Evolutionary novelty in gravity sensing through horizontal gene transfer and high-order protein assembly

Horizontal gene transfer (HGT) can promote evolutionary adaptation by transforming a species’ relationship to the environment. In most well-understood cases of HGT, acquired and donor functions appear to remain closely related. Thus, the degree to which HGT can lead to evolutionary novelties remains unclear. Mucorales fungi sense gravity through the sedimentation of vacuolar protein crystals. Here, we identify the octahedral crystal matrix protein (OCTIN). Phylogenetic analysis strongly supports acquisition of octin by HGT from bacteria. A bacterial OCTIN forms high-order periplasmic oligomers, and inter-molecular disulphide bonds are formed by both fungal and bacterial OCTINs, suggesting that they share elements of a conserved assembly mechanism. However, estimated sedimentation velocities preclude a gravity-sensing function for the bacterial structures. Together, our data suggest that HGT from bacteria into the Mucorales allowed a dramatic increase in assembly scale and emergence of the gravity-sensing function. We conclude that HGT can lead to evolutionary novelties that emerge depending on the physiological and cellular context of protein assembly.

The ability to sense gravity allows plants and fungi to orient the growth of shoots and roots, and fruiting bodies, respectively. This response, known as gravitropism, depends on sedimentation of dense cytoplasmic bodies [20][21][22], which generate cell elongation-promoting signals at the cell cortex. Plant gravity sensing is mediated by starch bodies that form within specialized plastids [20]. In the fungi, gravitropism has been demonstrated in the multicellular Basidiomycota [21] and the Mucorales [22]. However, gravity-sensing organelles have only been examined in the Mucoralean Phycomyces blakesleeanus [23] where giant single-celled sporangiophores exhibit gravitropism through a combination of buoyant lipid globules and sedimenting protein crystals that form within vacuoles [24]. A crystal-less mutant grows normally, but displays defective gravitropism, indicating that the crystals indeed serve as gravity sensors [24][25][26]. Similar structures have been observed in other members of the Mucorales [22], suggesting that this function arose early in this lineage. However, its basis and evolutionary origin remain unknown.
Here, we identify the octahedral crystal matrix protein (OCTIN). Phylogenetic analyses indicate that octin was acquired from a gram-negative bacterium. Both Phycomyces crystals and bacterial OCTIN form disulfide-bonded high-order oligomers, suggesting that they share elements of a conserved assembly mechanism. Given the size of bacterial cells, thermal fluctuations are expected to dominate the movement of OCTIN oligomers. This precludes any speculated role in bacterial gravity sensing. We conclude that HGT of a bacterial octin into the common ancestor of the Mucorales is likely to have relieved constraints on OCTIN oligomer size, allowing evolution of the gravity-sensing function. The data exemplify a general mechanism for the evolution of adaptations based on HGT and high-order protein assembly.

Results and discussion
To determine the molecular basis of gravity sensing, we purified vacuolar crystals from P. blakesleeanus sporangiophores using the method of Ootaki and Wolken (Fig 1) [27]. As previously observed, a highly purified crystal fraction contains two major proteins, p55 and p14 (Fig 1C) [28]. Mass spectrometry indicates that peptides from these bands are derived from the N-(p14) and C-terminus (p55) of a single predicted protein, which we named OCTIN (Fig 1D). Edman degradation defines the N'-termini of p14 and p55 and full-length octin transcript is detected exclusively in sporangiophores (Figs 1D and S1A). These data indicate that p14 and p55 are derived through proteolytic processing of an OCTIN precursor. Furthermore, sequencing the octin gene from the crystal-less mutant reveals a stop codon at W326 (Figs 1D and S1B). Together, these observations identify two OCTIN-derived proteins as primary components of Phycomyces gravity-sensing crystals.
Full-length OCTIN is sporadically present in eukaryotes and bacteria (Figs 1E and S2). In the fungi, OCTIN is found exclusively in members of the Mucoromycotina, suggesting that it was acquired early on in this lineage. Homologs are also found in the protozoan Stramenopiles, including all sequenced Oomycetes, the Pelagophyceae diatom Aureococcus anophagefferens and both sequenced Haptophytes (the brown alga Emiliania huxleyi and the phytoplankton Chrysochromulina). OCTIN also occurs sporadically in diverse bacterial clades, where it is found in Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes ( Fig 1E). Mucorales octin sequences do not encode a predicted signal sequence, suggesting localization through the cytoplasm-to-vacuole targeting pathway, which has been associated with the import of oligomeric vacuolar resident proteins [29]. Predicted signal sequences are found in OCTIN homologs from gram-negative bacteria and the Oomycetes, suggesting that these proteins are directed to the periplasm and secretory pathway, respectively. The sporadic distribution of OCTIN in eukaryotes ( Fig 1E) could be explained by an early origin followed by extensive gene loss. However, both maximum likelihood (ML) and Bayesian analyses provide strong support for independent acquisition of OCTIN by the Mucoromycotina and Oomycetes through HGT from bacteria. In the ML tree, the Mucorales and Oomycetes each have a distinct sister bacterial group (Figs 2 and S3 and S4), while in the Bayesian tree, the Mucorales are nested within a clade of acido-and proteobacteria (S5 Fig). Enforcing eukaryote monophyly on the ML OCTIN phylogeny results in a topology significantly less likely than the unconstrained phylogeny as judged by the Shimodaira's Approximately Unbiased (AU) test (p-value = 0.021, S1 Table). The trees further suggest HGT among bacteria: acidobacteria and proteobacteria, as well as actinobacteria and proteobacteria, are interspersed to form distinct well-supported monophyletic groups (Figs 2 and S3, S4 and S5), while the constrained topology consistent with vertical transmission is significantly less likely (AU test pvalue = 0.009, S2 Table). OCTIN is found in a large number of species in deep branching clades in the proteobacteria and acidobacteria (S6 and S7 Figs), suggesting an ancient origin in bacteria. Together, the phylogenetic analyses support an origin for the gravity-sensing protein crystal through HGT from a gram-negative bacterium.
The OCTIN C-terminus contains a full-length formylglycine-generating enzyme (FGE) domain ( Fig 3A). In metazoans, FGE catalyzes the oxidation of cysteine to Cα-formylglycine to activate sulfatase enzymes in the endoplasmic reticulum (ER). In humans, its loss-of-function causes the fatal genetic disorder multiple sulfatase deficiency (MSD) [30]. Alignment between OCTIN from diverse species and human FGE reveals high overall sequence conservation, with many residues mutated in MSD being conserved in the OCTIN FGE domain. However, key FGE catalytic cysteines are absent in OCTIN sequences, suggesting that OCTIN does not function in sulfatase activation (S8 Fig). Interestingly, many other bacterial FGE domaincontaining proteins lack FGE catalytic residues, and like OCTIN, have N-terminal sequence extensions (S9 Fig). In some cases, these extensions show similarity to known domains, which include Kinase, Caspase, DinB, NATCH, and PEGA domains. DinB-FGE has been shown to function as a sulfoxide synthase. This activity depends on DinB catalytic residues that form contacts with the FGE domain [31,32]. Together, these data identify a bacterial superfamily of OCTIN-related proteins. The extent to which these function through structural or enzymatic mechanisms remains to be determined.
The position and number of OCTIN cysteine residues show significant variation between the diverse OCTIN-containing clades. However, within clades, cysteine residues can be well conserved ( Fig 3A), suggesting that they tailor OCTIN to its taxa-specific functions. When Phycomyces crystals are analyzed by SDS-PAGE under non-reducing conditions, p55 shifts to a highmolecular-weight species that migrates as smear around 250 kDa. By contrast, the migration of p14 is unchanged. These data indicate that p55 forms a disulphide-bonded sub-assembly ( Fig  3B). Rapid swelling and disintegration of crystals upon treatment with DTT (dithiothreitol) reveal the importance of disulphide bonds for crystal lattice stability. (Fig 3C and S1 Movie). Centrifugation confirms this effect-p55 and p14 are pelleted by centrifugation at 100,000 x g, whereas DTT treatment shifts both into the supernatant fraction. Together, these data further show that p14 associates with p55 through non-covalent interactions. Crystals also swell and dissolve upon addition of the protein denaturant sodium dodecyle sulfate (SDS) (S2 Movie). Neither DTT nor SDS fully solubilizes p55. However, when combined, they synergize to promote disassembly ( Fig 3D). Together, these data show that disulphide-bonded p55 sub-assemblies form a crystal lattice through additional non-covalent interactions. p14 is physically associated with the p55 lattice. However, its role in stabilizing this structure is unclear.
The origin of a gravity-sensing crystal through HGT from a gram-negative bacterium raises the important question of how bacterial OCTIN might be predisposed to this function. Bacteria descended from the likely octin donor are not currently genetically manipulable. To investigate this question, we expressed OCTIN from the gram-negative acidobacterium Terriglobus saanensis (OCTIN T ) in Escherichia coli. OCTIN T encodes a predicted signal sequence (SS T ) and a SS T -mCherry fusion protein is targeted to the periplasm as indicated by a fluorescent ring around the cell periphery. By contrast, a full-length OCTIN T -mCherry fusion protein produces punctate fluorescence at the cell periphery ( Fig 4A). Both proteins are released upon lysis of the outer membrane, indicating that they are indeed periplasmic ( Fig 4B). However, only OCTIN T can be pelleted by centrifugation, suggesting that patches seen by fluorescence represent stable high-order oligomers ( Fig 4C). Non-reducing SDS-PAGE shows that like Phycomyces OCTIN, OCTIN T forms intermolecular disulphide bonds (Fig 4D). Furthermore, as with Phycomyces OCTIN, SDS and DTT synergize to promote OCTIN T oligomer disassembly ( Fig 4E). Compared with Phycomyces OCTIN, DTT alone has little effect, suggesting that these assemblies rely more on non-covalent interactions. Nevertheless, these data support a related underlying mechanism of self-assembly for Phycomyces and bacterial OCTIN.
Phycomyces sporangiophores are approximately 100 μm in diameter [33] and OCTIN crystals have an average edge length of 5 μm [27]. By contrast, octin-containing bacteria whose and absence (−) of the reducing agent 2-ME. P55, but not p14, migrates as a high-molecular-weight smear in the absence of 2-ME. Note that p46 also shifts in the absence of 2-ME, suggesting that it is a processing variant of p55. (C) Stills taken from a video recording the disassembly of Phycomyces OCTIN crystals by the reducing agent DTT (S1 Movie). (D) Synergistic disassembly of sizes are known have diameters ranging from 0.3 to 0.8 μm [34][35][36][37][38][39]. To the best of our knowledge, bacterial gravitropism has not been observed. Moreover, assuming an OCTIN assembly size of 1 μm or less, and taking into account cytoplasmic viscosity [40], the density of OCTIN crystals [41], and the bacterial cytoplasm [41], an estimation of sedimentation velocity based on Stokes' law indicates that bacterial OCTIN oligomers would be too small to function as gravity sensors. The low ratio of particle movement by gravitational force relative to Brownian motion (Péclet number, [42]) for oligomers in this size range further demonstrates that their movements would be dominated by thermal fluctuations (S10 Fig and S1 Text) [22]. While the function of OCTIN in bacteria remains unknown, its ability to form high-order oligomers is likely to have predisposed neo-functionalization towards a role in gravity sensing in the Mucorales. This is likely to have required the accumulation of mutations relating to crystal Phycomyces OCTIN crystals by SDS and DTT. While SDS alone is sufficient to completely shift p14 to the supernatant after centrifugation at 100,000 x g, only the combination of SDS and DTT has the same effect on p55. 2-ME, 2-Mercaptoethanol; DTT, dithiothreitol; FGE, formylglycine-generating enzyme; OCTIN, octahedral crystal matrix protein; P, pellet; S, supernatant; SDS, sodium dodecyl sulfate; T, total.
https://doi.org/10.1371/journal.pbio.2004920.g003 lattice assembly, vacuole targeting, and proteolytic processing. If primitive assemblies were too small to function as gravity sensors (S10 Fig), what factors could account for the retention of octin? Phycomyces OCTIN crystals are found in clusters (Fig 1A), which increases their effective size and sedimentation velocity [24]. Similarly, early OCTIN oligomers could have acted as sensors by clustering. Other scenarios involving neutral selection or another function could also have played a role in the evolutionary transition. In the latter scenario, we note that presently available information does not preclude an enzymatic activity for OCTIN.
The periplasm of gram-negative bacteria and the eukaryotic secretory pathway are both oxidizing environments that share a related machinery for translocation of proteins from the cytoplasm [43]. Indeed, OCTIN T -mCherry is targeted to the ER when expressed in mammalian tissue culture cells (Fig 4F). To determine whether Phycomyces OCTIN (OCTIN P ) can self-assemble upon heterologous expression, we expressed an ER-targeted version in mammalian cells. This version of OCTIN co-localizes with an ER lumenal marker, but does not display a punctate signal, suggesting an absence of self-assembly. Western blotting further shows an absence of proteolytic processing (Fig 4G). This indicates that OCTIN crystal assembly is likely to require taxa-specific processing activities. Many vacuolar hydrolases are synthesized as auto-inhibited precursors, which are activated upon delivery to the vacuole through processing by resident proteases [44]. We speculate that the region between p14 and p55 functions to inhibit crystal lattice formation through an analogous mechanism (see S11 Fig for a model of OCTIN assembly). Phycomyces has yet to be transformed [33], and this limits its use as a model system. Thus, understanding the control of crystal assembly will require the identification of OCTIN processing factors and reconstitution in a genetically amenable model system.
Phylogenetic analyses strongly support the acquisition of bacterial OCTIN by the Mucorales ancestor through HGT (Figs 2 and S3, S4 and S5). Through its signal sequence, this protein would have been targeted to the endomembrane system ( Fig 4F). In this context, the size constraint on OCTIN oligomers was relieved, allowing eventual increase in assembly scale and emergence of the gravity-sensing novelty. The case of OCTIN exemplifies how HGT of a protein undergoing high-order assembly can lead to a novel function that emerges depending on a combination of cellular potentialities and physiological imperatives.

OCTIN identification and phylogenetic analyses
P. blakesleeanus wild-type strain NRRL155 [25] and crystal-less mutant strain C2 [24] were grown as previously described [41]. Octahedral crystals were purified as previously described [27]. Bands corresponding to p14 and p55 were analyzed by mass spectrometry and Edman degradation (Alphalyse A/S, Odense, Denmark). Peptides p14, p46, and p55 identified the same P. blakesleeanus protein (National Center for Biotechnology Information [NCBI] accession: XP_018295118.1). The search for OCTIN homologs was performed with BLASTP [45] against the NCBI nonredundant database [46] using the OCTIN-specific N-terminal domain (amino acids 1-500) as the query. HMMER3 [47] was used to confirm the presence of the FGE domain (PF03781) [48] in BLAST hits. The accessions of these hits are reported in S3 Table. The extended bacterial species trees (S2 and S9 Figs) were constructed based on a previously reported microbial phylogeny [49]. The original tree, which contains multiple strains from the same species, was pruned to retain 1 strain per species whose annotated genome is available in the NCBI Reference Sequence Database (RefSeq, ftp.ncbi.nlm.nih.gov/refseq/). PhyloPhlAn [49] was used to insert additional octin-possessing species that are not present in the original tree (S4 Table). All other species trees (S4, S6 and S7 Figs) were constructed from 400 conserved protein sequences by PhyloPhlAn using RefSeq bacterial proteomes. The presence of signal sequence was predicted using Phobius [50]. Phylogenetic trees were visualized with ETE3 [51].
To construct OCTIN protein trees (Figs 2 and S3 and S5), OCTIN sequences from the NCBI reference protein database were used. MAFFT [52] with the option E-INS-i was used to obtain sequence alignments, which were trimmed using Trimal [53] at a gap threshold of 70%. ML bootstrap analysis was performed with RAxML [54] using the automatic bootstrapping option [55] (300 replicates) and the PROTGAMMAILGX model as suggested by ProtTest [56]. The human FGE sequence, which serves as the outgroup (Fig 2), was placed on the ML tree a posteriori using the RAxML option -f v [57]. Bayesian trees were constructed using MrBayes [58], run with 12 chains, temperature 0.05, sampling every 500th generation for 300,000 generations. Convergence was assessed using RWTY [59]. The ML and Bayesian phylogenies, as well as the matrix used to derive them are accessible under the identifier S22330 at TreeBASE (https://treebase.org/). To compare the ML trees with and without the monophyly constraint, the best-scoring tree with monophyly constraint was constructed using RAxML with the same parameters specified above for the construction of unconstrained trees. Phylogenetic hypothesis testing using the resampling estimated log-likelihood (RELL) test, Shimodaira-Hasegawa (SH) test, Kishino-Hasegawa (KH) test, and AU test was then performed with the PAML package 'codeml' [60] and CONSEL [61].

Identification of FGE domain-containing proteins
The search for FGE domain-containing proteins was performed with HMMER3 [47], using the FGE alignment (PF03781) downloaded from http://pfam.xfam.org. The search was performed on RefSeq proteomes of species present in the bacterial phylogeny shown in S2 Fig. Sequences containing at least 100 amino acids upstream of the FGE domains were selected. Annotated domains within these sequences were identified using the hmmscan function of HMMER3 [47]. Homologs of the gliding motility protein GldK, whose N-terminal domain is not annotated, were manually added based on similiarity to the known GldK sequence from Flavobacterium johnsoniae (NCBI accession: AAW78679.1).

Recombinant OCTIN expression
T. saanensis octin was codon-optimized for expression in E. coli and the synthetic sequence was obtained from Genscript. Full-length octin P was amplified by reverse transcription polymerase chain reaction (RT-PCR) from Phycomyces sporangiophore total RNA. Octin sequences and mCherry fusions were integrated into the pETDuet-1 vector (Novagen, cat #71146) for transformation in E. coli strain HMS174 (Novagen, cat #69453). Primers used in generating the expression plasmids are listed in S5 Table. E. coli periplasmic extract was obtained following a previously described protocol [62] with modifications. The induced culture was centrifuged at 2,500 x g and 4 o C for 10 minutes. The pellet was then gently resuspended in ice-cold PE buffer (20% sucrose, 1 mM EDTA, 50 mM Tris pH 7.4) and placed on a nutating mixer at 4 o C for 15 minutes. This was followed by centrifugation at 2,500 x g and 4˚C for 10 minutes. The supernatant was transferred to a clean tube and supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher 78440). This extract was aliquoted and flashfrozen for disassembly assays and western blot. OCTIN T and mCherry variants were detected by western blotting using horseradish-peroxidase-conjugated rat anti-HA antibodies (ROCHE, cat# 12013819001) or mouse anti-mCherry (SAB2702286 SIGMA) and secondary goat anti-mouse IgG (SAB4600004 SIGMA). Blot images were acquired using the ChemiDoc Touch Imaging System (Bio-Rad).

OCTIN crystal and bacterial oligomer disassembly
Crystals suspended in Tris-buffered saline buffer (TBS; 10 mM Tris pH 7.2, 150 mM NaCl) were mounted on a microscope slide. DTT or SDS was added to one side of the coverslip to a final concentration of 50 mM or 0.1%, respectively. Crystal disassembly was recorded using an epifluorescence microscope (BX51; Olympus) and a digital camera (Coolsnap HQ; Photometrics) controlled by Metamorph.
Synergistic disassembly of Phycomyces crystals (Fig 3D) and bacterial OCTIN oligomers (Fig 4E) by SDS and DTT was performed by incubating the crystals or periplasmic extract with the indicated combinations of SDS and DTT for 30 minutes at 25 o C. This was followed by centrifugation at 100,000 x g for 30 minutes at 25 o C. The total sample and the resulting supernatant and pellet fractions were analyzed by SDS-PAGE.

E. coli imaging
Overnight cultures of transformed HMS174 cells were diluted into fresh media and allowed to grow to OD600 of 0.7 before induction with 1 mM IPTG. After 4 hours, 5 μl of the suspension was diluted into 1 ml of fresh LB media and 300 μl was placed on a 35-mm microscopy dish (Matek P35G-1.5-10-C) that had been pre-treated with 50 μg/ml poly-D-lysine (Sigma P7886). After 1 hour the media was removed and replaced with 2 ml of fresh media. Imaging was carried out with a Leica SP8 inverted laser-scanning confocal microscope fitted with a white-light laser and 100x lens of numerical aperture (NA) 1.4. Each image is composed of 4 averaged frames taken at 1% laser power at 587-nm excitation with a scan speed of 400 MHz.

Mammalian cell culture
HeLa cells cultured in 6-well dishes or 8-well chamber slides were transiently transfected with the indicated plasmids using lipofectamine 3000 (ThermoFisher) and cultured for 48 hours before fixing for microscopy or harvesting for western blot analysis. Cells were fixed with 4% Paraformaldehyde (EMS #15700) in phosphate buffered saline (PBS) and then kept in 90% glycerol PBS for imaging. Imaging was carried out using a Leica SP8 fitted with a 63x objective NA of 1.4. The white-light laser was set to 488 nm and 587 nm for GFP and mCherry, respectively. Images are a single z plane taken with 8 line averages at 5% laser power, with a scan speed of 200 MHz, 50% gain and a pixel size of 70 nm. To extract protein for western blotting, HeLa cells were lysed in RIPA buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1% Triton-X100, 0.1% Sodium Deoxycholate, 1% SDS) supplemented with Halt protease and phosphatase inhibitor cocktail. Insoluble material was pelleted at 10,000 x g and the supernatant fraction boiled in SDS-PAGE loading dye. 10 μg of total cell extract was run per lane. Western blotting was carried out as stated above. Members of the FGE subfamily possess catalytic residues and do not contain other domains. FGE domain-containing proteins whose N-terminal extension shows similarity to a known domain are color-coded according to the legend. Those containing novel domains are indicated by black bars. All of these lack FGE catalytic cysteine residues. FGE, formylglycine-generating enzyme; OCTIN, octahedral crystal matrix protein.

Supporting information
(TIF) S10 Fig. Estimated sedimentation properties of hypothetical OCTIN assemblies of varying  diameters. (A) Sedimentation velocity estimated based on Stokes' law, taking into account cytoplasmic density and viscosity (S1 Text). The cross indicates the reported sedimentation velocity of Phycomyces crystal clusters [26]. The estimated particle size corresponding to this sedimentation velocity is in agreement with actual cluster size [26]. The inset shows estimated sedimentation velocity of sub-micron particles in μm/minute. Grey region indicates the size range of cytoplasmic particles in OCTIN-possessing bacteria, given the cell diameter range of 0.3-0.8 μm (S1 Text). (B) Péclet number of hypothetical OCTIN assemblies. The cross indicates the Péclet number corresponding to the Phycomyces crystal cluster documented in reference [26]. Note that thermal fluctuations dominate the movement of assemblies in the size range of bacterial cytoplasmic bodies. OCTIN, octahedral crystal matrix protein.
(TIF) S11 Fig. Model for formation of the OCTIN crystal lattice. The boxed cartoon depicts different regions of full-length OCTIN. The formation of a 3-dimensional protein lattice requires a minimum of 3 intermolecular contacts. For simplicity, the disulphide crosslinked p55 subassembly is depicted as a trimer and the lattice is depicted in two dimensions. We speculate that non-covalent contacts required for assembly are shielded by the region between p14 and p55, which is removed by proteolytic processing. The folding and processing events could occur simultaneously or in the opposite order to that depicted. Note that the role of p14 in lattice assembly remains unclear. OCTIN, octahedral crystal matrix protein.