bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 made available under aCC-BY-NC-ND 4.0 International license.

1 Iso-propyl stilbene: A life-cycle signal?

2 Alexia Hapeshi1*, Jonatan Mimon Benarroch1, David James 3 Clarke2, Nicholas Robin Waterfield1 4 5 1Microbiology and Infection Unit, Division of Biomedical Sciences, Warwick 6 Medical School, University of Warwick. 7 2School of Microbiology and APC Microbiome Institute, University College 8 Cork, Cork, Ireland 9 10 *For correspondence: [email protected] 11 12 Keywords: 3,5-dihydroxy-4-isopropyl-trans-stilbene, Photorhabdus, secondary 13 metabolites 14 15 Repositories: Sequencing data has been deposited to NCBI under BioProject 16 PRJNA509896 (SRA accessions SRR8306869-SRR8306874 and 17 SRR8307122). 18

19 ABSTRACT 20 21 Members of the Gram-negative bacterial genus Photorhabdus are all highly 22 insect pathogenic and exist in an obligate symbiosis with the 23 entomopathogenic worm . All members of the 24 genus produce the small molecule 3,5-dihydroxy-4-isopropyl-trans-stilbene 25 (IPS) as part of their secondary metabolism. IPS is a multi-potent compound 26 that has antimicrobial, antifungal and anticancer activities and also plays an 27 important role in symbiosis with the nematode. In this study we have 28 examined the response of Photorhabdus itself to exogenous ectopic addition 29 of IPS at physiologically relevant concentrations. We observed that the 30 had a measureable phenotypic response, which included a decrease 31 in bioluminescence and pigment production. This was reflected in changes in 32 its transcriptomic response, in which we reveal a reduction in transcript levels bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 made available under aCC-BY-NC-ND 4.0 International license.

33 of genes relating to many fundamental cellular processes, such as translation 34 and oxidative phosphorylation. Our observations suggest that IPS plays an 35 important role in the biology of Photorhabdus bacteria, fulfilling roles in 36 quorum sensing, antibiotic-competition advantage and maintenance of the 37 symbiotic developmental cycle. 38 39 INTRODUCTION

40 41 Stilbenoids are natural products with important biological properties. Most are 42 synthesised by plants in response to pathogenic attack. However, 43 Photorhabdus and Bacillus bacteria engaged in symbioses with 44 entomopathogenic also produce certain stilbene derivatives (1,2). 45 Photorhabdus is an entomopathogenic bacterium, which forms a symbiotic 46 relationship with Heterorhabditis nematodes. Infection of insect larvae by the 47 nematodes results in regurgitation of the bacteria into the insect (3). 48 Photorhabdus then produces a large set of toxins and digestive enzymes to 49 kill and utilise the insect as a food source (4). Additionally, it releases multiple 50 antimicrobial factors to eliminate competition in the insect carcass by other 51 bacteria or fungi found in the soil (5,6). 3,5-dihydroxy-4-isopropyl-trans- 52 stilbene (IPS) (figure 1) is a small molecule produced by Photorhabdus, which 53 has been shown to have antimicrobial (1), antifungal (7,8) and anticancer (9) 54 activities and also seems to play an important role in symbiosis (10).

55 IPS is produced by or encoded in the genomes of all Photorhabdus strains 56 investigated so far (1,7,8,11–13). Additionally, depending on the strain and 57 the growth phase of the bacteria other stilbene derivative compounds may 58 also be synthesised (1,8). Joyce et al (2008) demonstrated that Photorhabdus 59 utilises a pathway distinct from that used in plants for IPS biosynthesis. The 60 Photorhabdus biosynthetic pathway is composed of two branches that 61 converge, with one originating in phenylalanine metabolism and the other in 62 branched-chain amino acid metabolism. Additionally, one of the stilbene 63 precursors is involved in the production of branched chain fatty acids and it is 64 thus possible that fatty acid oxidation can contribute to IPS production. bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

65 66 In vitro, the expression of stlA, which encodes a phenylalanine-ammonium 67 lyase that catalyses the first step in stilbene biosynthesis is induced by 68 nutrient limitation (14). Activity of the stlA promoter seems to negatively 69 correlate with the amount of nutrients in the media and shows distinct 70 changes at the different growth phases (14,15). The production of the 71 alarmone molecules (p)ppGpp is required for this as deletion of both relA and 72 spoT results in a shutdown of stlA promoter activity (16). Additionally, post- 73 transcriptional regulation plays a role in controlling stlA expression and this is 74 mediated via the BarA/UvrY two-component system and CsrA/csrB (17). 75 However, a uvrY deficient strain showed no significant difference in the actual 76 amounts of stilbenes produced (18). Moreover, it has been established that 77 the production of IPS is repressed by the LysR-type regulator HexA that 78 controls the synthesis of secondary metabolites in general (18,19) and is itself 79 regulated by the action of Hfq (20). This illustrates that multiple levels of 80 regulation are present which affect the levels of stilbenes. 81 82 In vivo, IPS production is associated with entry of the bacteria into the 83 stationary phase of growth following replication in the insect host. In an 84 infection model using larvae, the production of IPS by 85 Photorhabdus is detectable 24 h post infection after which the levels of IPS 86 remain stable for several days (21). It should be noted that by 24 h, the 87 population of Photorhabdus within Galleria increases to a very high density of 88 109 bacteria/g of wet larvae (21). Actual concentrations of IPS vary depending 89 on the entomopathogenic complex used and subsequently the Photorhabdus 90 symbiont strain. Levels can range from ~600 to ~4000 µg/g wet insect tissue 91 (21,22). Additionally, Eleftherianos et al. (2006) have estimated that in the 92 haemolymph of caterpillars that have been infected with 93 subsp. laumondii TT01 the levels of IPS can 94 reach up to 275 – 550 µg/ml (15). 95 96 One suggested function of the stilbenes, as a result of their antimicrobial and 97 antifungal properties, is the protection of the insect carcass from competition. 98 Another possible role is in the establishment of a successful symbiosis. A bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

99 Photorhabdus stilbene-negative mutant is severely impaired in its ability to 100 promote infective juvenile recovery into adult hermaphrodites in an in vitro 101 symbiosis assay (10). However, the presence of IPS in itself is not sufficient 102 for the induction of IJ recovery (10) indicating that other factors produced by 103 the bacteria are also necessary for the process. With this in mind, we 104 examined the effect of IPS on P. luminescens itself. It has been previously 105 shown that high concentrations of IPS (>100 µg/ml) are toxic to the bacteria 106 (23) but the mechanism behind this has not been investigated in detail. We 107 demonstrate that the presence of IPS actually has a profound effect on central 108 metabolism, secondary metabolism and consequently the phenotype of the 109 bacteria. We propose that the timing of IPS production in an insect infection 110 plays a crucial role in orchestrating the relationship between the bacteria and 111 the entomopathogenic nematode host. 112 113 RESULTS 114 115 In order to investigate the effect of IPS on Photorhabdus bacteria we first 116 provided exogenous IPS at various concentrations from the beginning of 117 growth in batch culture. At this growth phase the bacteria do not normally 118 synthesize the compound. Under these conditions P. luminescens shows a 119 clear dose dependent response to ectopic IPS, with growth rate reducing as 120 the concentration of the compound added increases (figure 2A). A clear dose 121 response in bioluminescence was also observed, the levels of which 122 decreased with higher IPS concentrations. This holds true until the later 123 stages of growth when the levels of bioluminescence, in the presence of IPS, 124 actually become higher than in the control cultures (figure 2B). 125 126 Based on these findings we subsequently chose a concentration of 32 µg/ml 127 and investigated whether addition of IPS to bacteria that were already growing 128 exponentially, for approximately 5 h, would have the same effect as above. 129 Indeed, we observed that early on after treatment there were significantly 130 lower (q value < 0.01) levels of bioluminescence in the cultures that were 131 treated with the compound compared to those treated with the carrier solvent bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

132 DMSO only. Interestingly, again by 24 h this effect was reversed (q value < 133 0.001). Furthermore, subsequent growth was also moderately negatively 134 affected, which was significant even at 30 min post treatment (q value <

135 0.001), with IPS treated cultures having a mean OD600 at 600 nm of 0.49,

136 while DMSO treated cultures had a mean OD600 of 0.54 (figure 3). 137 138 Additionally, we noted that by the end of the experiment (24h growth), the 139 cultures of P. luminescens were seen to have very little pigmentation when 140 prematurely exposed to IPS, compared to untreated cultures which showed 141 the typical orange/red pigmentation (figure 4a). This observation was 142 corroborated by spectrophotometric measurements of the cultures (data not 143 shown). P. luminescens normally produces increasing amounts of 144 anthraquinone pigments at late stages of its growth phase. The absence of 145 the red colour suggests that production of anthraquinones is either directly or 146 indirectly inhibited by IPS. Interestingly in a mutant strain where IPS 147 production is prevented, through disruption of stlA by transposon insertion 148 (24), the resulting colonies are hyper-pigmented, also suggesting a 149 deregulation of anthraquinone production (figure 4b). 150 151 Since IPS appears to affect aspects of P. luminescens secondary metabolism 152 we decided to investigate its role on the formation of a biofilm by P. 153 luminescens. The ability to form a biofilm has been implicated in the 154 successful establishment of symbiosis with the Heterorhabditid nematode (25) 155 and may be part of a pathogenic / symbiotic life-cycle switch in Photorhabdus. 156 Furthermore, previous work has associated Photorhabdus secondary 157 metabolites with a role in nematode symbiosis (20,26). We performed this 158 experiment using LB media as this was previously shown to support better 159 biofilm formation in P. temperata than BHI or SOC media (27). It should be 160 noted that wild-type Photorhabdus does not form very thick biofilms in vitro, 161 and the biofilms they do form in standard microplate assays appear to occur 162 mostly on the liquid-air interface. Supplementation of the cultures with IPS did 163 not have a significant effect on the total amount of biofilm formed (figure 5a). 164 However, when this assay was repeated with the stlA mutant, which forms a bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

165 much thicker biofilm, addition of IPS resulted in a significant reduction in the 166 amount of biofilm suggesting that IPS plays a modulatory role (figure 5b). 167 168 In order to elucidate whether the phenotypic observations followed a more 169 systemic pattern we performed an RNA-seq transcriptomic study on P. 170 luminescens TT01 following addition of exogenous IPS to mid log planktonic 171 cells. At 32 µg/ml of IPS we were able to see a detectable growth delay and 172 reduction in bioluminescence. We therefore used this concentration to 173 investigate the cause of the growth delay, which we reasoned would be 174 reflected in changes in gene expression. Furthermore, P. luminescens TT01 175 would be expected to be exposed to at least this IPS concentration (and likely 176 higher) during insect infection (see above). For this experiment IPS was again

177 added to cultures at exponential phase (OD600 ~ 0.45) and samples for RNA 178 extraction were harvested 30 min later. Bioluminescence measurements 179 confirmed the inhibitory effect of the IPS in the samples used for RNA 180 extraction (Figure S1). 181 182 The response of Photorhabdus to IPS was systemic with 258 genes 183 significantly up-regulated and 463 genes down-regulated, when only

184 considering the padj threshold (table S1). Of those, 52 genes had at least 2- 185 fold higher expression whilst 84 genes had at least 2-fold lower expression in 186 the cultures treated with IPS compared to those treated with DMSO. For the 187 purposes of this analysis, we also used the small RNA identification tool 188 toRNAdo (28), to identify putative non-coding RNAs and examine if their 189 expression changes in the presence of IPS. From the output of toRNAdo, 80 190 sRNAs were added to the annotation of the genome and included in the 191 differential gene expression analysis. Of those, 4 were significantly 192 overexpressed and 10 had significantly lower levels of expression in the 193 presence of IPS. One putative sRNA was among the top 20 overexpressed

194 genes (log2 fold change of 2.0). This is located opposite a tRNA-Phe, whose 195 levels however appear to be unchanged. Interestingly, we observed that 21 196 tRNA encoding-sequences were moderately down-regulated following 197 treatment and so were various tRNA modifying enzymes. This is intriguing as 198 it was recently shown that tRNAs can get rapidly degraded shortly after bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

199 starvation (29) or stress (30), presumably to prevent mistranslation and the 200 accumulation of damaged or misfolded proteins. No tRNAs were up-regulated 201 in the presence of IPS. 202 203 Secondary metabolism 204 Consistent with our previous observations we observed IPS dependent down- 205 regulation of the transcription of certain genes involved in secondary 206 metabolism. In particular, the biosynthetic gene cluster antA-antI (plu4186- 207 4194) encoding the type II polyketide synthase and modifying enzymes 208 involved in anthraquinone production (31) were the most down-regulated 209 genes in our dataset. This explains the absence of a pigment in IPS treated 210 cultures of Photorhabdus that was observed (see above). Additionally, among 211 the genes with the most marked reduction in transcript levels were genes 212 belonging to the Photorhabdus variable region plu1434–plu1448 (32), some of 213 which are responsible for antibiotic production. Similarly, cpmA and cpmD, 214 encoding enzymes involved in carbapenem biosynthesis, were down- 215 regulated. It is of note, however, that contrary to other secondary metabolites 216 that are most highly produced at stationary phase, expression of the cpm 217 genes is actually maximal during exponential phase (5). Another highly down- 218 regulated cluster of genes is that located in the region plu1409-plu1415. 219 Perhaps surprisingly, the expression of the IPS biosynthesis genes stlA, stlB, 220 stlCDE and bkdABC was not significantly reduced. Note, these genes are 221 encoded across the genome rather than in a single operon, suggesting the 222 pathway regulon was not affected. However, as this was during a time point 223 when the bacteria would be producing low amounts of IPS, if any, it is 224 possible that these genes cannot be repressed further or that there is no 225 feedback acting to control their expression. On the other hand, transcript 226 levels for plu3042 and plu3043, which encode a transaminase and a 227 prephenate dehydratase respectively, are significantly reduced. The products 228 of these genes are likely to be involved in the production of 2,5- 229 dihydrophenylananine, which is the precursor to another cryptic stilbene 230 derivative, 2,5-dihydrostilbene (33). Contrary to our phenotypic observations 231 that addition of IPS causes a rapid drop in bioluminescence the transcript 232 levels of the lux operon itself were not affected. Previous work has also bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

233 suggested that bioluminescence activity appears to be under post- 234 transcriptional control (19). Also, in contrast to the processes discussed 235 above, which were repressed by IPS, transcription of some other secondary 236 metabolite genes was actually up-regulated. For example, this was the case 237 with plu3533, encoding a non-ribosomal peptide synthase (NRPS) that 238 potentially produces a gramicidin/tyrocidin antibiotic. 239 240 Basic metabolic processes 241 In large part the effect of ectopic IPS addition on the bacterial transcriptome 242 resembles a classic stringent response (Figure 6). In many bacteria this is 243 reflected in the up-regulation of amino acid biosynthesis, with the concurrent 244 down-regulation of many aspects of ribosome biogenesis and protein 245 translation (34,35). In particular, in our dataset 52 out of 55 genes encoding 246 proteins that form the ribosomal subunits, or are otherwise associated with 247 ribosomes, were significantly down-regulated. RNA polymerase subunits also 248 exhibit reduced transcript levels. Furthermore, a large number of genes 249 encoding enzymes involved in the Tri-Carboxylic Acid (TCA) cycle are also 250 negatively affected. The response to IPS seen in the transcriptome is however 251 more extensive than we would expect from stringent response alone. 252 Specifically we also see repression of the transcription of genes involved in 253 Oxidative Phosphorylation, including subunits of the F-type ATPase, 254 succinate dehydrogenase and NADH dehydrogenase. In addition, the 255 pathways of purine and pyrimidine metabolism are also strongly affected. In 256 particular, various genes encoding purine and pyrimidine de novo biosynthetic 257 enzymes are down-regulated by ectopic IPS exposure. On the contrary, the 258 gene that codes for CpdB, which is involved in nucleotide catabolism is up- 259 regulated and so are plu4417 encoding uridine phosphorylase, nudE 260 encoding an ADP compounds hydrolase, and plu0661 encoding a putative 261 pyrimidine/purine nucleotide 5'-monophosphate nucleosidase. 262 263 In other Gram-negative bacteria, the stringent response is controlled by the 264 levels of the (p)ppGpp alarmone, which is in turn regulated by the opposing 265 enzymic activities of the relA and spoT gene products. Interestingly, we see 266 no change in the transcription of the orthologous relA (plu0910) and spoT bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

267 (plu0272) genes, although this does not rule out any post-transcriptional or 268 post-translational regulation of their activities. To this effect, we observed that 269 the transcript level of plu4550, which encodes the essential protein 270 ObgE/CgtA are lower in the presence of IPS. In E. coli and Vibrio cholera 271 CgtA was shown to bind SpoT and it has been suggested that it promotes its 272 (p)ppGpp hydrolase activity (36,37). Hydrolysis of (p)ppGpp would otherwise 273 suppress the stringent response. 274 275 Interestingly, amongst the most highly IPS induced genes were those 276 encoding for enzymes involved in sulfur metabolism as well as components of 277 the sulfate/thiosulfate ABC transporter. As the control cultures were treated 278 with an equivalent amount of the IPS carrier solvent DMSO, this cannot be 279 attributed solely to the presence of DMSO in the media. Note however that 280 some of these enzymes are involved in the cysteine and methionine 281 metabolism, which relates to the stringent response-like changes discussed 282 above. Nonetheless, among the overexpressed genes involved in sulfur 283 metabolism was the sufABCDSE operon that encodes the Suf pathway 284 proteins, unrelated to amino acid synthesis. The Suf pathway is an iron-sulfur 285 (Fe-S) cluster biogenesis system that operates under stress conditions. SufS 286 is a cysteine desulfurase, which then serves as a donor for the assembly of 287 the Fe-S cluster, which is eventually transferred to other proteins. The Isc 288 system is another Fe-S cluster assembly system and we observed that genes 289 iscRSUA encoding the enzymes of the system and the transcription factor 290 IscR, as well as hscR, encoding a co-chaperone are also overexpressed. We 291 note that disruption of the TCA cycle in an mdh mutant (26) also resulted in an 292 up-regulation of these pathways (unpublished data). 293 294 Transporters 295 Of note is the significant reduction in transcript levels for putP, which encodes 296 a proline (sodium) transporter. On the other hand there was no effect on the 297 expression of proVXW encoding the ProU ABC transporter. L-proline plays an 298 important role in the biology of Photorhabdus, influencing production of 299 secondary metabolites and affecting the proton motive force. Interestingly, a bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

300 ΔputP mutant had abolished stilbene production but up-regulated 301 anthraquinone (38). 302 303 The transcript levels of genes encoding transporters for iron and zinc are 304 increased. For example, plu3940 and plu3941 encoding ExbD and ExbB 305 respectively are both overexpressed. Furthermore, genes plu2320-2321 306 involved in the biosynthesis of a siderophore related to the yersiniabactin from 307 Yersinia enterocolitica are also up-regulated in addition to gene plu2316, 308 which encodes a receptor protein that is also predicted to be involved in iron 309 uptake. Iron uptake was previously shown to be important in the virulence of 310 Photorhabdus, whilst depending on the bacteria-nematode complex it may 311 also play a role in symbiosis (39,40). On the contrary, the transport of 312 dipeptides, oligopeptides, glutamate/aspartate, arginine and long chain fatty 313 acids is repressed. Similarly, tctA and tctC that encode subunits of the 314 tripartite tricarboxylate transporter are repressed. We also note that the AcrAB 315 components of the enterobacterial AcrAB-TolC efflux pump are 316 transcriptionally up-regulated. The AcrAB-TolC tripartite system is known to 317 provide resistance to antibiotics but also has a role in pathogenicity (reviewed 318 in (41)). Other affected genes include tatA and tatB of the twin-arginine 319 translocation system and which are up-regulated, while secY, secF and yidC 320 that encode components of the Sec pathway of protein export are repressed. 321 Finally, genes plu0634 and plu1333 (rtxB) encoding subunits belonging to the 322 RTX toxin family of ABC transporters are also repressed. 323 324 Bacterial surface components 325 Genes plu2656-plu2660 which belong to the pgbPE operon that is required for 326 O-antigen synthesis and assembly (42) are also down-regulated. The pgbPE 327 operon is required for Photorhabdus virulence as well as colonization of the 328 nematode gut (42). Additionally, a group of genes belonging to the wbl cluster 329 whose products are likely to be involved in lipopolysaccharide biosynthesis 330 are down-regulated. These are wblT, wblR, wblP and wblU. Similarly, genes 331 plu4658-plu4663 encoding enzymes RffG, WecABC and WzzE involved in the 332 synthesis of the enterobacterial common antigen (ECA) are also repressed. 333 This could be significant as in addition to the pgbPE operon other genes bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

334 involved in LPS biosynthesis were also previously shown to be necessary for 335 Photorhabdus virulence and transmission to the nematode (43). Furthermore, 336 motility and/or attachment to surfaces may also be affected by the presence 337 of stilbene as we observed a mild drop in transcript levels of fliT (plu1951), fliC 338 (plu1954), fliD (plu1953), encoding flagellar proteins. Additionally, we noted a 339 reduction in transcript levels of plu00928 and of the operon plu2156-plu2159, 340 which are predicted to be involved in the production of fimbriae. 341 342 Transcriptional regulators 343 A large cluster of genes, which encode PAS4-LuxR solo transcriptional 344 regulators are almost all significantly down-regulated (plu2001-plu2016). 345 These are LuxR receptors that lack a cognate LuxI synthase and it has been 346 suggested that they are involved in host hormone-like signal detection (44). 347 Additionally, plu4444 encoding one of the putative alternative sigma factors 348 FecI, was up-regulated, whilst none of the other homologues of known sigma 349 factors was differentially expressed. This was also the case for lrp and tyrR, 350 whose products are known to affect the expression of stlA (14) and for genes 351 encoding the global regulators HexA or Hfq. 352 353 Other differentially expressed genes 354 355 Other genes affected by IPS treatment encode for products that are involved 356 in competition or defense mechanisms, further suggesting that the bacteria 357 are sensing and responding to stress. In particular, amongst the most highly 358 up-regulated genes are plu2816 and plu2817, involved in the biosynthesis of 359 rhabduscin, a phenoloxidase inhibitor which provides protection against the 360 insect immune system (45). Additionally, genes plu0884-plu0888, plu1893- 361 plu1894, plu4175 and plu4177 encoding either S-type pyocins or their 362 immunity proteins were up-regulated in the presence of IPS. Furthermore, we 363 observed an up-regulation of genes encoding subunits of the Photorhabdus 364 Tc toxins, in particular tccA1 (plu4169), tccB1 (plu4168), tccC2 (plu0960) and 365 tccC4 (plu0976). Finally, we noted the up-regulation of CRISPR related genes 366 such as plu1791 and plu1795, whose products are annotated as a CRISPR- bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

367 associated nuclease/helicase of the Cas3 type and the CRISPR-associated 368 protein Csy3 respectively. 369 370 DISCUSSION 371 372 Photorhabdus bacteria have a complex life cycle that includes an insect 373 pathogenic stage and a nematode symbiotic stage. The two stages are 374 associated with distinct metabolic states. More specifically, the virulent stage 375 is associated with active primary metabolism and rapid growth, whilst the 376 symbiotic stage requires secondary metabolism and this shift is reliant on a 377 functional TCA cycle (26). Crucially, mutants in key TCA cycle enzymes are 378 unable to produce stilbenes, light or anthraquinone pigments and are also 379 unable to support IJ recovery in vitro. Importantly, even though these mutants 380 are still virulent towards insects, they give rise to a population within the insect 381 cadaver that has altered characteristics compared to the wild-type bacteria 382 (26). Production of the alarmone (p)ppGpp following nutrient limitation in the 383 insect cadaver, is crucial for survival, induction of infective juvenile recovery 384 and re-establishment of symbiosis (16). Photorhabdus commonly exhibits two 385 dominant phenotypic variant types, the primary variant produces light, 386 anthraquinone and other “symbiosis factors” whilst in the secondary variant 387 these factors are absent (46). Both types are virulent but only the primary 388 variant can establish symbiosis. In addition, virulence of the secondary variant 389 was attenuated when secondary metabolism was de-repressed, through 390 mutation of the regulator HexA, even though the ability of the bacteria to form 391 a symbiotic relationship with the nematode was restored (47). It is conceivable 392 that HexA acts to repress premature secondary metabolism, as this would 393 have a detrimental effect on virulence. HexA is a LysR-type regulator with a 394 putative but as yet unknown small molecule ligand (19). 395 396 The important role of the TCA cycle in the lifecycle switch of Photorhabdus is 397 highlighted by our findings, as the TCA cycle is repressed in the presence of 398 the inappropriate ectopic levels of IPS used. This, and the observation that 399 light and anthraquinone pigment production is inhibited by addition of stilbene bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

400 to WT P. luminescens bacteria indicate the presence of a negative feedback 401 loop that controls secondary metabolism. This is also supported by the 402 observation that the stlA- mutant appears hyper-pigmented and so likely has 403 deregulated production of anthraquinones (48). The effects of IPS on 404 Photorhabdus suggest that the bacteria can actually detect it in a dose 405 dependant manner, as well as produce it. This could either be the result of a 406 direct effect of IPS on the concentration of other metabolic compounds or it 407 could occur through the binding of IPS to one of the LuxR solo receptors. 408 Photorhabdus lacks a typical Gram-negative acyl-homoserine lactone quorum 409 sensing (QS) circuit. However it has been shown that one of the LuxR 410 receptors does recognise photopyrones, which are synthesised by 411 Photorhabdus, regulating a clumping phenotype (49). Alternatively, IPS could 412 act as the ligand for HexA. However, a previous proteomic study has shown 413 that the TCA cycle is actually up-regulated in secondary variants (50), in 414 which the levels of HexA are higher (19). 415 416 In addition to the precise regulation of bacterial metabolism that occurs during 417 the insect infection, it has been demonstrated that following establishment of 418 symbiosis, P. temperata bacteria residing in the Heterorhabditis nematode 419 also experience large transcriptional changes (51). This transcriptional 420 rewiring has the potential of affecting the bacterial metabolism in such a way 421 as to slow down growth and overcome starvation and oxidative damage. This 422 process again needs to be controlled and so far the LysR-type regulator HdfR 423 has been implicated in this (52). 424 425 It is of note that the levels of IPS in the Galleria infection model remain high 426 for the entire duration of infective juvenile development (21). It has previously 427 been proposed that in addition to its the antimicrobial, antifungal and non- 428 cognate nematode repellent effects, IPS might be acting as the food signal for 429 nematode development. However, it is possible that it is also acting as a 430 signalling molecule for the bacteria to switch to the symbiotic lifestyle. This is 431 supported by the observation that IPS on its own cannot induce IJ recovery 432 (10). It is thus possible that IPS exerts its effects on the bacteria in a way that 433 makes them “primed” for transmission to the nematode and may also induce bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

434 the release of other compounds that are also necessary for IJ recovery. The 435 actual concentration of IPS may also be important in the effect it has on 436 Photorhabdus. Compared to the amounts that can accumulate in the insect 437 cadaver, we used a moderate concentration in this study. We would also like 438 to point out however that it is not known what the intracellular levels of IPS 439 actually are at different stages of the bacterial cycle. This could be modulated 440 by differential changes in membrane permeability or degradation. It is 441 tempting to speculate that mechanisms such as epoxidation or light 442 production might be used as methods to inactivate IPS in the cytoplasm. It 443 was shown for example that an epoxide derivative of IPS has reduced toxicity 444 towards Photorhabdus (23). Furthermore, it is known that trans-stilbenes, 445 such as resveratrol can easily be converted into the less active cis form by 446 exposure to light (53,54). 447 448 This would not be the first instance of a compound with antimicrobial 449 properties that can act as a signalling molecule or have an effect on the 450 producer bacteria. It has been actually proposed that the majority of small 451 molecules synthesised by bacteria do indeed play such a role (55). 452 Interestingly, specific acyl-homoserine lactone molecules known to be 453 involved in quorum sensing can also display antimicrobial activities (56). On 454 the other hand, phenazine antibiotics appear to promote survival of 455 Pseudomonas in low oxygen conditions (57) whilst pyocyanin was also shown 456 to be the signal for the activation of genes that are controlled by quorum 457 sensing (58). In summary, we provide evidence of another multipotent small 458 molecule, which is able to modulate the phenotype of the producer bacteria. 459 460 MATERIALS AND METHODS 461 462 Bacterial strains and reagents 463 464 The strains used in this study were P. luminescens subsp. laumondii TT01 465 (WT) (59) a rifampicin resistant derivative of this strain, P. luminescens spbsp. 466 laumondii TT01 (RifR) (60) and a rifampicin resistant stlA disruption mutant, bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

467 BMM901 (TT01 stlA::Kn) (24). The strains were grown in Lysogeny broth (LB) 468 or in Lysogeny agar (LBA) plates at 28 ˚C unless otherwise stated. Purified 469 IPS was a gift from Helge Bode (Goethe Universität Frankfurt, Germany) and 470 additional compound was purchased from iChemical (Shanghai, China). In 471 both cases, purity was confirmed by HPLC. 472 473 Growth, bioluminescence and spectral measurements 474 475 Growth (absorbance at 600 nm) and luminescence were monitored using a 476 microplate reader (Omega Fluostar, BMG Labtech). Overnight cultures of 477 Photorhabdus were diluted in either Mueller Hinton broth (MH) or LB to an

478 OD600 of 0.1. The diluted cultures were then aliquoted in black flat-bottomed 479 µClear® 96-well plates (CELLSTAR®, Greiner) and an equal volume of media 480 containing IPS at double the desired concentration was added to the wells. 481 The plates were then incubated at 28 ˚C in the microplate reader, with double 482 orbital shaking at 300 rpm and measurements of absorbance at 600 nm as 483 well as luminescence were taken every 30 min. To study the effect of IPS 484 addition during exponential growth, the cultures of Photorhabdus were diluted

485 to an OD600 of 0.05 and aliquoted in a 96 well plate, 145 µl per well. Growth 486 and luminescence were monitored as described above for 5.5 h until the

487 OD600 reached ~ 0.5. At this point, 5 µl of 960 µg/ml IPS dissolved in DMSO, 488 or DMSO only were added to the wells and the plate was returned to the 489 microplate reader. To compare the levels of luminescence between the two 490 treatments, luminescence was divided by absorbance at 600 nm. Then, 491 multiple t-tests were performed on GraphPad Prism version 8.0.0 (GraphPad 492 Software, available at www.graphpad.com) using the Benjamini, Krieger and 493 Yekuteili method (61) for controlling the false discovery rate. To monitor 494 differences in pigment production, the experiment above was repeated and at 495 the end of the growth curve (t = 24 h) the absorbance of the cultures at the 496 whole spectrum of 250 – 650 nm was measured. 497 498 Biofilm assays 499 bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

500 Biofilm was measured using a microplate assay and crystal violet staining. 501 Briefly, bacteria grown overnight were diluted to an OD of 0.05 in LB 502 supplemented with either DMSO or 32 µg/ml IPS. 500 µl aliquots were used 503 to inoculate the wells of a FalconTM 24-well tissue culture treated polystyrene 504 plate and this was then incubated stationary at 28 ˚C. After 24 h the 505 supernatant from each well was discarded and the wells were washed three 506 times with PBS to ensure that non biofilm-associated bacteria are removed. 507 The plate was left to dry for 1 h and then 600 µl 0.2 % crystal violet was 508 added to each well. Following a 30 min incubation excess crystal violet was 509 removed by washing the wells three times with PBS. A solution consisting of 510 70 % ethanol and 10 % isopropanol was used to lyse the cells and release the 511 crystal violet. The amount of dye was quantified using a FLUOstarR Omega 512 microplate reader and measuring absorbance at 570 nm. For each strain, the 513 experiment was performed with four biological replicates and for each of those 514 four technical replicates were used. To assess significance, a paired ratio t- 515 test was performed. 516 517 RNA extraction and sequencing 518

519 Overnight cultures of P. luminescens TT01 were diluted to OD600 of 0.05 in 520 two tubes containing 15 ml LB and incubated at 28 ˚C, 180 rpm until the

521 OD600 reached a value of approximately 0.45. Then, to one culture 32 µg/ml of 522 IPS were added, whilst the other culture was treated with an equivalent 523 volume of DMSO. Samples (500 µl) were harvested following incubation for 524 30 min and treated with RNAprotect (Qiagen). The bacteria were lysed using 525 the enzymatic lysis method described in the RNAprotect handbook in TE 526 buffer containing 20 µl of Qiagen Proteinase K and 1 mg/ml lysozyme. 527 Extraction was performed using the miRNeasy Mini kit (Qiagen) following the 528 manufacturer’s protocol. DNA removal was performed using the Invitrogen 529 TURBO DNA-free kit with Superasin RNAse inhibitor added in the reaction. 530 The samples were then further purified using the Norgen RNA Clean-Up and 531 Concentration kit. The absence of DNA contamination was confirmed by PCR 532 using universal 16S primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 533 1429R (5’-CGGTTACCTTGTTACGACTT-3’) (62) and the Agilent Bioanalyser bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

534 with a Prokaryote Total RNA Pico kit. RNA samples were treated with the 535 bacterial Ribo-Zero rRNA (Bacteria) Removal Kit (Illumina) to remove 536 ribosomal RNA using 2.5 µg of input RNA and 8 µl of Ribo-Zero removal 537 solution per sample. Clean up of depleted RNA was performed using ethanol 538 precipitation. Libraries for sequencing were prepared using the Illumina 539 Truseq Stranded mRNA kit with a slightly modified protocol. Specifically, 5 µl 540 of rRNA depleted RNA were used as input and this was directly mixed with 13 541 µl of the Fragment, Prime, Finish Mix. Following this, the procedure was as 542 described in the TruSeq Stranded mRNA Sample preparation guide. 543 Sequencing was performed using the Illumina MiSeq Next Generation 544 Sequencer on two MiSeq v3 cartridges with 75 bp paired-end reads. Three 545 sample libraries were sequenced on each cartridge.

546 547 Genome sequencing and Bioinformatic analyses 548 549 The genome of P. luminescens TT01 used in this study was sequenced to 550 account for any differences between lab strains and hence the published 551 genome (GenBank assembly GCA_000196155.1) (63). Sequencing was 552 performed on an Illumina MiSEQ platform with 250 bp paired-end reads. 553 Reads were trimmed using Sickle (https://github.com/najoshi/sickle) and a de 554 novo assembly of the genome was performed with SPAdes v3.0.0 run in 555 ‘careful’ mode (64). Only contigs that were at least 500 bp and had coverage 556 of at least 10 fold were kept for further analysis. The contigs were reordered 557 using Mauve Contig Mover (65) based on the reference assembly 558 GCA_000196155.1. The reordered contigs were artificially joined to create a 559 single fasta sequence that was used as the reference for mapping RNAseq 560 reads. The sequence was annotated using prokka v1.11 (66). Bowtie2 (67) 561 was used for mapping, in ‘very-sensitive’ mode. To identify putative small non- 562 coding RNAs we used the toRNAdo script [28; available at 563 https://github.com/pavsaz/toRNAdo] obtaining a list of ncRNAs for each 564 replicate sample. Putative ncRNAs identified in all three replicates of each 565 condition or in at least 4 samples in total were added to the annotation of the 566 genome. The artificially concatenated and annotated genome is available on bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

567 figshare (doi: 10.6084/m9.figshare.7473065). Following this, differential 568 expression analysis was performed using DeSeq2 (68) with the file outputs of 569 BEDtools coverageBed (69). A multi factor analysis was performed to 570 measure the effect of IPS treatment whilst accounting for sample pairing. A 571 False Discovery Rate of α = 0.05 was used. To identify the corresponding 572 alleles in the published assembly of P. luminescens TTO1 (63), a USEARCH 573 global alignment was performed using USEARCH version 8.0 (70) with the 574 protein sequences of assembly GCF_000196155.1 (63) as a database and 575 with a minimum identity threshold of 0.9. Additionally, a whole genome 576 alignment was done using blastn and visualized with the Artemis Comparison 577 Tool (71) to account for any alleles not identified by USEARCH. To investigate 578 the pathways affected, the Uniprot identifiers of proteins encoded by the 579 differentially expressed genes were used as input into STRING database 580 version 10.5 (72) in order to obtain a list of overrepresented KEGG pathways. 581 Multiple testing is corrected for using the method of Benjamini and Hochberg 582 (73). A total of 664 genes could be matched to items in the database. This 583 process was repeated for the up-regulated and down-regulated genes 584 separately, with 422 and 242 items matching to the database respectively. 585 586 AUTHOR STATEMENTS 587 588 Authors and contributions 589 Alexia Hapeshi: Conceptualisation, Investigation, Formal Analysis, and 590 Writing – Original Draft Preparation. 591 Jonathan Bennaroch: Investigation. 592 David Clarke: Resources, Writing – Review and Editing. 593 Nick Waterfield: Conceptualisation, Project Administration, Writing – Review 594 and Editing, Funding and Resources. 595 596 Conflicts of interest 597 The authors declare that there are no conflicts of interest. 598 599 Funding information bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

600 This work was funded by Warwick Medical School, University of Warwick. 601 602 Acknowledgements 603 We would like to thank Helge Bode (Goethe Universität Frankfurt, Germany) 604 for providing purified IPS. 605 606 607 REFERENCES 608 609 1. Paul VJ, Frautschy S, Fenical W, Nealson KH. Antibiotics in microbial 610 ecology - Isolation and structure assignment of several new 611 antibacterial compounds from the insect-symbiotic bacteria 612 Xenorhabdus spp. J Chem Ecol. 1981;7(3):589–97. 613 doi:10.1007/bf00987707 614 2. Kumar SN, Siji JV, Rajasekharan KN, Nambisan B, Mohandas C. 615 Bioactive stilbenes from a Bacillus sp. N strain associated with a novel 616 rhabditid entomopathogenic nematode. Lett Appl Microbiol. 2012 617 May;54(5):410–7. doi:10.1111/j.1472-765x.2012.03223.x 618 3. Ciche TA, Ensign JC. For the Insect Pathogen Photorhabdus 619 luminescens, Which End of a Nematode Is Out? Appl Environ Microbiol. 620 2003 Apr 1;69(4):1890–7. doi:10.1128/aem.69.4.1890-1897.2003 621 4. Daborn PJ, Waterfield N, Blight M a., Ffrench-Constant RH. Measuring 622 virulence factor expression by the pathogenic bacterium Photorhabdus 623 luminescens in culture and during insect infection. J Bacteriol. 624 2001;183(20):5834–9. doi:10.1128/jb.183.20.5834-5839.2001 625 5. Derzelle S, Duchaud E, Kunst F, Danchin A, Bertin P. Identification, 626 Characterization, and Regulation of a Cluster of Genes Involved in 627 Carbapenem Biosynthesis in Photorhabdus luminescens. Appl Environ 628 Microbiol. 2002 Aug 1;68(8):3780–9. doi:10.1128/aem.68.8.3780- 629 3789.2002 630 6. Sharma S. The lumicins: novel bacteriocins from Photorhabdus 631 luminescens with similarity to the uropathogenic-specific protein (USP) 632 from uropathogenic Escherichia coli. FEMS Microbiol Lett. 2002 Sep bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.

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881 FIGURES 882 883 Figure 1. The structure of 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) 884 885 Figure 2. Dose dependent effect of IPS on A) growth and B) bioluminescence 886 of P. luminescens TT01. Bacteria were grown in LB supplemented with either 887 DMSO or different concentrations of IPS. Shown are the averages of 4 888 biological replicates from 3 independent experiments ± standard error. 889 890 Figure 3. The effect of IPS addition at mid-exponential on growth (A) and 891 luminescence (B) of P. luminescens TT01. The bacteria were grown for ~5.5 892 h and 32 µg/ml IPS was added to the cultures. Shown are the averages from 893 three biological replicates ± standard error. 894 895 Figure 4. The influence of IPS on pigment production. A) Cultures of P. 896 luminescens following addition of either the DMSO solvent alone or 32 µg/ml 897 IPS (in DMSO) and growth for 24 h. B) P. luminescens TTO1 RifR and a stlA- 898 mutant strains streaked out on LB agar and incubated for 96 h. 899 900 Figure 5. The effect of IPS on P. luminescens biofilm. The amount of biofilm 901 formed by A) P. luminescens TTO1 RifR and B) P. luminescens stlA- as 902 measured using a crystal violet assay in the presence of either the DMSO 903 solvent or 32 µg/ml IPS in DMSO. Results are the average means of four 904 biological replicates ± standard error. ** p < 0.01. 905 906 Figure 6. KEGG pathways affected by the presence of ectopic IPS. 907 Overrepresented pathways were identified using STRING version 10.5 using 908 either all the differentially expressed genes in our dataset or the up and down-

909 regulated genes separately. Bars correspond to the –log10(FDR), whereby 910 multiple testing correction is done using the Benjamini and Hochberg method 911 (73), and circles indicate the number of affected genes in each pathway. 912 913 bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/498857; this version posted December 18, 2018. The copyright holder for this preprint (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 made available under aCC-BY-NC-ND 4.0 International license.