Page 1 of 16 ACS Synthetic Biology Tunable protein pistons 1 2 3 4 5 1 A tunable protein piston that breaks membranes to release 6 7 2 encapsulated cargo 83 9 10 4 Jessica K. Polka1,2 and Pamela A. Silver*1,2 11 12 5 1. Department of Systems Biology, Harvard Medical School, Boston MA 02115 13 6 2. Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115 14 15 7 Running title: Tunable protein pistons 16 8 17 9 * To whom correspondence should be addressed: pamela_silver@hms.harvard.edu. 200 Longwood 18 10 Avenue, Warren Alpert Building, Boston, MA 02115 19 20 11 21 22 23 12 24 25 13 ABSTRACT 26 27 28 14 Movement of molecules across membranes in response to a stimulus is a key component of cellular 15 programming. Here, we characterize and manipulate the response of a protein-based piston capable of 29 16 puncturing membranes in a pH-dependent manner. Our protein actuator consists of modified R bodies 30 17 found in a bacterial endosymbiont of paramecium. We express and purify R bodies from in E. coli; these 31 18 pistons undergo multiple rounds of rapid extension and retraction. We developed a high throughput 32 19 screen for mutants with altered pH sensitivity for tuning of the extension process. We show that the R 33 20 bodies are capable of acting as synthetic pH-dependent pistons that can puncture E. coli membranes to 34 35 21 release the trapped content. As such, these protein machines present a novel way to selectively rupture 36 22 membrane compartments and will be important for programming cellular compartmentalization. 37 38 39 23 Keywords: Bioengineering, biotechnology, protein assembly, conformational change, refractile bodies, 24 Kappa particles 40 25 41 42 43 26 INTRODUCTION 44 27 R bodies (Type 51 refractile bodies) are ribbon-like protein polymers that are naturally expressed in the 45 28 cytoplasm of Caedibacter taeniospiralis, an endosymbiont of “killer” strains of Paramecium tetraurelia 46 47 48 29 (reviewed in 1). These bacteria, also called kappa particles, confer to their host the ability to kill other 30 strains of Paramecium. This killing is dependent on ingestion of the R body-containing bacteria 2 that are 49 31 shed into the environment by the killer strain 3,4. Inside the food vacuole of the non-killer paramecium, 50 32 acidic conditions cause the R body to unroll from a coil 500nm in diameter to form a tube 165nm in 51 33 diameter and up to 20µm long (Figure 1A-C). This extension deforms and punctures the membrane of 52 34 the food vacuole, mixing contents of the bacteria with the paramecium’s cytoplasm 2,5. The subsequent 53 54 35 death of the paramecium presumably results from the release of unidentified toxins from the bacteria 55 56 36 into the cytoplasm, as killing does not occur when sensitive strains are fed purified R bodies or E. coli 37 expressing R bodies 6–8. Thus, R bodies themselves are not lethal, but rather they have been proposed to 57 58 59 60 1 ACS Paragon Plus Environment ACS Synthetic Biology Page 2 of 16 Tunable protein pistons 1 2 3 38 act as delivery devices for other molecules (Figure 1A). As such, we speculated that the natural behavior 4 5 39 of R bodies could be used as a tunable device for cell engineering applications. 6 40 Type 51 R bodies are natural molecular machines that switch between extended and retracted forms in 7 8 9 41 response to changes in pH; they can revert to their contracted form when the pH is raised 9. 42 Additionally, they resist harsh conditions including salt, detergents, and heat 10. These structures can be 10 43 expressed in E. coli 7,10,8 from an operon of four open reading frames, rebA-D, two of which (RebA and 11 44 RebB) are major structural proteins 11. The process of R body extension is a simple, brute-force solution 12 45 to the challenge of membrane disruption, and it therefore holds promise as a research or clinical tool for 13 46 selective release or penetration of membrane compartments in a programmable manner. 14 15 47 Here, we develop pH tunable R bodies as protein-based devices for the synthetic biology tool box. As 16 48 such, we show that R bodies can undergo many sequential cycles of extension and contraction in vitro. 17 18 19 49 We also describe a simple assay that enables quantitation of the pH response of R bodies and its 50 application in a screen for mutant R bodies that switch conformations at lower pH. Informed by this 20 51 screen, we designed additional mutants that switch conformations at higher pH, thereby making them 21 52 useful for a diversity of cell-based designs as well as in vitro applications. Finally, we demonstrate that R 22 53 bodies can release cytoplasmic contents from E. coli by rupturing the cell membrane. 23 24 25 54 RESULTS 26 55 Production of functional R bodies in E. coli 27 56 We expressed the reb locus 12 in E. coli cells and purified R bodies based on their ability to sediment. R 28 57 bodies from our E. coli expression system (Methods) behave as predicted 7,9: at high pH, they resemble 29 58 coils of ribbon (Figure 1B) by negative stain transmission electron microscopy. At low pH, they instead 30 59 form extended, hollow tubes with pointed ends (Figure 1C). 31 32 60 R body extension results in secondary structure changes and a macroscopic phenotype 33 61 R bodies display two distinct circular dichroism spectra at high and low pH (Figure 1D). Analysis of this 34 35 62 data suggests that R bodies are dominated by helical secondary structure, slightly more so at low pH 36 63 than at high pH (Figure 1E). We also found that R body solubility depends on their extension state. R 37 64 bodies in the contracted, high pH state will sediment after several hours at room temperature, while 38 65 those in the extended, low pH state remain in solution (Figure 1B and C). This difference can be rapidly 39 66 appreciated with the naked eye in tubes or in 96-well plates. 40 41 67 R bodies can undergo many pH dependent cycles of extension and contraction 42 68 R bodies are capable of undergoing many dozens of cycles of extension and contraction without any 43 69 apparent loss of function. Using phase contrast microscopy and flow cells that permitted on-stage buffer 44 45 70 changes, we observed that single R bodies undergo multiple cycles of extension and contraction in 46 71 response to pH modulation (Figure 1F). A full-frame movie of this process is provided as Supplemental 47 72 Movie 1. 48 49 73 To further probe the limits of this reversibility, we sequentially altered the pH of a solution of R bodies 50 74 for 120 cycles by adding small volumes of HCl or KOH in the absence of buffer (Figure 1G), reserving 51 75 aliquots of R bodies at each step to confirm their extension state. We found that the first and last 52 76 aliquots showed no detectable difference in their ability to respond to either high or low pH (pH 7.0 and 53 77 5.0, respectively) as measured by phase contrast microscopy (Figure 1H). Thus, R body function remains 54 78 robust over many activity cycles. 55 56 57 58 59 60 2 ACS Paragon Plus Environment Page 3 of 16 ACS Synthetic Biology Tunable protein pistons 1 2 3 79 Measuring R body extension in varied ionic strength solutions with a high-throughput assay 4 5 80 We used the differential solubility of extended and contracted R bodies described above to develop a 6 81 rapid assay for R body extension state that is amenable to quantification. In a plate reader, the pellets 7 82 formed by contracted R bodies create a higher absorbance value than the same concentration of R 8 83 bodies in solution. This difference can be enhanced by programming the plate reader to agitate the 9 84 plate before the reading; this reproducibly focuses the material toward the center of the well (Figure 10 85 2A). In this fashion, R body state can be measured across many conditions in a short period of time. The 11 12 13 86 assay is subject to drift over time as R bodies settle; therefore we waited at least 4 hours before reading 87 the plates and kept the plate covered to reduce edge effects due to evaporation during this time. We 14 88 first used this method to confirm that the sequential pH changes described above indeed cause R bodies 15 89 to change state (Figure 1G). 16 17 90 Using this assay, we studied the impact of ionic strength on R body extension state. At high salt 18 91 concentrations, the highest pH at which R bodies are completely extended is approximately 5.4. 19 92 Meanwhile, at very low ionic strength, R bodies remain completely extended above pH 6.2. These data 20 93 also reveal that within any given series, absorbance increases with pH, creating a sigmoidal curve with a 21 94 median value that we refer to as the conversion pH (Figure 2B). 0M KCl was excluded from subsequent 22 23 95 as it does not have a conversion pH; all 0M wells lacked a visible pellet (Figure 2A). For the remaining 24 96 conditions, we interpolated the pH value at 50% maximum absorbance for each individual trace. These 25 97 values were used as inputs in a one-way ANOVA with Tukey-Kramer test, finding statistically significant 26 98 differences between 0.063M KCl and 0.25M-2M KCl. We verified that the observed transfer curve 27 99 reflects actual morphological changes in R bodies by visualizing their state with phase contrast 28 100 microscopy (Figure 2C). 29 30 101 A screen for R bodies that extend at variable pHs identifies changes in a region of RebA 31 102 We adapted our plate-based assay as a screening tool for R bodies with differential pH responses. We 32 103 amplified a region spanning the rebA and rebB open reading frames with error-prone PCR and cloned 33 34 104 this region into a plasmid backbone containing an unmodified copy of the remainder of the reb operon, 35 105 which includes rebC and rebD (Figure 3A). Single colonies resulting from a transformation of this library 36 106 into C43 E. coli cells were then selected for growth in a 96-well plate format. R bodies were expressed 37 107 and purified in this format, then transferred to an optically-clear plate in buffer at pH 5.5. Under these 38 108 conditions, wild type R bodies will remain soluble, but mutants that require a lower pH to extend will 39 109 sediment (Figure 3A, right panel). We therefore visually selected wells with a dense, visible pellet (like 40 41 42 110 111 those highlighted in Figure 3B) as putative hits. These were subsequently confirmed by pH titration (Figure 3C) and classified according to their approximate conversion pH. 43 112 Out of a library of 1728 clones, we identified 60 isolates defective in pH response (representative 44 45 113 isolates shown in figure 3C, sequences shown in Supplemental figures 1 and 2). Of these, 13 did not 46 114 extend below pH 3.0 (Figure 3E), though mutants from this class resemble normally assembled R bodies 47 115 by negative stain electron microscopy (Figure 3D). These clones possessed a total of 16 unique 48 116 mutations, 9 of which fall into a 20 base pair region at the C-terminus of RebA. Five of these clones have 49 117 a single amino acid change in this region as their sole mutation. Taken together, these results provide 50 118 evidence that this region is a key controller of the extension process. 51 52 119 Rational design produces mutants with increased pH sensitivity 53 120 Three of the unique mutations in the C-terminal portion of RebA identified above were conversions to 54 55 121 proline. Because proline residues are known to destabilize alpha helices13, we constructed a series of 56 122 mutants with either individual residues or tracts of residues replaced with alanines or prolines (Figure 57 123 4A). We identified two alanine mutants that enable R bodies to extend at higher pH than wild type, with 58 59 60 3 ACS Paragon Plus Environment ACS Synthetic Biology Page 4 of 16 Tunable protein pistons 1 2 3 124 the more dramatic being RebA G90A (Figure 4B). We interpolated each trace’s pH value at 50% 4 5 125 maximum intensity and used these as inputs in unpaired two-sample t-tests assuming equal variance, 6 126 finding both Reb 1 vs Reb65 and Reb1 vs 221 to be significantly different. The increased conversion pH 7 127 phenotypes arealso evident by phase contrast microscopy (Figure 4C). 8 9 128 R bodies are capable of rupturing E. coli spheroplasts 10 129 R bodies can act as synthetic membrane-breaking devices. We constructed a plasmid encoding 11 130 functional fluorescent R bodies as well as a soluble fluorescent protein, mCherry (Methods). After 12 131 expressing this construct in E. coli, we treated cells with lysozyme and imaged them in flow chambers to 13 132 which we added low pH buffer that contained salts (methylamine hydrochloride and potassium 14 133 benzoate) shown to disrupt E. coli’s otherwise robust pH homeostasis 14 (Figure 5A). When spheroplasts 15 16 134 containing functional (wild type) R bodies were exposed to low pH buffer, 60% of the cells lysed (ie, lost 17 135 their m-Cherry fluorescence) 10 minutes after the addition of the low-pH buffer (Figure 5B). The loss of 18 136 fluorescence was often accompanied by dramatic protrusions of R bodies that distended cells (Figure 5E 19 137 and G, right side of Supplemental Movie 2). R bodies sometimes extended inside cells before the 20 138 membrane ruptured (Figure 5G, leftmost 4 cells), suggesting that extension causes lysis. By contrast, 21 139 when cells from the same culture were not treated with lysozyme, only 9.1% lysed. When cells were 22 23 140 treated with lysozyme, but thesalts to disrupt pH homeostasis were omitted, ( only 9.8% lysed. Finally, 24 141 when spheroplasts containing R bodies with the RebA S88P mutation, which are incapable of extension, 25 142 were treated with both salts and lysozyme, only 11.5% lysed. (Figure 5B-F, left side of Supplemental 26 143 Movie 2). Based on a one-way ANOVA with Tukey-Kramer test, cells containing functional R bodies 27 144 treated with lysozyme and salts lyse significantly more frequently than any of the control conditions. 28 145 However, none of the controls are significantly different from one another. Thus, we conclude that R 29 146 bodies lyse cells by extension and membrane disruption at low pH. 30 31 32 147 DISCUSSION 33 148 R bodies as reversible protein machines 34 149 Our data show that type 51 R bodies are capable of multiple rounds of extension and contraction, 35 36 150 suggesting that all of the energy for their transformations comes from chemical changes in the buffer 37 151 instead of from other sources such as protein folding. Using these properties, we have demonstrated 38 152 that upon pH-activated extension, R bodies will puncture cell membranes causing release of the 39 153 contents. 40 41 154 Mechanism of action 42 155 We speculate that R body extension requires the formation or extension of a helix in RebA. Such 43 156 rearrangements would have precedent in the loop-to-helix transition that drives the pH-dependent 44 157 rearrangement of viral hemagglutinin 15. Analysis of our R body circular dichroism data (Figure 1D) 45 158 suggests a slightly greater contribution of helices to the low pH spectrum than to the high pH spectrum 46 47 159 (Figure 1E). Though this difference accounts for only 2% of the residues, these may bridge the small, 48 160 unstructured regions predicted in the C-terminal region of RebA (PSIPRED predictions shown in Figure 49 161 3E). Many of the mutations identified in the screen introduce proline residues, which can disrupt helices 50 162 13. Therefore, these mutations may prevent the pH-dependent formation of a helix in this C-terminal 51 163 region. Conversely, alanine residues can stabilize helices, so it is unsurprising that some of the rationally 52 164 designed alanine mutants we produced bias R bodies toward an extended conformation. 53 54 165 We have shown that when R bodies are in buffers of high ionic strength, a lower pH is required to 55 166 contract them than is needed at low ionic strength (Figure 2B and C). As K+ and Cl- fall relatively early in 56 57 167 the Hofmeister series, high concentrations of salt may function to increase surface tension and 58 59 60 4 ACS Paragon Plus Environment Page 5 of 16 ACS Synthetic Biology Tunable protein pistons 1 2 3 168 therefore strengthen interactions between hydrophobic residues. These hydrophobic interactions may 4 5 169 play a role in R body contraction. 6 170 Implications for bioengineering 7 8 171 Because R bodies are robust to buffer changes and capable of functioning in a cell-independent fashion, 9 172 they should be functional in a wide variety of biotechnology applications. We show that R bodies are pH- 10 173 dependent protein actuators and propose that upon extension can penetrate membranes to effect 11 174 release and delivery of trapped contents and thereby alter cell states or local conditions. Moreover, R 12 175 body activity is completely protein based and is therefore fast and reversible unlike devices that depend 13 176 on new transcription and translation. 14 15 177 The ability to engineer R body pH sensitivity expands their potential utility in a range of diverse contexts, 16 178 especially in delivering molecules across biological barriers. For example, in an application that parallels 17 179 their proposed natural function, R bodies could be used to enhance phagosomal delivery of DNA, RNA, 18 19 20 180 181 or other bioactive molecules. In particular, some cell types do not severely acidify the phagosomal compartment 16. In this case, R bodies engineered with increased pH sensitivity (such as the RebA G90A 21 182 mutant) would be advantageous. In addition, R bodies could be used as biological pistons or switches in 22 183 MEMS devices; the pH mutants could be used to build devices that assume many different states as a 23 184 function of pH. 24 25 185 Type 51 R bodies are just one among several described varieties of R bodies, all of which have been 26 186 reported to have varied properties (reviewed in 1). Given that genes encoding R body homologs have 27 187 recently been identified in a wide variety of bacteria 17, we will be able to produce actuators of different 28 29 188 sensitivities and strengths by harnessing this natural diversity. 30 31 189 METHODS 32 190 Cloning 33 191 The Reb locus 12 was Gibson assembled into pETM11 using gBlocks from Integrated DNA Technology 34 35 192 under the control of the vector’s T7 promotor. Site directed mutagenesis was performed with New 36 193 England Biolab’s Q5 kit. To screen for pH variant R bodies, the RebA and RebB coding sequences were 37 194 amplified with error-prone PCR (EP-PCR) using Taq polymerase in the presence of 62.5 and 125uM 38 195 MnCl2 to introduce errors. This product was then cloned into an unmutated backbone containing the 39 196 remainder of the reb operon. To make fluorescent R bodies, we fused mNeonGreen 18 to the N-terminus 40 197 of a second copy of RebB under the control of a weak RBS, BBa_B0033 from the Registry of Standard 41 42 198 Biological Parts. This was cloned downstream of an mCherry ORF with a strong RBS, which itself was 43 199 cloned downstream of the other ORFs in the operon. A list of constructs used in this study is available in 44 200 Table 1. 45 46 47 201 202 R body expression and purification Plasmids containing R bodies were transformed into C43 cells 19, a derivative of BL21 with attenuated T7 48 203 expression 20. Cells were grown in TPM to OD 600 0.2-0.6 and induced with 1mM IPTG. Expression 49 204 proceeded for 18 hours at 37° C. 50 51 205 To purify R bodies, cell pellets were flash-frozen in liquid nitrogen, then thawed. Cells were resuspended 52 206 in 25mM Tris pH 7.5, 100mM NaCl, and 2mM EDTA. Egg white lysozyme was added to a concentration of 53 207 approximately 17 µg/ml, and cells were incubated at 37° C for 1 hour. After this time, the buffer was 54 208 adjusted to contain 10mM MgCl2, 10mM CaCl2, and approximately 15 µg/ml DNAse from bovine 55 56 209 pancreas. Cells were once again incubated at 37° C for 20 minutes. Next, the buffer was adjusted to 57 210 contain 1% SDS. After manual mixing, cells were spun at 4,000 RPM in a tabletop centrifuge for 20 58 59 60 5 ACS Paragon Plus Environment ACS Synthetic Biology Page 6 of 16 Tunable protein pistons 1 2 3 211 minutes to pellet the R bodies. The R body pellet was then washed three times by resuspension in 4 5 212 water, followed by spins, as above. 6 213 R bodies were stored at 4° C or at -80° C after flash-freezing in liquid nitrogen in the presence of 25mM 7 8 214 Tris pH 7.5, 100mM KCl, and 15% glycerol. 9 215 Electron microscopy 10 11 216 200 mesh formvar and carbon-coated copper grids (Electron Microscopy Sciences) were glow discharged 12 217 for 30 seconds before the application of R bodies. These were washed by applying the grids sequentially 13 218 to three drops of buffer and three drops of either 0.75% uranyl formate or 1% uranyl acetate, wicking 14 219 with filter paper between each wash. Grids were visualized on either a JEOL 1200EX or a Tecnai G2 Spirit 15 220 BioTWIN. 16 17 221 Circular dichroism 18 222 R bodies at a concentration of 0.2mg/ml (calculated by Bradford) were washed into 0.1M sodium 19 223 phosphate buffer at pH 6.2 and 4.7. Data was collected on a JASCO J-815 Circular Dichroism 20 224 Spectropolarimeter. Data was analyzed with DichroWeb 21,22 predictions based on the CDSSTR method 21 22 225 23,24 and reference set 3 25. 23 226 Light microscopy 24 25 227 Flow cells used in spheroplast (see below) and kinetics experiments were constructed from a 22x22mm 26 228 coverslip adhered to a 22x60mm coverslip with Scotch double-sided tape. For kinetics experiments, 27 229 coverslips were heated at 50° for 4 hours in 1M HCl, then washed and sonicated in water, then ethanol 28 230 for 30 minutes each. Completed flow cells were then incubated for 5 minutes in 10µl of PBS containing 29 231 1mg/ml BSA (1/20th of which was labeled with biotin). After washing with PBS, cells were incubated with 30 232 25µg/ml streptavidin and washed again. At this point, wild type R bodies that had been nonspecifically 31 32 33 233 234 labeled with maleimide-biotin (15 minutes at room temperature with 1mM EZ-link maleimide-PEG2biotin, Thermo Fisher Scientific) were added and left to incubate for 5 minutes. The chambers were 34 235 washed with more PBS followed by 1mg/ml BSA/BSA-biotin solution. Finally, during imaging, pH was 35 236 changed by flowing 30-50µl of citric acid-Na2HPO4 buffer at either pH 5.2 or pH 6.2 through the flow cell. 36 37 237 Static images of purified R bodies were obtained from simple wet mounts on untreated coverslips and 38 238 slides. 39 40 239 All images were acquired on a Nikon TE2000 microscope equipped with a 100x phase objective, Perfect 41 240 Focus, and an Orca ER camera. Images were processed with Fiji. 42 43 241 Spectrophotometric assay 44 242 The absorbance of R bodies (100µl in each well of a 96-well plate) at 600nm or 590nm was read on a 45 243 Perkin Elmer Victor3V plate reader following 15 seconds of agitation by the plate reader. R bodies were 46 244 allowed to settle out of solution for at least 4 hours ahead of each reading, and wells were covered with 47 245 parafilm or plate lids to reduce evaporation during this time. 48 49 246 For statistical tests, the pH value at 50% maximum intensity was linearly interpolated from the two 50 247 nearest points for each individual trace. These were used as inputs for a t-test assuming equal variance 51 52 248 or one-way ANOVA with Tukey-Kramer tests. 26 53 249 Screen for mutants defective in pH response 54 55 56 250 251 The error-prone PCR-generated library described above (Cloning) was transformed into E. coli C43 cells, and single colonies were picked to 1ml of TPM in 96-well assay blocks. R bodies were expressed and 57 252 purified as described above. After washing R bodies in water sequentially, they were resuspended in 58 59 60 6 ACS Paragon Plus Environment Page 7 of 16 ACS Synthetic Biology Tunable protein pistons 1 2 3 253 250mM MES pH 5.5 and 250mM KCl. Hits were visually identified by the sedimentation of R bodies in 4 5 254 the respective well and confirmed by measuring the behavior against a pH series as in Figure 2C. 6 255 Sequence analysis 7 8 9 256 Sequences were aligned with Geneious version 8.1 (Biomatters Ltd). Secondary structure prediction was 257 done with PSIPRED 27,28. 10 11 258 Spheroplasting 12 259 Because R body expression takes several hours, we employed a spheroplasting method described 13 260 previously that works even in stationary phase 29. Briefly, cells expressing R bodies that had been 14 261 growing at 37° for 4-12 hours after induction were harvested and washed in 200mM Tris pH 8.0, then 15 262 resuspended in the same buffer. This was diluted 1:1 with 200mM Tris pH 8.0 containing 1M sucrose 16 263 and 1mM EDTA. 10ul of a solution containing 7 mg/ml lysozyme was added, and the mixture was 17 18 19 264 265 incubated at room temperature for 20 minutes. The buffer was adjusted to 20mM MgCl2 and cells were loaded into flow cells (see Microscopy). The flow cell was washed with buffer containing 200mM Tris pH 20 266 8.0, 0.5M sucrose, and 0.5mM EDTA prior to the start of imaging. The buffer was then swapped to 21 267 contain 100mM MES pH 4.9 with or without 40mM methylamine hydrochloride and 40mM potassium 22 268 benzoate, a combination that has been previously used to destroy E. coli’s pH homeostasis 14. 23 24 269 The experiment was repeated three times. To calculate fraction of lysed cells, the number of cells with 25 270 mCherry fluorescence in the resulting movies were tallied before and 10 minutes after adding the low- 26 271 pH buffer. The difference between the values represented cell lysis. The fractions of lysed cells were 27 272 used as inputs in a one-way ANOVA with Tukey-Kramer test.26 28 29 30 273 SUPPORTING INFORMATION 31 274 Supplemental Figure 1 - Sequences of RebA identified in screen for low-pH mutants 32 33 275 Supplemental Figure 2 - Sequences of RebB identified in screen for low-pH mutants 34 35 276 Supplemental Movie 1 - Reversible R body kinetics 36 37 277 Supplemental Movie 2 - RebA S88P and WT R bodies in spheroplasts 38 39 278 AUTHOR INFORMATION 40 279 Address correspondence to Pamela A. Silver, pamela_silver@hms.harvard.edu. 200 Longwood Avenue, 41 280 Warren Alpert Building, Boston, MA 02115 42 43 281 Conflicts of interest 44 282 The authors have no conflicts of interest to declare. The funders of this study had no role in the decision 45 283 to publish. 46 47 284 Author contributions 48 285 JKP and PAS designed the study. JKP performed all experiments and analyzed the data. JKP and PAS 49 50 286 wrote the paper. 51 52 287 ACKNOWLEDGEMENTS 53 288 We are grateful to Timothy Mitchison (HMS), Jeff Way (HMS), Michael Baym (HMS), Ethan Garner 54 289 (Harvard), Justin Kollman (UW), Rob Phillips (Caltech), Dan Fletcher (UC Berkeley) and Michael Vahey 55 56 290 (UC Berkeley) for helpful discussions. We thank Cameron Myhrvold (HMS) for advice about statistics. We 57 58 59 60 7 ACS Paragon Plus Environment ACS Synthetic Biology Page 8 of 16 Tunable protein pistons 1 2 3 291 are grateful to Genevieve Dobihal (Harvard), Nathan Rollins (Harvard), and Brendan Cruz (Harvard) for 4 5 292 constructive feedback on the manuscript. 6 293 This work was supported by a Jane Coffin Childs Postdoctoral Fellowship to JKP and an Office of Naval 7 8 294 Research MURI Grant N00014-11-1-0725. 9 10 295 REFERENCES 11 296 (1) Pond, F. R., Gibson, I., Lalucat, J., and Quackenbush, R. L. (1989) R-body-producing bacteria. 12 297 Microbiol. Rev. 53, 25–67. 13 14 15 298 299 (2) Mueller, J. A. (1965) Vitally stained kappa in Paramecium aurelia. J. Exp. Zool. 160, 369–372. (3) Sonneborn, T. M., Jacobson, W., and Dippell, R. V. (1946) Paramecin 51, an antibiotic produced by 16 300 Paramecium aurelia; amounts released from killers and taken up by sensitives; conditions protecting 17 301 sensitives. Anat. Rec. 96, 514. 18 302 (4) Austin, M. L. (1946) Contributions towards an analysis of the killing action of variety 4 killers in 19 303 Paramecium aurelia. Anat. Rec. 96, 514. 20 304 (5) Jurand, A., Rudman, B. M., and Preer, J. R. (1971) Prelethal effects of killing action by stock 7 of 21 22 305 Paramecium aurelia. J. Exp. Zool. 177, 365–387. 23 306 (6) Preer, L. B., Jurand, A., Preer, J. R., and Rudman, B. M. (1972) The Classes of Kappa in Paramecium 24 307 Aurelia. J. Cell Sci. 11, 581–600. 25 308 (7) Quackenbush, R. L., and Burbach, J. A. (1983) Cloning and expression of DNA sequences associated 26 309 with the killer trait of Paramecium tetraurelia stock 47. Proc. Natl. Acad. Sci. U. S. A. 80, 250–254. 27 310 (8) Schrallhammer, M., Galati, S., Altenbuchner, J., Schweikert, M., Görtz, H.-D., and Petroni, G. (2012) 28 29 30 311 312 Tracing the role of R-bodies in the killer trait: Absence of toxicity of R-body producing recombinant E. coli on paramecia. Eur. J. Protistol. 48, 290–296. 31 313 (9) Preer Jr., J. R., Hufnagel, L. A., and Preer, L. B. (1966) Structure and behavior of R bodies from killer 32 314 paramecia. J. Ultrastruct. Res. 15, 131–143. 33 315 (10) Kanabrocki, J. A., Quackenbush, R. L., and Pond, F. R. (1986) Organization and expression of genetic 34 316 determinants for synthesis and assembly of type 51 R bodies. J. Bacteriol. 168, 40–48. 35 317 (11) Heruth, D. P., Pond, F. R., Dilts, J. A., and Quackenbush, R. L. (1994) Characterization of genetic 36 37 318 determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116. J. Bacteriol. 38 319 176, 3559–3567. 39 320 (12) Jeblick, J., and Kusch, J. (2005) Sequence, Transcription Activity, and Evolutionary Origin of the R- 40 321 BodyCoding Plasmid pKAP298 from the Intracellular Parasitic BacteriumCaedibacter taeniospiralis. J. 41 322 Mol. Evol. 60, 164–173. 42 323 (13) Pace, C. N., and Scholtz, J. M. (1998) A helix propensity scale based on experimental studies of 43 324 peptides and proteins. Biophys. J. 75, 422–427. 44 45 325 (14) Martinez, K. A., Kitko, R. D., Mershon, J. P., Adcox, H. E., Malek, K. A., Berkmen, M. B., and 46 326 Slonczewski, J. L. (2012) Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and 47 327 Bacillus subtilis observed by fluorescence ratio imaging microscopy. Appl. Environ. Microbiol. 78, 3706– 48 328 3714. 49 329 (15) Harrison, S. C. (2008) Viral membrane fusion. Nat. Struct. Mol. Biol. 15, 690–698. 50 330 (16) Canton, J., Khezri, R., Glogauer, M., and Grinstein, S. (2014) Contrasting phagosome pH regulation 51 52 53 331 332 and maturation in human M1 and M2 macrophages. Mol. Biol. Cell 25, 3330–3341. (17) Raymann, K., Bobay, L.-M., Doak, T. G., Lynch, M., and Gribaldo, S. (2013) A Genomic Survey of Reb 54 333 Homologs Suggests Widespread Occurrence of R-Bodies in Proteobacteria. G3 GenesGenomesGenetics 55 334 3, 505–516. 56 57 58 59 60 8 ACS Paragon Plus Environment Page 9 of 16 ACS Synthetic Biology Tunable protein pistons 1 2 3 335 (18) Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., 4 5 336 Day, R. N., Israelsson, M., Davidson, M. W., and Wang, J. (2013) A bright monomeric green fluorescent 6 337 protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409. 7 338 (19) Miroux, B., and Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts 8 339 that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 9 340 289–298. 10 341 (20) Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Högbom, M., van Wijk, K. 11 12 13 342 343 J., Slotboom, D. J., Persson, J. O., and de Gier, J.-W. (2008) Tuning Escherichia coli for membrane protein overexpression. Proc. Natl. Acad. Sci. U. S. A. 105, 14371–14376. 14 344 (21) Whitmore, L., and Wallace, B. A. (2004) DICHROWEB, an online server for protein secondary 15 345 structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32, W668–W673. 16 346 (22) Whitmore, L., and Wallace, B. A. (2008) Protein secondary structure analyses from circular 17 347 dichroism spectroscopy: Methods and reference databases. Biopolymers 89, 392–400. 18 348 (23) Compton, L. A., and Johnson, W. C. (1986) Analysis of protein circular dichroism spectra for 19 20 349 secondary structure using a simple matrix multiplication. Anal. Biochem. 155, 155–167. 21 350 (24) Manavalan, P., and Johnson Jr., W. C. (1987) Variable selection method improves the prediction of 22 351 protein secondary structure from circular dichroism spectra. Anal. Biochem. 167, 76–85. 23 352 (25) Sreerama, N., and Woody, R. W. (2000) Estimation of Protein Secondary Structure from Circular 24 353 Dichroism Spectra: Comparison of CONTIN, SELCON, and CDSSTR Methods with an Expanded Reference 25 354 Set. Anal. Biochem. 287, 252–260. 26 355 (26) McDonald, J. (2015) Handbook of Biological Statistics. Sparky House Publishing. 27 28 356 (27) Jones, D. T. (1999) Protein secondary structure prediction based on position-specific scoring 29 357 matrices. J. Mol. Biol. 292, 195–202. 30 358 (28) Buchan, D. W. A., Minneci, F., Nugent, T. C. O., Bryson, K., and Jones, D. T. (2013) Scalable web 31 359 services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Res. 41, W349–357. 32 360 (29) Witholt, B., Boekhout, M., Brock, M., Kingma, J., Heerikhuizen, H. V., and Leij, L. D. (1976) An 33 361 efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia 34 35 362 coli. Anal. Biochem. 74, 160–170. 36 37 363 TABLES 38 364 Table 1. Plasmids used in this study. The following plasmids with the exception of Reb_206 and 207 are 39 365 available through Addgene. 40 41 Addgene ID PAS J. Polka Description 42 number designation 43 44 71224 PAS619 Reb_1 Expresses wild type R bodies from C. taeniospiralis in 45 pETM11 backbone 46 71225 PAS620 Reb_79 Reb_1 with RebA(Q91R) 47 71226 PAS621 Reb_80 Reb_1 with RebB(I97T) 48 71227 PAS622 Reb_81 Reb_1 with RebB(T28A) 49 71228 PAS623 Reb_82 Reb_1 with RebB(S82P) 50 51 71229 PAS624 Reb_83 Reb_1 with RebA(D89G) 52 71230 PAS625 Reb_140 Reb_1 with RebA(S88P) 53 71231 PAS626 Reb_65 Reb_1 with RebA(Y82A) 54 55 71233 PAS628 Reb_221 Reb_1 with RebA(G90A) 56 57 PAS629 Reb_206 Expresses wild type R bodies with mNeonGreen-tagged 58 second copy of RebB under a weak RBS downstream as 59 60 9 ACS Paragon Plus Environment ACS Synthetic Biology Tunable protein pistons 1 2 3 4 5 6 366 PAS630 Reb_207 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 10 well as soluble mCherry Reb_206 with RebA(S88P) ACS Paragon Plus Environment Page 10 of 16 Page 11 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Synthetic Biology For TOC only 273x148mm (72 x 72 DPI) ACS Paragon Plus Environment ACS Synthetic Biology Page 12 of 16 A 1 2 Killer strain Sensitive strain shedding B CContracted Extended 3 4 5 6 7 feeding 8 9 10 pH 6 pH 5 11 12 13 R body toxins 14 15 Toxin release D 40 30 20 pH 6.2 pH 4.7 CD (a.u.) 16 10 17 0 18 19 20 21 22 23 24 E25 Food vacuole acidification R body extension Death of sensitive Paramecium Helix1 Helix2 -10 -20 -30 185 195 205 215 225 235 Wavelength (nm) Strand1 Strand2 Turns Unordered 245 255 Total 26 pH 6.2 (NRMSD: 0.001) 0.43 0.12 0.11 0.11 0.06 0.18 1.01 27 28 pH 4.7 (NRMSD: 0.000) 0.43 0.14 0.10 0.10 0.06 0.18 1.01 29 F30 0’ 2’ 4’ 6’ 31 32 33 8’ 10’ 12’ 14’ 34 G 1.4 1.2 1 0.8 A600 pH 7.25 6.75 A600 35 0.6 6.25 36 16’ 18’ 20’ 22’ 37 38 0.4 0.2 5.75 39 24’ 26’ 28’ 30’ 0 5.25 40 0 20 40 60 80 100 120 140 160 180 200 220 240 41 Measurement number H42 32’ 34’ 36’ 38’ Measurement 1 Measurement 240 43 44 45 40’ 42’ 44’ 46’ 46 47 48’ 50’ 52’ 54’ 48 49 pH 5 pH 7 pH 5 pH 7 50 51 52 53 54 55 56 57 58 59 60 pH ACS Paragon Plus Environment Page 13 of 16 A 1 pH 2 [KCl] 3 4 5 6 7 8 9 10 11 12 13 B14 15 1.1 16 1 17 18 0.9 19 0.8 Normalized A590 20 0.7 21 22 0.6 23 0.5 24 0.4 25 26 0.3 27 0.2 28 0.1 29 30 0 31 5.2 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2M KCl 1M KCl 0.5M KCl 0.25M KCl 0.125M KCl 0.063M KCl 0M (not norm.) 5.4 5.6 ACS Synthetic Biology C [KCl] 2M pH 5.16 1M pH 5.31 0.5M pH 5.20 0.125M pH 5.17 0M 5.8 6 6.2 pH pH 5.19 ACS Paragon Plus Environment pH 5.62 pH 6.10 pH 5.75 pH 6.21 pH 5.67 pH 6.14 pH 5.62 pH 6.09 pH 5.66 pH 6.12 A Error prone PCR (~750bp) 1 2 3 4 5 6 pET vector 7 8 9 10 11 C12 1 13 Normalized A590 14 0.8 15 16 0.6 17 18 0.4 19 20 0.2 21 22 0 23 3.7 24 E25 26 RebA 27 28 29 30 31 32 33 34 35 RebB 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4.2 4.7 5.2 pH BACS Synthetic Biology Expected behavior in pH 5.5 buffer WT Mutant 5.7 6.2 Reb79: RebA(Q91R) Reb82: RebB(S82P, D88G) Reb83: RebA(D89G), RebB frameshift Reb81: RebB(T28A) Reb80: RebB(I97T) Reb1: wild type ACS Paragon Plus Environment D Page 14 of 16 500nm Page 15 of 16 ACS Synthetic Biology A 1 Clone 80 | B90 100 Approx. | | pH_ 2 Wild type …IMYTLSAASDGQAVSTVDNST… 5.7_ 31 4 Reb79 …IMYTLSAASDGRAVSTVDNST… <3 5 Reb140 …IMYTLSAAPDGQAVSTVDNST… <3 6 Reb65 …IMATLSAASDGQAVSTVDNST… 5.8 0.8 Normalized A590 7 Reb66 …IMYTLAAASDGQAVSTVDNST… 5.7 8 Reb67 …IMYTLSAAADGQAVSTVDNST… 5.8 0.6 9 Reb68 …IMYTLSAASAGQAVSTVDNST… 5.6 10 Reb221 …IMYTLSAASDAQAVSTVDNST… 6.1 11 Reb69 …IMYTLSAASDGQAVATVDNST… 5.6 0.4 12 Reb217 …IMAAAAAAAAAAAAAAAAAAT… N/A 13 Reb218 …IMAAAAAAAAAAAVSTVDNST… <3 14 Reb219 …IMYTLSAAAAAAAVSTVDNST… 5.3 15 Reb220 …IMYTLSAASDGQAAAAAAAAT… <3 16 Reb222 …IMYTLSAASPGQAVSTVDNST… <3 17 Reb235 …IMYTLSAASDGQAVSPVDNST… <3 18 Reb236 …IMYTLSAASDGQAVSTPDNST… 4.4 19 Reb237 …IMYTLSAASDGQAVSTVPNST… 4.2 20 Reb238 …IMYTLSAASDGQAVSTVDPST… 5.3 21 Reb239 …IMYTLSAASDGQAVSTVDNPT… 5.5 22 23 Reb240 …IMYTLSAASDGQAVSTVDNSP… 5.5 0.2 0 5.2 24 25 C26 27 28 29 Reb1: Wild type 30 31 32 Reb65: 33 RebA 34 (Y82A) 35 36 37 38 Reb221: 39 RebA 40 (G90A) 41 42 43 pH 5.16 5.62 5.73 5.85 5.91 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 5.7 pH Reb1: wild type Reb65: RebA(Y82A) Reb221: RebA(G90A) 6.2 6.10 6.23 ACS Synthetic Biology 30 second intervals  1A 2 3 + Sucrose, +/- lysozyme G 4 5 6 7 8 Induction + Low pH buffer, +/- salts 9 B10 11 12 1 0.8 * * * Fraction lysed cells 13 0.6 14 0.4 15 16 0.2 17 0 18 -0.2 19 Genotype WT WT WT S88P 20 Lysozyme - + ++ 21 Salts +- ++ 22 Exp. 1 fraction .004 (257) .115 (156) .750 (124) .031 (160) 23 Exp. 2 fraction .029 (103) .108 (65) .755 (237) .162 (277) 24 Exp. 3 fraction .168 (375) .087 (297) .401 (262) .115 (266) 25 Total fraction .091 (735) .098 (518) .605 (623) .115 (703) 26 (# of cells) 27 28 29 30 C31 32 WT - + Before 10’ after buffer change 33 D34 35 WT + - 36 37 38 E39 WT + + 40 F41 42 S88P + + 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 16 of 16