Physiological and molecular responses of a newly evolved auxotroph of Chlamydomonas to B12 deprivation

The corrinoid B12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions and we previously demonstrated that experimental evolution of the non-requiring alga Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupted C1 metabolism, caused an accumulation of starch and triacylglycerides and a decrease in photosynthetic pigments, proteins and free amino acids. B12 deprivation also caused a substantial increase in reactive oxygen species (ROS), which preceded rapid cell death. Surprisingly, survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B12 limitation and an increase in B12 use-efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B12-producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly adapted to B12 deprivation, association with B12-producers can ensure long-term survival whilst also providing the environment to evolve mechanisms to better tolerate B12 limitation.


Abstract 21
The corrinoid B 12 is synthesised only by prokaryotes yet is widely required by eukaryotes as 23 an enzyme cofactor. Microalgae have evolved B 12 dependence on multiple occasions and we 24 previously demonstrated that experimental evolution of the non-requiring alga 25 Chlamydomonas reinhardtii in media supplemented with B 12 generated a B 12 -dependent 26 mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology 27 of a nascent B 12 auxotroph. Our analyses demonstrate that B 12 deprivation of metE7 28 disrupted C1 metabolism, caused an accumulation of starch and triacylglycerides and a 29 decrease in photosynthetic pigments, proteins and free amino acids. B 12 deprivation also 30 caused a substantial increase in reactive oxygen species (ROS), which preceded rapid cell 31 death. Surprisingly, survival could be improved without compromising growth by 32 simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. 33 Significantly, we found further improvements in survival under B 12 limitation and an increase 34 in B 12 use-efficiency after metE7 underwent a further period of experimental evolution, this 35 Introduction 46 47 Over 50% of algal species require an exogenous source of B 12 for growth (1), yet large areas of the ocean are depleted of this vitamin (2, 3). Eukaryotic algae cannot 49 synthesise B 12 , but must instead obtain it from certain prokaryotes that can (1). Indeed, 50 whilst dissolved B 12 concentrations are positively correlated with bacterioplankton density (4, 51 5), they have been found to negatively correlate with phytoplankton abundance (6, 7). 52 Furthermore, nutrient amendment experiments suggests B 12 limits phytoplankton growth in 53 many aquatic ecosystems (8-10). Despite this, understanding of the physiological and 54 metabolic adaptations that B 12 -dependent algae employ to cope with B 12 deprivation is rather 55 limited. 56 57 In many algae B 12 is required as a cofactor for the B 12 -dependent methionine 58 synthase enzyme (METH) (11), although some algae encode a B 12 -independent isoform of 59 this enzyme (METE) and do not require B 12 for growth. Bertrand et al (12), showed that the 60 B 12 -dependent marine diatom Thalassiosira pseudonana, which encodes only METH, 61 responds to B 12 scarcity by increasing uptake capacity and altering the expression of 62 enzymes involved in C1 metabolism. Heal et al (13) found that despite these responses B 12 63 deprivation disrupted the central methionine cycle, transulfuration pathway and polyamine 64 biosynthesis. Phaeodactylum tricornutum, a marine diatom which uses but does not depend 65 on B 12 (encoding both METE and METH), responds similarly to T. pseudonana (12) but can 66 also rely on increasing expression of METE to maintain the production of methionine. 67 Phylogenetic analysis of the METE gene among diatoms shows no simple pattern of gene 68 loss or gain, as indeed is the case across the eukaryotes (14, 15), but there is a clear link 69 between the lack of a functional copy of the METE gene and B 12 -dependence (11, 16). 70 As with the diatoms, the phylogenetic distribution of METE within the Volvocales (a 71 family of green freshwater algae) points to gene loss on several independent occasions. culture containing metE7 at a density roughly 20 times greater than the alga. The 96 well 187 plates were incubated at 25°C, under continuous light at 100 µE·m -2 ·s -1 , on a shaking 188 platform at 120 rpm. Each week the cultures were diluted: Those in TAP +1000 ng·l -1 B 12 189 were diluted 10,000-fold, TAP +25 ng·l -1 B 12 = 100-fold, and TP = 5-fold. Every three weeks 190 10 µl of serial dilutions of each culture was also spotted onto TAP agar + Ampicillin (50 191 µg·ml -1 ) and Kasugamycin (75 µg·ml -1 ) and TAP agar + 1000 ng·l -1 B 12 to check for B 12 -192 independent C. reinhardtii, or bacterial contaminants and to act as a reserve in the case of 193 contamination. If cultures were found to be contaminated, then at the next transfer they were 194 replaced by colonies from the same well that had grown on the TAP agar plates. At four 195 points during the 12-month evolution period all cultures were transferred to TAP agar plates 196 where they were stored for 2 weeks during an absence from the lab, meaning that the total 197 Methionine synthase plays a central role in the C1 cycle (Fig. 1A), and thus facilitates 206 nucleotide synthesis and production of the universal methyl donor S-adenosylmethionine, 207 which is essential for many biosynthetic and epigenetic processes (25, 26). Wild-type (WT) 208 C. reinhardtii can operate these cycles in the absence of B 12 using the methionine synthase 209 variant METE, but metE7 relies solely on the B 12 -requiring METH isoform. Before 210 investigating the effect of B 12 deprivation on C1 metabolism in metE7 we first wanted to 211 eliminate the possibility that other mutations in the experimentally evolved metE7 line might 212 account for its B 12 dependent phenotype. We therefore generated an independent METE 213 mutant line (metE4) using CRISPR/Cpf1 (21). This mutant has an in-frame stop codon ( methionine levels were raised 6-fold, which was somewhat unexpected given that 229 methionine synthase activity was impeded. SAH levels were also significantly elevated, 230 whereas there was no effect on SAM. Consequently, the SAM:SAH ratio decreased by 10-231 fold to 3:1 under B 12 deprivation. We then studied the dynamics of these changes by 232 measuring metabolites and RNA abundance at several points during 3 days of B 12 233 deprivation and then for 2 days following add-back of 1000 ng·l -1 B 12 . The transcripts for all 234 six tested C1 cycle genes increased rapidly in the first 6 h and then plateaued; reintroduction 235 of B 12 led to an immediate reduction to near initial amounts (Fig. S2A). Similar profiles were 236 seen for the metabolites SAM and SAH, although the peak occurred later at 24 h (Fig. S2B). than 1 within 24 h. A subsequent gradual increase occurred over the next 2 days, and 240 resupply of B 12 increased this ratio further over the following 2 days. The likelihood therefore 241 is that many cellular processes would be impacted in B 12 -deprived metE7 cells. 242

B 12 deprivation significantly impacts cell physiology and biochemical composition 244
Our data demonstrate a substantial impact of B 12 limitation on the expression of C1 245 metabolic genes as well as the abundance of C1 metabolites. To elucidate downstream 246 consequences of perturbed C1 metabolism we also characterised broader physiological 247 responses to B 12 deprivation. As has been documented previously (17), growth of metE7 248 cells was significantly impaired in B 12 -deprived conditions (Fig. S3A). However, by day 2 the 249 B 12 deprived cells had a 36% larger diameter resulting in a 150% increase in volume ( Fig. 2A  250 and Fig. S3B), indicating that cell division was more restricted than overall growth. Moreover, 251 cell viability, which was assayed by the ability of cells to form colonies when plated on B 12 -252 1 0 replete TAP agar, decreased to below 25% within 4 days of B 12 limitation (Fig. S3C). This 253 was preceded by a reduction in photosystem II maximum efficiency (Fv/Fm) (Fig. S3D), an 254 often-used indicator of algal stress (27, 28). 255 The biochemical composition of C. reinhardtii cells is altered considerably and 256 similarly under various nutrient deprivations and so we hypothesised that B 12 limitation would 257 also induce broadly the same responses (20, 29, 30). Therefore, metE7 cells were 258 precultured as before in 200 ng·l -1 B 12 , then washed and resuspended in TAP with (1000 259 ng·l -1 ) or without B 12 and cultured mixotrophically for 4 days. Cultures were visually inspected 260 by microscopy ( Fig. 2A) and the amounts of various cellular components were measured on 261 day 2 and 4 ( Fig. 2B). Chlorophyll levels declined considerably under B 12 deprivation so that 262 by day four the cells had a bleached appearance with an 85% lower concentration than the 263 B 12 replete cells. Similarly, free fatty acids (FFA), polar lipids and proteins were at least 50% 264 lower under B 12 deprived conditions on day 4. Starch content on the other hand, showed the 265 largest absolute increase from B 12 replete to B 12 deprived cells (Fig S3), and triacylglycerides 266 were 10-fold higher in B 12 -deprived cells (Fig 2B), which effectively balanced the loss of 267 polar lipids and free fatty acids so that overall lipid levels were roughly 8-10% of dry mass in 268 both treatments. To look in more detail, quantification of free amino acids and fatty acid 1 1 composition of all lipid classes was carried out (Fig S4). By day 4 most of the amino acids 270 decreased significantly under B 12 deprivation. Particularly noteworthy is the reduction in 271 methionine, in contrast to its elevation at an earlier timepoint, and the increase in glutamine, 272 the only amino acid to be more abundant in B 12 deprived cells. Overall the degree of fatty 273 acid saturation was higher under B 12 deprivation, due mainly to an increase in the dominant 274 saturated fatty acids palmitate (16:0) and stearate (18:0) (Fig. S5B), although levels of 275 several unsaturated fatty acids, in particular 16:2, 16:3 (7,10,13) , 18:1 and 18:2, were also 276 elevated. 277 278

Responses to nitrogen deprivation improve survival under B 12 deprivation 279
Our results demonstrate that B 12 deprivation of metE7 causes several changes in 280 biochemical composition akin to those exhibited following nitrogen deprivation of WT C. 281 reinhardtii. To further investigate this comparison we measured growth, viability, and 282 photosynthetic efficiency under both conditions over a timecourse (Fig. S6). metE7 culture 283 density increased more under B 12 than nitrogen deprivation (Fig. S6A), but started to decline 284 after day 2, unlike under nitrogen deprivation where growth continued more slowly over 4 285 days. For cell viability, both conditions caused a decline, but while loss of viability continued 286 in B 12 deprived cells, under nitrogen deprivation the initial loss was followed by recovery (Fig.  287   S6B). Maximum photosynthetic efficiency of photosystem II, however, did not recover under 288 either condition, and its decline was more rapid in nitrogen-deprived cells (Fig. S6C). 289 The increased viability of metE7 under nitrogen compared with B 12 deprivation 290 suggested to us that either the metabolic role of B 12 would make it intrinsically more difficult 291 to cope without or that the evolutionary naivety of metE7 to B 12 dependence would mean it 292 had little time to evolve protective responses to B 12 limitation. We therefore tested whether 293 responses to nitrogen deprivation could afford some protection against B 12 deprivation. 294 Viability measurements were monitored over several days, and cultures lacking nitrogen or 295 B 12 behaved as previously (Fig 3A). However, metE7 cells deprived of both nitrogen and B 12 296 simultaneously were more similar to those starved on nitrogen: there was an initial decrease 297 in viability followed by recovery to a level significantly higher than in B 12 deprivation alone. 298 As total growth in B 12 and nitrogen deprivation was not significantly different from B 12 299 deprivation alone (Fig. S7) this apparent protective mechanism in response to nitrogen 300 deprivation is not simply a result of inhibiting growth and hence avoiding severe B 12 301 starvation. 302 In C. reinhardtii, as in many photosynthetic organisms, the absorption of light energy 303 in excess of that required for metabolism can increase the production of reactive oxygen 304 species (ROS) (31). To investigate whether the cell death observed under B 12 deprivation of 1 2 diacetate was incubated with cells at different timepoints during nutrient deprivation. We 307 found that ROS levels increased in all nutrient deprived conditions in the first two days but 308 were highest in those cells deprived of B 12 alone (Fig. 3B). This peak coincided with the start 309 of the substantial decline in cell viability (Fig. 3A). The combination of B 12 and nitrogen 310 deprivation reduced ROS levels to similar amounts to those seen in the nitrogen-deprived 311 cells, and so may be a factor behind reduced cell death. 312 313

Natural B 12 auxotroph Lobomonas rostrata fares better under B 12 limiting conditions 314 than metE7 315
Considering that metE7 quickly lost viability in the absence of B 12 while nitrogen 316 starvation invoked protective responses independent of B 12 status, it is possible that as a 317 novel auxotroph metE7's response to B 12 deprivation is simply underdeveloped. To test this 318 we compared the B 12 physiology of metE7 with Lobomonas rostrata, a naturally B 12 -319 dependent member of the same Volvocaceae family of chlorophyte algae (32, 33). Cell 320 viability was significantly greater in L. rostrata cells compared to the metE7 line after 2-4 days of B 12 deprivation despite also growing to a greater density (Fig S7A). Moreover, a B 12 322 dose-response experiment, in which the two species were each cultured mixotrophically in a 323 range of B 12 concentrations, revealed that L. rostrata reached a higher optical density than 324 metE7 at all B 12 concentrations below 90 ng·l -1 , while the inverse was true above 90 ng·l -1 325 ( Fig 4A). This indicates that L. rostrata has a lower B 12 requirement than metE7. 326 In the natural environment the ultimate source of B 12 is from prokaryotes since they 327 are the only known B 12 producers (34). In separate studies it was shown that B 12 -dependent 328 growth of L. rostrata and metE7 can be supported by the B 12 synthesising bacterium 329 1 4 higher density than L. rostrata under axenic, B 12 -supplemented conditions, it grew less well 333 in coculture with M. loti (Fig. 4B), indicating B 12 provision from the bacterium is less effective 334 at supporting the growth of metE7 than of L. rostrata, perhaps simply due to their different 335 B 12 requirements, but possibly due to more sophisticated symbiotic interactions. 336 337 Experimental evolution in coculture improves B 12 -use efficiency and resilience to B 12 338 deprivation 339 Together our data suggest that the newly evolved metE7 line is poorly adapted to 340 coping with B 12 deprivation, but we wanted to determine whether the metE7 line could evolve 341 improved tolerance to B 12 limiting conditions, so we employed an experimental evolution 342 approach. We designed three distinct conditions, referred to as H, L and C. Condition H 343 (TAP medium with high (1000 ng·l -1 ) B 12 ) was a continuation of the conditions that had 344 initially generated metE7 (17). Condition L (TAP medium with low (25 ng·l -1 ) B 12 ) was chosen 345 so that B 12 would limit growth. Condition C (coculture with M. loti in TP medium) was a 346 simplification of an environmental microbial community. Eight independent cultures for each 347 condition were established from a single colony and then subcultured once per week over a 348 total period of 10 months. To account for the different growth rates in the three conditions, 349 we applied the following dilution rates of 10,000, 100, and 5 times per week in condition H, L 350 and C respectively (Fig S8). After 10 months under selective conditions all 24 cultures had 351 survived and were then treated with antibiotics to remove the M. loti from condition C and to 352 ensure that there were no other contaminating bacteria. We then subcultured the lines in 353 mixotrophic conditions with TAP + 200 ng·l -1 B 12 three times over nine days to ensure they 354 were all acclimated to the same conditions. The behaviours of the algal populations, 355 hereafter referred to as metE7H, metE7L, and metE7C, were then compared alongside the 356 progenitor metE7 line, which had been maintained on TP agar with 1000 ng·l -1 B 12 without 357 subculturing. 358 Under high levels of B 12 (320 ng·l -1 ) a similar optical density was reached by the 359 progenitor metE7 strain and the metE7H and metE7C populations, whereas metE7L growth 360 was somewhat compromised (Fig. S10A). When grown across a range of B 12 concentrations 361 to determine a dose response, the metE7C populations reached a significantly higher optical 362 density at the lower concentrations of 20 and 40 ng·l -1 B 12 than the other lines (Fig. 5A). The 363 concentration of B 12 required to produce half the maximum growth (EC 50 ) of metE7C was 364 therefore much lower than the progenitor metE7 or metE7H (Fig. S10B) and this was 365 reflected in the higher B 12 use efficiency i.e. the maximal increase in yield (OD 730 ) that results 366 from an increase in B 12 concentration (Fig. 5B). However, the maximal growth rate of 367 metE7C was significantly lower (Fig. S10C), and it is tempting to conclude that this is a 368 necessary trade-off. We also compared the viability of the experimentally evolved lines 1 5 during B 12 deprivation (Fig. 5C). Fig. 5C shows that although all lines lost viability during B 12 370 deprivation, metE7L and metE7C survived substantially better, with a median survival time 371 more than a day longer (Fig. 5D) than both the progenitor metE7 and metE7H. 372

373
To elucidate which factors contributed to the improved survival, we performed a multi-374 parameter physiological analysis (Fig. S11). 16 parameters were measured across the 32 375 metE7 populations and the dataset visualised in three ways. Fig S11A presents the data as 376 a heatmap with the most similar populations, which generally were those exposed to the 377 same evolution conditions, clustered together to form a phylogenetic tree. Fig. S11B displays 378 the first two components of a principal component analysis of the data, which confirmed that 379 the experimental evolution populations tended to form separate clusters. Fig. S11C is a  380 correlation matrix of the parameters to reveal those pairs that are most positively or 1 6 negatively correlated with one another. A more definitive statistical approach was then used 382 to determine the most important parameters for predicting survival time during B 12 383 deprivation: Using stepwise minimisation of the Bayesian information criterion of the full 384 linear model the 15 other parameters were reduced to just three. So, it was concluded that 385 higher B 12 use efficiency, lower ROS levels and lower maximal growth rate were sufficient to 386 explain longer survival time under B 12 deprivation of the metE7 populations. 387

388
Comparison of the growth of the evolved lines when cocultured with M. loti showed, 389 perhaps unsurprisingly, that the metE7C lines grew better than the others (Fig 5E), and at 390 the end of the growth period had a significantly higher number of algae supported per 391 bacterium (Fig 5F). This algal:bacterial ratio was also optimally predicted by three 392 parameters: higher algal B 12 use efficiency and lower algal maximal growth rate, as for 393 survival time, but also lower algal B 12 uptake capacity. Together these results indicate that 394 experimental evolution in coculture not only improves growth in coculture but also increases 395 synthesis and could cause the observed reduction in protein abundance (Fig. 2B). However, 423 methionine levels increased between 12 and 24h of B 12 deprivation (Fig S1B), suggesting a 424 reduction in its use, proteolysis, or increased synthesis due to higher METH expression or 425 via alternative pathways such as the S-methylmethionine cycle, as documented in plants 426 (39). 427 METE transcript abundance showed a much higher dynamic range than METH during 428 B 12 deprivation and add-back (Fig. S2A) Chlorosis is a common symptom of nutrient deficiency in C. reinhardtii, evident in 444 nitrogen, sulfur, iron, and zinc limiting conditions and so it is not surprising that B 12 445 deprivation of metE7 caused a substantial decline in total chlorophyll (Fig. 2B) (48-50). The 446 decrease in total protein content occurred more slowly and was less substantial (50% 447 reduction over four days) than reported under nitrogen and sulphur deprivation (80% 448 reduction within one day) (51). During nitrogen and iron starvation in C. reinhardtii 449 membrane lipids decrease drastically concomitant with the increase in TAGs (52,53). This is 450 very much like what we observed for metE7 under B 12 deprivation, although here the level of 451 free fatty acids and polar lipids decreased by a roughly similar amount to the increase in 452 TAGs indicating there is little to no de novo fatty acid synthesis. In addition, B 12 deprivation 453 causes similar shifts in fatty acid composition to nitrogen and iron deprivation, most notably a 454 substantial increase in palmitic acid (16:0) and decrease in polyunsaturated 16:4 fatty acid 455 (53,54). Despite these similarities, B 12 deprivation may elicit an increase in TAGs by a 456 different pathway due to disrupted C1 metabolism, as has been observed in several 457 organisms (55)(56)(57). This is thought to be due to a reduction in the methylation potential 458 limiting membrane lipid synthesis and hence diverting more lipids towards TAGs (57,58). 459 Therefore, B 12 deprivation could provide a complementary approach to other nutrient 460 deprivation experiments in improving our understanding of lipid metabolism in C. reinhardtii 461 and other algae. 462 From an evolutionary perspective, the prevalence of vitamin B 12 dependence among 463 algae appears somewhat at odds with the severe fitness penalties that would be incurred 464 given limiting dissolved B 12 concentrations, particularly when the fitness benefit in replete B 12 465 is marginal (17). However, relative to optimal axenic laboratory conditions in which the 466 metE7 line evolved, in the environment multiple nutrients may colimit growth perhaps even 467 eliciting responses that mitigate against B 12 deprivation, as we observed here, and B 12 -468 producing bacteria may not simply co-occur with algae but also actively engage in 1 9 mutualistic interactions (1, 35, 59, 60). Furthermore, our evidence suggests that selection 470 under coculture conditions led to the newly evolved B 12 auxotroph developing increased B 12 471 use efficiency and becoming better adapted to tolerating B 12 limitation, which could make 472 this line more robust to the unreliable B 12 supply in the natural environment. However, these 473 improvements appeared to come at the expense of maximal growth rate in B 12 replete 474 conditions (Fig. S10C), which is not unexpected in light of previous experimental evolution 475 studies in C. reinhardtii (61). As one of the conserved responses of C. reinhardtii upon 476 detecting depletion of various nutrients is to decrease cell division, it is possible that slower 477 growth might even be selected for under B 12 deprivation. Indeed, a low growth rate was 478 found to be a significant predictor of greater survival time under B 12 deprivation, alongside 479 low ROS levels and high B 12 use efficiency. 480 The fact that metE7 survived a 10-month period either with limited artificial 481 supplementation of B 12 or by relying completely on bacterial B 12 provision, does suggest that 482 even a newly evolved and poorly adapted B 12 auxotroph would have ample opportunity to 483 adapt further. What adaptations are likely to improve growth and survival under B 12 484 deprivation are not altogether clear, but it is not unreasonable to assume that exaptation of 485 existing nutrient limitation responses would play a major role. B 12 dependence is certainly a 486 risky evolutionary strategy, and one which may have ended in extinction countless times, but 487 our work suggests that even the simplest of symbioses with B 12 -producing bacteria may be 488 sufficient to ensure the survival and drive the continued evolution of B 12 -dependent algae. 489