 respectively, within the 16S and 23S rRNA [24]. 525 VapB4 antitoxin interacts with VapC4 toxin to inhibit its effect 526 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint To ascertain whether the inhibition caused by the VapB4 antitoxin occurs through the binding 527 of the antitoxin to the VapC4 toxin rather than the toxin's target (RNA), we performed toxin 528 pull-down experiment. To that end, both the VapC4 toxin with a His -tag and the VapB4 529 antitoxin with a StrepII tag were expressed in E. coli . These lysates were subjected to Ni -530 NTA affinity chromatography, which resulted in the elution of both proteins combined within 531 a single (elution 2) fraction ( Figure 6a). The size of the eluted proteins corresponded to the 532 migration pattern of both the VapC4 toxin and the VapB4 antitoxin. This co -purification 533 strongly indicates the formation of a complex between the toxin and antitoxin within the 534 cellular environment. This complex formation further suggests that the antitoxin hinders the 535 toxin's activity by strongly binding to the toxin’s active si te, fulfilling the type II TA system 536 criterion. A control set was also prepared using cell lysate from E. coli cells that expressed 537 only VapB4 antitoxin with a StrepII tag. When this lysate was subjected to Ni -NTA affinity 538 chromatography, no band corresponding to the VapB4 antitoxin was observed in the elution 2 539 fraction ( Figure S3). Verification through Western Blot analysis, using anti -His antibodies 540 and anti-Strep-II antibodies, confirmed that the observed protein bands indeed corresponded 541 to the VapC4 toxin and VapB4 antitoxin, respectively (Figure 6b). 542 To gain a quantitative understanding of the interaction between the VapC4 toxin and VapB4 543 antitoxin, a Fluorescent Resonance Energy Transfer (FRET) analysis was conducted. In this 544 experiment, the VapB4 antitoxin was labeled with the fluorescent dye Alexa -532 (TRITC), 545 while the VapC4 toxin was labeled with the fluorescent dye Alexa -488 (FITC) ( Figure 6c). 546 As the concentration of the VapB4 antitoxin was raised, an increase in energy transfer was 547 detected. The dataset was subjected to fitting using the binding equation outlined in the 548

section ( Figure 6d). The resulting dissociation constant (K d) derived 549 from the experiment was determined to be 40 ± 2 nM, highlighting a robust interaction 550 between the VapC4 toxin and VapB4 antitoxin. 551 VapC4 toxin enables the cells to withstand heat stress 552 The maximal upregulation of the vapBC4 operon during heat stress suggested an important 553 role in heat stress adaptation [ 15]. In the present study, we generated the S. acidocaldarius 554 deletion strains ΔvapC4, and ΔvapBC4 and subjected them to heat stress at 85℃ for different 555 time intervals and then determined the viability of the cells for each time point using an MTT 556 assay. Deletion strains Δ vapC4 and ΔvapBC4 showed no difference in growth at 75℃ 557 compared to the WT (Figure 7a). However, the vapC4 toxin complementation in the vapBC4 558 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint

strain led to a slower growth probably due to the toxic nature of VapC4 ( Figure 559 7a). 560 Furthermore, upon subjecting cells in the mid -log phase (OD 600 0.5) to heat stress at 85℃, a 561 noticeable growth impairment was evident in the ∆vapC4 and ∆vapBC4 strains, characterized 562 by an inability to divide compared to cells cultivated at the optimal growth temperature of 563 75℃ ( Figure 7b -c). Notably, despite exhibiting less efficient growth under heat stress 564 conditions compared to their optimal growth environment, both the wild -type (WT) and 565 ∆vapBC4 + vapC4 complement ed cells showed resilience to heat shock ( Figure 7b-c). As 566 OD values can include readings from both living and dead cells, we sought to specifically 567 assess the quantity of viable cells following heat stress. To achieve this, we conducted an 568 MTT assay for a period of one hour of heat stress. Using the MTT assay, we found that the 569 ΔvapC4 cells could not withstand heat stress (HS) compared to WT cells, and the Δ vapC4 570 complemented with vapC4 ( Figure 7d). The same decrease in the percentage survivability 571 was also observed for Δ vapBC4 cells ( Figure 7 e). Since we were unable to generate a 572 ΔvapB4 strain, we tried to complement the vapC4 gene into Δ vapBC4 background 573 (ΔvapBC4+vapC4) to check whether the toxic effect of VapC4 is indeed responsible for 574 rescuing the cells during heat stress. As expected, Δ vapBC4+vapC4 cells were resilient to 575 heat stress at 85℃ ( Figure 7 e). Therefore, our findings suggest that VapC4 might help S. 576 acidocaldarius form persister-like cells that can withstand heat stress. 577 The vapC4 toxin gene deletion aggravates biofilm formation. 578 Biofilm formation is a common survival strategy adopted by different bacteria and archaea in 579 response to environmental stresses [ 43-45]. Biofilms offer protection against adverse 580 environmental conditions, enhance nutrient availability, and facilitate genetic exchange [ 46]. 581 Understanding the relationship between VapBC4 and biofilm formation would show how this 582 TA system contributes to stress resistance and survival strategies in these thermoacidophiles. 583 We conducted a biofilm formation assay using the crystal violet staining method with the S. 584 acidocaldarius strains cultivated in 24 -well microtiter plates for 48 hours. It was observed 585 that the Δ vapC4 cells exhibited increased biofilm formation compared to WT and 586 ΔvapC4+vapC4 complementation strains ( Figure 8a ). A significant increase in biofilm 587 formation was also observed for Δ vapBC4 cells compared to WT and ΔvapBC4+vapBC4 588 complementation (Figure 8b). Further complementation of the vapC4 gene into the ΔvapBC4 589

led to decreased biofilm formation ( Figure 8b). Hence, this study reveal ed that 590 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint the VapC4 toxin plays an inhibitory role in forming biofilms. In a previous study, a 591 comparable phenomenon of biofilm overproduction was noted in both the ΔvapC14 and 592 ΔvapBC14 strains, where it was hypothesized that the VapC14 (Saci_2183) toxin targets 593 crucial biofilm -related RNAs, including the transcript of the recognized Lrs14 -like biofilm 594 activator (saci_1223) in S. acidocaldarius [25, 47]. We also investigated the impact of heat 595 stress for various time spans (15 mins, 30 mins, 45 mins, and 60 mins) on the biofilm 596 formation of S. acidocaldarius . It was observed that ΔvapC4 cells exhibited an increased 597 tendency to form biofilm when subjected to a minimum of 30 mins of initial heat stress 598 compared to WT and ΔvapC4+vapC4 complementation strains (Figure 8c) . Similarly, a 599 significant increase in biofilm formation was observed for ΔvapBC4 cells when exposed to a 600 minimum of 30 mins of initial heat stress (Figure 8d). Moreover, a decreased tendency of 601 biofilm production was seen in the heat -stressed ΔvapBC4 strain complemented with vapC4. 602 All mutant strains and complemented strains utilized underwent PCR verification to confirm 603 the presence or absence of the relevant genes ( Figure S4). From th ese observations, we can 604 speculate that heat stress -induced VapC4 -mediated persister cells may not preferentially 605 adopt a biofilm lifestyle as a coping strategy to heat stress, whereas heat stress -induced 606 VapC4-deficient cells show an increased tendency to form biofilm as a coping mechanism to 607 thermal stress. 608 609 4. DISCUSSION 610 The presence of a large number o f vapBC toxin-antitoxin (TA) systems within the group of 611 thermoacidocphilic archaea, including S. acidocaldarius, implies a possible role in adapting 612 to their specific environments. Because bacterial TA systems are involved in programmed cell 613 death or persister cell formation during stress conditions [2], we speculated that depending on 614 the stress conditions, S. acidocaldarius might activate or deactivate specific vapBC systems 615 to determine the fate of the cells. In our earlier research, we have demonstrated how the 616 vapBC system plays a part in adapting to various stresses. We observed distinct sets of vapBC 617 TA systems being upregulated in response to different stressors such as heat, oxidative 618 conditions, and nutrient deprivation [15]. 619 In this study, we showed that the heterologous expression of VapC4 in E. coli induced 620 bacteriostasis, suggesting that the toxic influence of VapC4 extends beyond the archaeal 621 domain. We demonstrated that VapC4 functions as a high-temperature catalyzed ribonuclease, 622 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint validating the existence of a PIN domain fold. The ribonucleolytic activity of VapC4 might be 623 involved in the inhibition of translation, which resulted in the repression of bacterial growth 624 during its overexpression . Additionally, we have demonstrated that the VapC4 toxin 625 hydrolyzes both rRNA and mRNA effectively in vitro. Further studies need to be performed 626 to ascertain whether the hydrolysis by VapC4 in S. acidocaldarius is specific to the RNA 627 sequence or structural folds. Also, subsequent in -vivo studies are required to ascertain 628 whether ribosomes have any inhibitory role on rRNA cleavage by VapC4. The toxic activity 629 of the VapC4 toxin is alleviated by its interaction with the cognate VapB4 antitoxin, resulting 630 in the formation of a stable VapBC4 toxin -antitoxin complex- a crucial criterion for type II 631 TA systems. This interaction might impede the access of the RNA substrate to the active site 632 of VapC, as previously reported [ 16, 48]. Remarkably, we have also demonstrated the 633 involvement of the vapC4 toxin in triggering persister-like cell formation in S. acidocaldarius 634 during heat stress. This VapC4 toxin -induced susceptibility to heat stress at 85℃ in S. 635 acidocaldarius can be attributed to the persister cell formation, where the cell enters a 636 dormant state, allowing them to temporarily withstand and survive the challenging conditions 637 posed by elevated temperatures, ensuring that the cells do not die. Persister cells in bacterial 638 populations are characterized by their capacity to withstand challenging conditions, including 639 heat stress, antibiotic exposure, and various environmental stressors [43 -45]. These cells 640 possess a unique capability to enter a dormant state, rendering them temporarily resistant or 641 tolerant to external stresses that would generally eliminate actively growing cells [46]. The 642 mechanisms underlying persister cell formation can involve various genetic, metabolic, and 643 environmental factors contributing to their resilience in unfavorable conditions. Numerous 644 studies have documented the induction of persister cell formation in bacteria through the TA 645 system, enabling these cells to endure various stresses encountered in their natural 646 environment [47 -49]. These persister cells exhibit metabolic activity while inhibiting cell 647 division, a strategic adaptation to cope with the imposed stress. The inhibition of translation 648 by the VapC4 toxin through its ribonucleolytic activity during heat stress might be a cellular 649 strategy aimed at conserving energy and resources while adapting to challenging conditions. 650 Heat stress can lead to protein misfolding and denaturation, posing a significant threat to 651 cellular functions [ 31, 49, 50]. By strategically inhibiting translation, the cell tries to 652 minimize the production of new proteins, reducing the load on the cellular machinery. This 653 reduction in protein synthesis serves several purposes. Firstly, it alleviates the burden on the 654 cellular ribosomes, which may be compromised or overburdened under heat stress conditions. 655 Secondly, limiting the production of new proteins helps prevent the accumulation of 656 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint misfolded or damaged proteins, which could contribute to cellular dysfunction. Entering a 657 persister state through translation inhibition might allow the cell to redirect its resources 658 toward stress response mechanisms, such as chaperone proteins that aid in protein folding and 659 repair [31, 49, 50]. This adaptive response helps the cell to better cope with the heat stress, 660 promoting survival until more favorable conditions are restored. The proposed mechanism of 661 persister cell formation by VapC4 toxin is illustrated as a model in ( Figure 9 ). However, 662 additional experiments are necessary to clarify the underlying physiology of this 663 phenomenon. The persister cell formation has also been documented in Haloferax volcanii in 664 response to starvation or exposure to lethal concentrations of different biocidal compounds 665 [51]. On the contrary, VapC4 was identified as a negative regulator of biofilm formation, 666 wherein the absence of the VapC4 toxin increased biofilm formation in S. acidocaldarius . 667 Therefore, the free cellular VapC4 toxin during heat stress could cue the cell to opt for 668 persister cell formation instead of biofilm formation. Persister cells exhibit temporary 669 resistance to stressors without needing structural adaptations, allowing the cell to endure 670 unfavorable conditions without forming complex structures like biofilms [52]. 671 However, limitations exist within this study, including the lack of structural determination for 672 VapC4 and the VapBC4 complex and the identification of the RNA cleavage site . In the 673 future, it would be also interesting to examine whether VapC5 affects transcripts related to 674 biofilm formation. Acknowledging these constraints, one could potentially unveil th e 675 underlying molecular mechanisms governing RNA substrate recognition and the binding 676 dynamics with its corresponding antitoxin. Additional investigations are necessary to 677 decipher the metabolic status and functionally characterize the persister cells of S. 678 acidocaldarius to obtain a comprehensive understanding. 679 680 Author contribution 681 A.G. and A.B. designed the study; A.B., A.R., C.B., J.D., and U.R.C. performed the 682 experiments. A.G., A.B., A.R., and S.V .A. analyzed the data. A.G. and A.B. drafted the 683 manuscript with inputs from all the authors. All the authors approved the submitted version. 684 Declaration of competing interest 685 The authors declare that they have no conflict of interest with the contents of this title. 686 687 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint 688 Acknowledgment 689 The authors express gratitude for the intramural funding received from Bose Institute, India. 690 AB acknowledges the fellowship support from CSIR (Council of Scientific and Industrial 691 Research), Government of India, under File No. 09/015(0525)/2017 -EMR-I. The authors 692 extend their appreciation to EMBO (European Molecular Biology Organization) for granting 693 AB the Scientific Exchange Grant (SEG 9915), enabling a 3 -month visit to Prof. Dr. Sonja - 694 Verena Albers' laboratory at the University of Freiburg, Germany. ACR was supported by a 695 Life Grant (AZ96727) by the VW Foundation awarded to SV A. 696 697 698 699 700 5. REFERENCES 701 1. Hayes, F. and L. Van Melderen, Toxins-antitoxins: diversity, evolution and function. 702 Crit Rev Biochem Mol Biol, 2011. 46(5): p. 386-408. 703 2. Jain, S., et al., Unravelling the physiological roles of mazEF toxin-antitoxin system on 704 clinical MRSA strain by CRISPR RNA -guided cytidine deaminase. J Biomed Sci, 705 2022. 29(1): p. 28. 706 3. Gerdes, K., S.K. Christensen, and A. Lobner -Olesen, Prokaryotic toxin -antitoxin 707 stress response loci. Nat Rev Microbiol, 2005. 3(5): p. 371-82. 708 4. Chan, W.T., M. Espinosa, and C.C. 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NPJ Biofilms Microbiomes, 818 2021. 7(1): p. 80. 819 820 821 822 Figure legend 823 Figure 1: Genomic organization of vapBC4 Toxin-antitoxin gene locus in Sulfolobus 824 acidocaldarius. a) Schematic representation of the genes saci_1812 (VapB4 antitoxin) and 825 saci_1813 (VapC4 toxin). The stop codon (TGA) of the vapB4 gene (marked in red) overlaps 826 the start codon (TTG) of the vapC4 gene (underlined). b) Agarose gel showing PCR 827 amplification of the respective region of the operon marked as 1, 2 and 3 in a), using cDNA 828 as template (1 - intermediate region of the saci_1812 ( vapB4) antitoxin and saci_1813 829 (vapC4) toxin gene; 2- region of the saci_1812 (vapB4) antitoxin; 3- region of the saci_1813 830 (vapC4) toxin. Negative control with no reverse transcriptase was used to verify that RNA 831 samples do not contain gDNA contamination. In the positive control, gDNA was used as a 832 template to amplify the intermediate portion of the saci_1812 and saci_1813 gene marked as 833 “1”. 834 Figure 2: The heterologous expression of VapC4 toxin (Saci_1813) inhibits growth in -835 vivo leading to persister cells (a) E. coli streaked M9 minimal media plates expressing 836 VapC4 toxin, VapB4 antitoxin, empty pET28a vector, VapC4 toxin + VapB4 antitoxin and 837 VapC4 toxin + empty pETDuet1 vector (b) Growth profiles of E. coli CBL21(DE3) cells 838 expressing the empty pET28a vector, VapC4 toxin, VapB4 antitoxin, both VapC4 toxin + 839 VapB4 antitoxin as well as VapC4 toxin + empty pETDuet1 vector in M9 minimal media 840 containing 0.5mM IPTG. 841 c) Bar graph showing the viability of cells expressing empty pET28a vector and VapC4 toxin 842 with increasing time interval. Experiments were performed in triplicates . The statistical 843 significance, P < 0.05, is indicated as *, P < 0.01 is indicated as **, and P < 0.001 is indicated 844 as ***; n.s.denotes not significant. 845 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint Figure 3: Purification of VapC4 toxin and vapB4 antitoxin a) Growth profile of E.coli 846 BL21(DE3)-RIL cells expressing VapC4 toxin in the presence of different IPTG 847 concentrations at 16 ℃ induction temperature. b) A 15% SDS PAGE shows the successful 848 purification of VapC4 toxin using Ni 2+-NTA affinity chromatography around 16 kDa. c) A 849 15% SDS PAGE shows successful purification of VapB4 antitoxin using Ni 2+-NTA affinity 850 chromatography around 9 kDa. 851 Figure 4: Ribonucleolytic Activity of VapC4 Toxin. 1% agarose gel demonstrating RNase 852 activity of VapC4 toxin at two distinct temperatures: a) 37℃ and b) 60℃ on S. 853 acidocaldarius total RNA. c) 1% agarose gel illustrating the inhibitory effect of VapB4 854 antitoxin on VapC4 toxin at two different temperatures, 37℃ and 60℃. d) Agarose gel 855 displaying RNase activity of VapC4 at different pH at 60℃. 856 Figure 5 : RNA specificity of VapC4 Toxin. a) Schematic showing different classes of 857 single-stranded RNA substrate generation by In -vitro- transcription (IVT) using pGEM3z 858 vector (PSP6 and P T7 denotes SP6 and T7 promoter respectively). The RNase activity of the 859 VapC4 toxin was examined at 60℃ using various classes of RNA substrates from S. 860 acidocaldarius, prepared through In -vitro transcription (IVT) reactions: b) 16S rRNA c) 23S 861 rRNA d) TetR mRNA e) 7S SRP RNA, and f) tRNAMet. 862 Figure 6 : Interaction between VapC4 toxin and VapB4 antitoxin a) 15% SDS -PAGE 863 showing co-elution of VapC4 toxin (at 16 kDa position) along with VapB4 antitoxin (at 9 kDa 864 position) in the Elution 2 fraction. b) Western Blot analysis with anti -His and anti -Strep-II 865 antibodies confirmed the presence of VapC4 toxin and VapB4 antitoxin, respectively. c) 866 VapC4 toxin tagged with Alexa -488 (FITC) was titrated with increasing concentration of 867 VapB4 antitoxin tagged with Alexa -532 (TRITC), resulting in FRET between TRITC 868 (acceptor) and FITC (donor). d ) Quantification of the binding of VapB4 antitoxin to VapC4 869 toxin using the equation outlined in the Material and Methods section revealed a dissociation 870 constant (Kd) of 40 ± 2.2 nM, indicating a strong and reliable binding affinity. 871 Figure 7. Impact of VapC4 Toxin on Heat Stress Survival a) Growth curve analysis of all 872 the Knock-out strains and vapBC4 + vapC4 at 75 ℃ in Brock medium at pH 3, supplemented 873 with 0.1% N -Z-amine,0.2% sucrose and 10 µg/ml uracil (if required). b) Growth curve 874 analysis of all the Knock -out strains and vapBC4 + vapC4 complementation strains at 75 ℃ 875 (optimum growth condition) from a starting OD600 0.5 measured for 6 hours. c) Growth curve 876 analysis of all the Knock-out strains and vapBC4 + vapC4 complementation cell at 85℃ (HS-877 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint heat stress) from a starting OD 600 0.5 measured for total duration of 6 hours. d) Graph 878 showing percentage survivability of ΔvapC4 cells vs. WT during HS at 85 ℃ at different time 879 intervals: Control (5 mins before HS), 15 mins HS, 30 mins HS, 45 mins HS, and 60 mins 880 HS. The ΔvapC4 + vapC4 indicates the vapC4 complementation strain e) Graph showing 881 percentage survivability of ΔvapBC4 cells vs. WT and ΔvapBC4 + vapC4 vs. WT during HS 882 at 85 ℃ at different time intervals: Control (5 mins before HS), 15 mins HS, 30 mins HS, 45 883 mins HS and 60 mins HS. The ΔvapBC4 + vapBC4 indicates the vapBC4 complementation 884 strain. 885 Figure 8. Impact of VapC4 toxin on biofilm formation a) Box plot depicting normalised 886 biofilm formation (OD 570/OD600) for ΔvapC4 cells vs. WT over two days. b ) Box plot 887 depicting normalised biofilm formation (OD 570/OD600) for ΔvapBC4 cells vs. WT and for 888 ΔvapBC4 + vapC4 cells vs. WT over two days. c) Box plot showing normalised biofilm 889 formation (OD 570/OD600) for heat stress induced ΔvapC4 cells vs. WT after varying time 890 points of heat stress over 2 days. d) Box Plot showing normalised biofilm formation 891 (OD570/OD600) for heat stress induced ΔvapBC4 cells vs. WT and ΔvapBC4 + vapC4 cells vs. 892 WT after varying time points of heat stress over 2 days. The statistical significance, P < 0.05, 893 is indicated as *, P < 0.01 is indicated as ** , and P < 0.001 is indicated as ***; n.s. denotes 894 not significant. 895 Figure 9. Proposed model showing the mode of action of the VapBC4 TA system during 896 heat stress. Under the usual conditions, the ribonucleolytic action of the VapC4 toxin is 897 blocked by the tight binding of VapB4 antitoxin, forming a stable VapBC4 TA complex 898 allowing the cells to grow and divide normally. During heat stress, activated stress -induced 899 protease (not yet known for archaea) might selectively cleave the Vap B4 antitoxin. The 900 unleashed VapC4 toxin, under stress conditions, can inhibit translation through two 901 mechanisms: it can either directly hinder translation by cleaving mRNAs, or it can indirectly 902 impede translation by cleaving 23S and 16S rRNAs, thereby destabilizing the interaction 903 between the 50S and 30S ribosomal subunits (not yet known for archaea). Inhibition of 904 translation serves as a vital mechanism for persister cell formation in response to stress 905 conditions. This process is a strategic response to conserve energy and prevent the synthesis 906 of potentially misfolded proteins under stress. The temporary halt in translation might 907 redirect resources to stress response mechanisms, aiding in protein folding and repair. 908 Persister cells, formed through this adaptation, enter a metabolically active but dormant state, 909 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 6, 2024. ; https://doi.org/10.1101/2024.06.06.597757doi: bioRxiv preprint allowing them to withstand challenging conditions until a more favorable environment is 910 restored. 911 912 913 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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