Functional diversity of isoprenoidal lipids in Methylobacterium extorquens PA1

Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate whether isoprenoid lipids play a complementary role in outer membrane ordering and cellular fitness. By genetically knocking out hpnE, and crtB we disrupted the production of squalene, and phytoene in Methylobacterium extorquens PA1, which are the presumed precursors for hopanoids and carotenoids, respectively. Deletion of hpnE unexpectedly revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in a pigmentation with a C30 backbone, rather than the previously predicted C40 phytoene-derived pathway. We demonstrate that hopanoids but not carotenoids are essential for growth at high temperature. However, disruption of either carotenoid or hopanoid synthesis leads to opposing effects on outer membrane lipid packing. These observations show that hopanoids and carotenoids may serve complementary biophysical roles in the outer membrane. Phylogenetic analysis suggests that M. extorquens may have acquired the C30 pathway through lateral gene transfer with Planctomycetes. This suggests that the C30 carotenoid pathway may have provided an evolutionary advantage to M. extorquens. Importance All cells have a membrane that delineates the boundary between life and its environment. To function properly, membranes must maintain a delicate balance of physical and chemical properties. Lipids play a crucial role in tuning membrane properties. In eukaryotic organisms from yeast to mammals, sterols are essential for assembling a cell surface membrane that can support life. However, bacteria generally do not make sterols, so how do they solve this problem? Hopanoids and carotenoids are two major bacterial lipids, that are proposed as sterol surrogates. In this study we explore the bacterium M. extorquens for studying the role of hopanoids and carotenoids in surface membrane properties and cellular growth. Our findings suggest that hopanoids and carotenoids may serve complementary roles balancing outer membrane properties, and provide a foundation for elucidating the principles of surface membrane adaptation.


Introduction 55
Microorganisms can withstand a diversity of environmental stresses ranging from extreme 56 temperatures to the immune defenses of multicellular organisms. The cellular surface membrane 57 serves as a first line of defense against environmental perturbations and the membrane's lipid 58 composition is critical for stress resistance. On the one hand the membrane must be robust enough 59 to withstand chemical and physical challenges. On the other hand, the membrane must be fluid 60 enough to support bioactivity. In eukaryotic organisms such as yeast, sterols play a crucial role in 61 achieving a fluid yet mechanically robust cell surface membrane 1 . However, bacteria generally do 62 not synthesize sterols with very few exceptions 2,3 . 63

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The absence of sterols from most prokaryotes suggests that alternate lipids may serve analogous 65 roles in surface membranes. All three domains of life possess isoprenoid synthesis pathways 66 derived from a common C5 isoprene building block which give rise to a broad suite of diverse lipid 67 classes including sterols, but also carotenoids and hopanoids, and the majority of archaeal lipids. 68 Because of their structural similarities that are derived from a common C5 isoprene building block, 69 resulting in rigid and often semi-planar structures, isoprenoid-derived lipids may share certain 70 biophysical features in membranes 3 . However, the mechanism and exact influence of isoprenoid 71 lipids on prokaryotic membrane properties and cellular fitness remains relatively unexplored. in vitro measurement on purified outer membranes 6 . Our findings showed that ∆shc, and ∆hpnE 168 mutant strains had a much lower GP which indicated less lipid packing as compared to the WT 169 strain even at their optimal growth temperature, whereas, ∆crtN, and ∆crtP strains had increased 170 lipid packing compared to the WT strain ( Figure 4A). These results imply that even in the native 171 state, the outer membrane lipids of the hopanoid knockout strains were less packed as compared 172 to the WT strain. In addition, the loss of carotenoids (∆crtN, ∆crtP) seemed to slightly increase 173 lipid packing. We then measured the GP at the maximum growth temperature of the hopanoid 10 mutants (32°C), and we showed that there is no marked change in GP (∆GP) for the WT strain as 175 opposed to ∆shc and ∆hpnE strains ( Figure 4B). We propose that cells preserve a certain range of 176 parameters to maintain their vitality and ability to survive challenging environmental 177 perturbations. Hence, the removal of hopanoids highly restricts their fitness at higher temperatures 178 by compromising the cellular adaptability. The phylogeny of HpnCDE enzymes showed monophyly of Alpha-and Gammaproteobacteria 26 , 192 but HpnCDE appeared more conserved than CrtB-CrtD-CrtI 26 (Figure 5A). This monophyly of 193 Alpha-and Gammaproteobacteria suggested that both squalene and C40 carotenoid biosynthesis 194 were ancestral in Proteobacteria. By contrast, the C30 FAD-dependent desaturase enzymes CrtN 195 and CrtP, displayed a more limited distribution in Alphaproteobacteria, particularly in 196 Rhodospirillales, Rhizobiales, Acetobacterales, Azospirillales orders (taxonomic orders according 197 to GTDB; Figure 5A). In addition, these sequences, including the M. extorquens ones, did not 198 branch close to, nor monophyletically with, the Gammaproteobacteria. Instead, the respective 199 alphaproteobacterial groups of CrtN and CrtP branched within the Planctomycetes (Figure 5B), 200 suggesting lateral gene transfer (LGT) from this group ( Figure 5B). Planctomycetes are a distant 201 bacterial phylum that had recently been proposed to produce C30 carotenoids via squalene synthesis 202 enzymes HpnCDE 26 . The similar topology between CrtN and CrtP branches ( Figure 5B) suggested 203 that these genes were transferred together i.e. in the same DNA fragment/locus. Therefore, unlike 204 CrtI-CrtD or HpnCDE enzymes which indicated an ancestral feature of Proteobacteria, the C30 205 carotenoid pathway in some Alphaproteobacteria orders suggest that they originated later by LGT 206 from Planctomycetes. 207 208

Discussion 209
In order to establish M. extorquens as a system to study the comparative role of carotenoids and 210 hopanoids in determining membrane properties, we first determined how to perturb biosynthesis 211 of the two pathways independently. Hopanoid biosynthesis in M. extorquens has been relatively 212 well described 27 , however the squalene synthase was never formally identified or confirmed by a 213 knockout strain. We identified and confirmed the function of hpnE as a key gene that would disrupt 214 squalene synthesis 19 , thereby disrupting hopanoids biosynthesis and preventing squalene 215 accumulation 28-30 . Under the assumption that carotenoids were derived from phytoene in M. 216 extorquens 25 , we targeted a phytoene synthase gene crtB. However, surprisingly deletion of crtB 217 showed no phenotype in pigmentation, whilst deletion of hpnE yielded non-pigmented mutant 218 strains that also lacked hopanoids. These unexpected results revealed that carotenoid synthesis was 219 derived from squalene rather than phytoene through a pathway that has recently been shown to 220 produce C30 carotenoids 20 . 221

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Having identified the genes required to independently disrupt the hopanoid and carotenoid 223 pathways, we investigated the effects of varying temperature on cellular growth rates in strains 224 deficient in either hopanoids or carotenoids. Hopanoids have been shown to modulate bacterial 225 membrane properties in a manner analogous to eukaryotic sterols 5,6 . In M. extorquens deletion of 226 hopanoid synthesis by deleting either shc (squalene hopene cyclase) or hpnE (hydroxysqualene 227 oxidoreductase) resulted in a large growth deterioration at higher temperatures. It has previously 228 been shown in other organisms that hopanoids are associated with sensitivity to high 229 temperatures 9-12,31 , and MD simulations also suggest that hopanoids could reinforce membranes 230 at higher temperatures 32 . Hopanoid biosynthesis deletion in both ∆shc and ∆hpnE mutants resulted 231 in a large decrease in lipid packing measured in vivo, consistent with our previous observations 232 with purified outer membranes 6 . Such low lipid packing, which is indicative of higher fluidity and 233 lower mechanical robustness, could render the outer membrane susceptible to destabilization at 234 higher temperatures, which can explain the growth impairment observed at higher temperatures. 235 Interestingly, the change in lipid packing between 27 and 32˚C was much higher for ∆hpnE and 236 ∆shc mutant strains relative to the WT strain, suggesting impaired homeoviscous adaptation in the 237 absence of hopanoids. 238 239 It has been hypothesized that carotenoids could share some of the lipid ordering properties of 240 sterols 8,33,34 . Since ∆hpnE deletion eliminated both hopanoid and carotenoid synthesis, we had to 241 target genes at a later stage of the carotenoid pathway that would allow to independently delete 242 13 carotenoid synthesis to study the impact on growth and lipid packing. We targeted the genes 243 involved in C30 biosynthesis; crtN and crtP, which both resulted in non-pigmented mutants that 244 still produced hopanoids. Neither of the carotenoid mutants showed a significant growth 245 impairment at any temperature from 10°C to 34˚C which is a phenotype similar to what has been 246 shown in Acholeplasma 35 , suggesting that in contrast to hopanoids, carotenoids are not critical for 247 temperature adaptation. However, it is also possible that carotenoid deletion can be compensated  production via HpnCDE implying that the production of precursor for carotenoid and hopanoid 282 biosynthesis in M. extorquens is controlled by the same genetic mechanisms. This fact also 283 supports the notion that the two isoprenoid lipid classes (hopanoids and carotenoids) serve 284 complementary roles in modulating membrane properties. We consequently hypothesize that the 285 observed overexpression of carotenoids 2,18,26 is due to squalene accumulation ( Table 1)

Evolutionary analyses for C30 and C40 carotenoid pathway 324
We performed protein searches of CrtI (P54980), CrtD (Q01671), CrtN (O07855) and CrtP 325 (Q2FV57) against NCBI database using phmmer 45 and with e-value threshold of 1e-5. We 326 combined all the sequences obtained and using GTDB taxonomy 46 , we removed redundant 327 sequences by taxonomic orders (from 90 up to 50% of identity threshold for the less and more 328 represented groups respectively). We then aligned this set of non-redundant sequences using 329 MAFFT 47 and performed a fast phylogenetic tree using FastTree 48 to exclude spurious sequences. 330 Once we obtained the final set of sequences, we re-aligned with MAFFT and removed those 331

In vivo Di-4 spectroscopy 394
Three biological triplicates of cells were grown at either 27°C or 32°C until cultures reached mid 395 exponential growth at around OD600 ~0.5. Cells were then diluted to OD600 0.2, washed and 396 resuspended in succinate-free media. Cells were then incubated with 80nM Di-4 ANEPPDHQ 397 (ThermoFisher, D36802) for 10 minutes at 950 rpm shaking on a thermomixer (Eppendorf, 398 ThermomixerC). Subsequently, cells were plated onto a black 96-well plate in analytical triplicates 399 per sample, and measured in a plate reader (TecanSpark M20). Excitation was set to: 485 nm, and 400 emission was recorded at 540 nm and 670 nm with a bandwidth of 20nm. 401 402

Strains, Construction of Plasmids, Generation of Mutants, and Gene complementation 403
Methylobacterium extorquens PA1 with cellulose synthase deletion was used in this study and 404 referred to hereafter as WT 43,44 , ∆shc was already available 6 . Genes for carotenoids biosynthesis 405 were identified based on M. extorquens gene annotations for phytoene desaturase and phytoene 406 synthases, and BLASTp was used to reconstruct the carotenoids biosynthesis pathway as shown 407 Mutants were constructed by unmarked allelic exchange as described 56,57 , for each gene primers 408 were designed to include 500bp upstream and 500bp downstream overhangs of the gene. The 409 produced PCR product was then used as a template for the construction of 2 plasmids one to delete 410 the gene and one for the inducible expression of the gene in the knockout strain as explained in 411 (Table 2). For gene deletion: plasmid pCM433 56 was linearized via restriction digestion using 412 enzymes NotI-HF, and SacI-HF (NEB, R3189, R3156 respectively), overhangs upstream and 413 downstream of the gene of interest were amplified (primers sequences available in Table S1) and 414 purified then cloned into linearized pCM433 using In-Fusion HD Cloning plus kit (Takara), primer 415 design was done using primer design tool (Takara). For inducible expression of the gene: plasmid 416 pLC291 was linearized using EcoRI-Hf, and KpnI-HF restriction enzymes (NEB R3101, R3142 417 respectively), gene was then PCR amplified and purified then cloned into plasmid pLC291 44 using 418 In-Fusion HD Cloning plus kit (Takara). All PCR products and linearized vector were purified 419 (Macherey and Nagel, Nucleospin PCR clean-up Gel extraction). 420 Deletion plasmids were introduced into WT via triparental conjugation. WT cells were mated with 421 E. Coli pRK2073 helper cells, and E. coli Stellar cells that carry the deletion/expression plasmid, 422 and the mating was done using a ratio of 5:1:1 Acceptor-strain : helper-strain : donor-strain. The 423 conjugation was done on NB-Agar plates at 30°C, overnight, then the cells were plated on hypho 424 media-agar plates with Tmp, and Tc. The clones were then grown for 9 hours in liquid media, then 425 plated on 10% sucrose plates for selection of mutants. Colony PCR was then performed on clones 426 from the sucrose plates, using Primers (Table S1)