Molecular complementarity between simple, universal molecules and ions limited phenotype space in the precursors of cells

Polyphosphate

PolyP is likely to have been abundant on the prebiotic earth because it can be produced readily from the dehydration of phosphate rock at high temperature. It was therefore a freely available source of energy that could have been used to activate the precursors of fatty acids, phospholipids, peptides and nucleic acids. In modern bacteria, polyphosphate kinase 1 or PPK1 is involved in functions that include quorum sensing, biofilm formation, motility and virulence whilst an exopolyphosphatase, PPX1, is involved in sporulation [14]. Defects in some of these functions may be related to a role for PolyP in the compaction of the nucleoid, which occurs in ppk1 mutants of Pseudomonas aeruginosa [27]; it may therefore be significant that PolyP binds in vitro to the histone-like, HU proteins of E. coli and P. aeruginosa since such binding might displace these proteins from the DNA [14]. The cellular functions of PolyP may also depend on its role as an energy donor. As well as PPK1, many bacteria have PPK2, a kinase that uses PolyP to catalyze the formation of nucleoside triphosphates and, in particular, the formation of GTP from GDP. Moreover, several bacteria have an NAD kinase that can catalyze the formation of NADP using either PolyP or ATP and an enzyme that can catalyze the formation of glucose-6-phosphate from glucose using PolyP [28],[29]. It has been proposed that the ancestor of glucose kinases in the hexokinase family was similar to glucomannokinase and used PolyP [30]. Another aspect of the role of PolyP in energy metabolism is in the response of cells to nutrient deprivation such as the lack of an amino acid; this deprivation can lead to a 100-fold increase in the level of PolyP which, in binding to the Lon protease, activates protein degradation (largely from inactive ribosomes) to yield a pool of amino acids that can be used to make other proteins [31]. During the nutrient starvation of Helicobacter pylori, PolyP binds to the principal sigma factor and, during such starvation, mutant strains defective in the interaction die, consistent with the authors’ suggestion that PolyP is a second messenger determining gene expression during stress in H. pylori and other pathogens [32].

PolyP has been implicated in the bacterial cell cycle. Addition of PolyP at a concentration of 0.05% led to the filamentation of Bacillus cereus with no septum formation but with apparently normal chromosome replication and segregation [33]. In Caulobacter crescentus, PolyP (and the alarmone ppGpp) inhibit the swarmer-to-stalked transition; moreover, in conditions of carbon depletion in which swarmer cells should not initiate chromosome replication, swarmer cells that are unable to make PolyP or ppGpp not only initiate chromosome replication but also cleave the replication inhibitor CtrA and develop polar stalks, consistent with PolyP being a major cell cycle regulator [34].

Finally, PolyP is believed to be central to the survival strategies of certain modern bacteria. At low temperatures, when phosphate is available but nitrogen is scarce, Yersinia pseudotuberculosis and Listeria monocytogenes store the phosphate as PolyP for future use [35]. In the cyanobacterium, Aphanizomenon ovalisporum, the huge increase in DNA as dormant cells are formed probably depends on phosphate provided by PolyP bodies; this increase is believed to allow long-term survival during stress and a subsequent rapid resumption of metabolism and cell division when conditions improve [36]. Similarly, Corynebacterium glutamicum stores PolyP when phosphate is available and the partitioning between cytosolic and granular forms of PolyP is dynamical [37].

Poly-(R)-3-hydroxybutyrate

PHB is a linear head-to-tail homopolymer of (R)-(3-hydroxybutyric acid). In a wide variety of bacteria, PHB is often known as a polymer of high molecular mass that constitutes a carbon store in the form of crystalline cytoplasmic granules that may constitute up to 90% of the cell’s dry weight [19]. However, a short chain form of PHB with a low molecular mass (<150 residues) has been found in all prokaryotes and eukaryotes examined to date. In E. coli, synthesis of this PHB depends on fatty acid metabolic pathways and short fatty acid catabolism, via the expression of genes in the atoDAEB operon as regulated by the AtoSC system [38]. AtoS is a membrane-bound histidine kinase that undergoes auto-phosphorylation in response to acetoacetate; AtoS then phosphorylates the response regulator AtoC which in turn activates transcription of the atoDAEB operon to allow growth on short-chain fatty acids.

The short chain form of PHB forms a non-covalent complex with PolyP in the membranes of bacteria and mitochondria [39]-[41] which functions as a calcium-selective channel as shown by both reconstitution experiments using cell extracts and in vitro experiments using chemically synthesized constituents [15]. This complex has many of the characteristics usually attributed to proteinaceous calcium channels, namely, selectivity for divalent over monovalent ions, a high permeance to calcium, strontium and barium ions, blockage by low concentrations of transition metal cations such as lanthanum, and, remarkably, voltage-activation and voltage-dependence [42]. PolyP and PHB were also found to be components of the calcium-transporting transient receptor potential channel TRPM8, a sensor of low temperature and cooling agents in the peripheral nervous system [43].

An ion channel role for the PHB/PolyP complex extends to potassium ions. In the potassium channel of Streptomyces lividans, KcsA, each of the constituent four polypeptides is covalently modified by very short chain PHB (<15 residues). These PHB-modified polypeptides surround a PolyP core that attracts, binds, and conducts K⁺ in response to an electrochemical stimulus whilst the polypeptides determine the selectivity for this ion by preventing access to the PolyP chain at the extracellular side (‘selectivity filter’) and by surrounding the PolyP chain with arginines at the cytoplasmic side to discourage binding of divalent cations [44]. A role for PolyP and PHB may extend to many other proteinaceous ion channels. For example, the mammalian calcium-transporting channel TRPM8, mentioned above, which forms a complex with PolyP [43], is modified by covalent attachment of very short chain PHB [45].

PHB/PolyP complexes are also involved in the uptake of DNA from the environment. When E. coli is subjected to the stresses leading to competence, it produces up to 20% more PHB/PolyP complexes than when growing exponentially [17]. Divalent cations such as Mg²⁺, Ca²⁺, Mn²⁺ and Sr²⁺ may cross-bridge phosphate residues of PolyP to phosphate residues of one strand of DNA. Following a transition back to normal growth conditions, the excess complexes are withdrawn from the membrane thereby transporting the bound single-stranded DNA through the PHB channel and into the cytoplasm. In support of this hypothesis, when DNA uptake is interrupted in E. coli, single-stranded DNA can be found complexed to PHB [42].

PHB/PolyP complexes may also be at the origin of pumps responsible for the efflux of ions from the cell [46]. Both PHB and PolyP are present in a very common pump, the Ca²⁺-ATPase of human erythrocyte membranes [16]. The putative mechanism is that the Ca(PolyP) chain is secreted through the PHB sheath by addition of phosphate from ATP to the PolyP chain at the cytoplasmic end and degradation of PolyP at the periplasmic face by exopolyphosphatases. This is a feasible mechanism since the ionic bonds between PolyP and Ca²⁺ are very strong whilst the ion-dipole bonds between PHB and Ca²⁺ are relatively weak.

Covalent modification of proteins by PHB is widespread. In E. coli, most PHB is associated with the ribosomal fraction. PHB-modified proteins include: the porins, OmpA, OmpF, OmpC and OmpW; one of the principal proteins of the outer membrane, Lpp; a subunit of the F1 ATP synthase, AtpD; elongation factors EF-Tu and EF-Ts; heat shock proteins, GroEL and DnaK; ribosomal proteins RplL and S1; histone-like protein, H-NS; RNA polymerase subunit, RpoA [17],[18]. It is likely that the covalent modification by PHB of proteins underlies many interactions between these proteins and other proteins or membranes or nucleic acids. One reason for this is that such modification would make the proteins less water-soluble and more lipid-soluble. Hence, the amphiphilicity and flexibility of PHB would facilitate the interaction of these proteins with nucleic acids and with membranes and, indeed, their interaction with one another as in the modern ribosome.

Like its PolyP partner, PHB is probably important for survival and for transitions between growth regimes. First, PHB can act as a carbon source to be used when growth conditions permit. Second, changes in ion levels often accompany transitions between growth and survival conditions (as mentioned above) and, here again, there is a clear role early in evolution for PHB at the heart of channels and pumps.

It is tempting to include PHB modification to many key proteins in a general role for PolyP/PHB in stress conditions and in transitions. If PHB modification is needed for the functioning of many proteins, it might be expected that mechanisms would have evolved to regulate such modification. An obvious mechanism would be the phosphorylation by protein kinases of the residues on proteins that would otherwise undergo PHB modification, coupled with the reciprocal action of phosphatases. In E. coli and other bacteria, phosphorylation on serine, threonine and tyrosine residues is common (in addition to the better-known phosphorylation on histidine) [47]-[49]. It is therefore significant that these residues are modified by PHB. For example, serine residues S163 and S167 of the sorting signal of OmpA are modified by PHB to allow OmpA to be incorporated as a narrow pore into lipid bilayers at room temperature [50]. Finally, we note that, as a phosphate donor, PolyP is often associated with PHB in proteins; early in evolution, this may have put PolyP in the right place at the right time to help modify a residue with a phosphate.

Polyamines

The synthesis of polyamines involves the conversion of L-arginine to ornithine via arginase followed by the decarboxylation of ornithine by ornithine decarboxylase to give the diamine putrescine which is the precursor for the triamine spermidine (via spermidine synthase) and tetramine spermine (via spermine synthase) [51]. Polyamines were believed to be present in all cells in the three kingdoms until recently when a few bacterial species, such as Staphylococcus aureus, were found to lack the genes needed for their synthesis and, in the case of S. aureus, to be able to grow in their absence [52]. Polyamines like putrescine, spermidine, spermine and cadaverine are required for the growth of E. coli where they mainly exist in the form of polyamine–RNA complexes [53]. Spermidine and spermine can also bridge the major and minor grooves of DNA, clamping together two different molecules or two parts of the same molecule [54]. In eukaryotes, lowering polyamine levels results in the partial unwinding of DNA and the unmasking of previously buried sequences that are potential binding sites for factors regulating transcription [55]. In addition to interacting with nucleic acids and binding to ribosomes, they block porins and decrease membrane permeability, and they stimulate the synthesis of proteins encoded by the “polyamine modulon” [20]. Many of these proteins are global regulators including the sigma factors RpoS, FecI, RpoN, and related RNA polymerase omega subunit RpoZ, the adenylate cyclase Cya, the transcription factor, Cra, which senses the glycolytic flux, SpoT which catalyzes both the hydrolysis and synthesis of (p)ppGpp, the effector of the stringent response, and the nucleoid-associated proteins Fis and H-NS. Recently, it has been proposed that polyamines play an important role in the stationary phase survival of E. coli because they stimulate the synthesis of SpoT, which leads to increased ppGpp levels in these conditions, and of RpoZ, which is needed for is necessary for the regulation of RNA synthesis with alternative sigma factors by ppGpp [56]. Polyamine functions in bacteria also include biosynthesis of siderophores, acid resistance, free radical scavenging, and biofilm formation [57],[58]. It should be noted here that, in plants, polyamines are implicated in protection against a wide variety of abiotic stresses including salt and drought stress, mineral deficiencies, chilling, wounding, heavy metals, UV, ozone and paraquat [21]. Note too that covalent modification of intracellular proteins by polyamines is one of the few modifications that add positive charges, as in the case of the stabilization of microtubules by transglutaminase-catalyzed polyamination of tubulins [59].

What is the relationship between polyamines and PHB/PolyP? AtoC is the response regulator of the AtoS-AtoC two-component system that activates atoDAEB to produce PHB as mentioned above. AtoC is also an antizyme responsible for the post-translational inhibition of polyamine biosynthetic enzymes [60]. Polyamines and PolyP interact directly [61]. Moreover, a speABC and cadA mutant of E. coli, which is deficient in polyamines, accumulates less PolyP during under conditions of amino acid starvation than the wild type whilst a speG mutant, which has higher levels of polyamines, accumulates more PolyP; given the increased stability in vitro conferred by polyamines on PolyP granules, the authors concluded that polyamines interact with PolyP and affect PolyP accumulation [62].

Interactions with lipids

Lipids have long been implicated in the origins of life [11],[63]-[65]. Their interactions with the other constituents of cells, such as ribosomes, have been shown to result in the accumulation of these constituents within liposomes [66]. In modern bacteria, interactions between phospholipids and the nascent peptides generated via coupling between transcription, translation and protein insertion into membrane are considered to structure the membrane into domains that help determine the phenotype [67]. Such interactions are a prime example of the importance of molecular complementarity.

Polyamines also interact with lipids. Interaction between polyamines and acidic phospholipids is an ionic one such that the strongest interaction is between the polyamine with the highest positive charges and the phospholipid with the highest content of negative groups [68]. By bridging proteins and lipids and by shielding the surface charges, interaction with polyamines may reduce the repulsive forces between negatively charged membrane components [69]. Molecular complementarity is based on interactions between molecules that protect them from degradation and lead to their accumulation. In line with this, exogenous polyamines help protoplasts and spheroplasts resist osmotic shocks [70]-[72]; intracellular physiological concentrations of spermine and spermidine increase the mechanical stability of resealed erythrocyte ghosts [73]; importantly, the binding of polyamines to the polar head-groups of the membranes helps protect them from lipid peroxidation [74],[75]. It has been suggested that polyamine binding to the acidic groups of phospholipids in membranes could lead to the clustering (and possibly phase-separation) of these phospholipids and even a bridging between acidic phospholipid domains of closely apposed membranes [69]. The latter possibility would give a major role to polyamines in the fission-fusion process between composomes in the prebiotic ecology scenario [11]. Finally, in this context, the maintenance of bilayer integrity must have been of fundamental importance early in the origins of life. in vitro, spermine stabilized a phospholipid membrane whilst putrescine and spermidine destabilized it [76]: in vivo, intracellular polyamines affected the flip-flop of phospholipids across the plasma membrane during apoptosis [77].

Interactions with calcium

Protocells would have had to cope with problems such as the solubility of phosphate posed by huge amounts of extracellular calcium and it may be that, in overcoming these problems, calcium became involved in many different processes as evidenced in modern bacteria [78],[79]. Moreover, calcium can interact with several of the simple constituents of modern cells that were likely constituents of protocells. For example, PolyP displays a strong preference for calcium over other physiological cations. Its preference for divalent over monovalent cations may be attributed to their higher binding energies, and the preference for calcium over magnesium ions to a lower hydration energy [80]. In the quest for solutions to the fundamental problem of generating coherent phenotypes, it could thus be argued that calcium was well-placed right from the start to play a unifying role by interacting directly or indirectly with PolyP, PHB, polyamines and phospholipids. This role continues in modern cells where calcium helps coordinate membrane and cytoskeletal structures, enzymic activity, and kinases and phosphatases.

One, unifying, role for calcium would be in helping provide a coherent response to stresses. A related role for calcium would be in the transitions between growth and survival strategies (where it might act as a kind of reset button to help cells escape from one state and progress to the next) [81]. Calcium would therefore serve as one of the factors involved in differentiation required for life on the scales [6]. Consistent with this, in bacteria: (1) calcium is essential for heterocyst formation [82] and for sporulation 69, (2) calcium affects growth rate via ATP levels [83] thereby putting it in a position to generate metabolic heterogeneity within a population [6], (3) calcium is involved in biofilm formation and dispersal [84]-[86], (4) calcium increases the expression of genes or the functioning of proteins involved in chemotaxis, swarming, virulence and motility [87]-[89], (5) calcium is implicated in the response to nitrogen starvation [90] and to environmental pollutants [91].

It might be argued that polyamines are the cell-made version of inorganic ions such as calcium. Both can condense as counterions onto charged membranes and filaments in vitro, where they can diffuse freely [92]-[94], and it is conceivable that such condensation also occurs in vivo [95],[96]. Polyamines do differ significantly from ions like calcium because of their polycationic structure with positive charges distributed at fixed positions along a flexible carbon chain, thereby allowing polyamines to have different interactions from those of the metal cations [69]. Cells may have rapidly evolved systems of regulation that exploit these differences and that allow coordination of the multiple functions of the primordial molecules. Spermine and spermidine play an important role in the regulation of calcium transport in mitochondria (for references see [69]). This raises the question of the reciprocal relationship - does calcium affect polyamine levels? There is circumstantial evidence that it might. High extracellular calcium maximizes PHB accumulation via AtoS-AtoC [97] – and AtoC is the antizyme which inhibits the synthesis of polyamines [60]. Moreover, calcium channel blockers, which alter PHB levels and distribution (so as to alter, presumably, calcium influx through PHB/PolyP channels) inhibit signal transduction through AtoS-AtoC [98].

Calcium is implicated in PolyP metabolism. Acidocalcisomes, for example, are organelles that are rich in calcium, pyrophosphate and PolyP, and that contain pumps, antiporters, and channels as well as enzymes involved in the synthesis and degradation of pyrophosphate and PolyP [25]. Acidocalcisomes and the related volutin granules of PolyP are conserved across the phyla [23],[24] and are thought by some to be the first, calcium-containing, acidic compartment [25]. As mentioned above, PolyP granules may interact with polyamines (note that even if those isolated from E. coli did not contain calcium the phoU mutant studied had 1000-fold higher levels of PolyP than the wild type [62]). The functions attributed to acidocalcisomes include the regulation of osmolarity, intracellular pH, and calcium levels as well as PolyP metabolism, the storage of cations and phosphorus, and the response to light [23]. In mitochondria, decreasing the level of PolyP increases the capacity of mitochondria to accumulate calcium as well as increasing NADH levels and decreasing the inner membrane potential [99].

Calcium can interact with anionic lipids to form domains and to drive membrane domain dynamics [100]-[102], as may also be the case for polyamines (see above). The formation of membrane domains of anionic phospholipids is intimately linked with the timing and positioning of cell division [67],[103],[104]. This raises the question of whether calcium plays a role, via membrane dynamics, in cell division. It may therefore be significant that addition of calcium (or magnesium) reverses the inhibition by exogeneous PolyP of cell division and even promotes minicell formation [33] since this might be attributed not only to the effect of calcium on polymerization of the key protein in bacterial division, the tubulin-like FtsZ [105], but also to the effect of calcium on membranes containing anionic phospholipids. One model system that might reveal such effects are bacterial L-forms, which are bacteria that grow without their peptidoglycan walls and which can usefully be considered as a throwback to the first cells [106],[107]. An L-form of E. coli was found to have a five-fold lower level of FtsZ than its parent [108] whilst an engineered L-form of Bacillus subtilis manages to divide without FtsZ at all [109]. If the division of L-forms represents a return to the putative, membrane domain-based, division of protocells, it would also be significant that division in an E. coli L-form depends on calcium [108].

Extracellular calcium has been found to have a surprising effect on the growth rate of E. coli with a 10 mM concentration resulting in a 10% reduction in generation time [110]. What might be the mechanism? This calcium concentration also led to a 30% increase in the concentration of ATP [110]. Given that ATP can be generated from PolyP and that both calcium influx and efflux can be generated by PHB/PolyP complexes in the membrane, it might be imagined that these SUMIs were responsible. That said, deletion of the genes coding for AtoA (likely to be involved in PHB synthesis) and for PPK (involved in PHB synthesis) did not result in defective calcium efflux [110]. Another mechanism might comprise calcium condensing onto DNA, as mentioned above [92] and, significantly, calcium binds to DNA gyrase to result, it is proposed, in relaxation [111]. There may, of course, be alternative or complementary explanations involving, for example, calcium effects on the ATP synthase [112].

From composome to hyperstructure (and back again)

At a very early stage of the evolution of the prebiotic ecology, equilibrium composomes could have exploited the free energy associated with the attraction of peptides to the boundaries of lipid domains [113] to drive polymerization. Such boundaries can concentrate, align and orientate monomers, reduce hydrolysis and increase condensation. Indeed, a model of abiotic catalysis shows the free energy change from the redistribution of peptides within domains is sufficient to drive the formation of the peptide bond; the resulting polymerization is significant and the equilibrium distribution of polymer lengths is shifted towards longer peptides. This model is supported by the observation of shifts in equilibria in reactions in organic solvents [114],[115] and by the 10¹¹ increase in reaction rates due to orientation effects [116].

In our hypothesis, boundaries or interfaces between combinations of SUMIs and other molecules abounded in the equilibrium composomes which dominated early prebiotic evolution. Catalysis on these interfaces resulted from the dynamic effects on the composomes of changes in the physical and chemical nature of their environment, and division to generate a network on interacting polymers. This process was guided by the molecular complementarity between the SUMIs and between the monomers and polymers with the SUMIs. Is there evidence for the existence of equilibrium composomes in the modern world of hyperstructures?

The equilibrium type of hyperstructure is well-represented by the acidocalcisome (see above). Is it reasonable to hypothesize that acidocalcisomes are the vestiges of SUMI-based composomes essential to the emergence of life? More specifically, are acidocalcisomes the descendants of composomes that were able to accumulate phosphates and store energy while regulating calcium and other ion concentrations and fluxes thereby surviving environmental stresses? Could the molecular complementarity between SUMIs involved in acidocalcisome metabolism have resulted in the formation of self-organizing composomes in which integrative interactions between SUMIs (in the appropriate conditions) converted energy into biologically useful forms such as polymerization? Universality and conservation are criteria that may help answer these questions. Acidocalcisomes are found in archaea, protista, bacteria, animalia, plantae and fungi [117],[118] and this universality suggests that their origin predates any “Last Universal Common Ancestor” and that they are one of the absolute essentials for the emergence of cellular life [14]. This conjecture is supported by the finding that a 57 amino acid segment of the acidocalcisome protein PF03030, a V-H + PPase, is almost completely conserved across all branches of life [118]. Moreover, acidocalcisomes reveal the ongoing importance of the molecular complementarity between the SUMIs insofar as they are rich in PolyP and calcium and have interactions with polyamines (see above).

In our hypothesis of prebiotic evolution, the composome population progressively became enriched in non-equilibrium composomes in which the interactions between the constituents were weaker and more dynamic than in equilibrium composomes. These relatively fragile composomes may have been kept together by the forces generated by an early form of coupled transcription-translation in which the nascent products interacted with extensive structures such as a membrane. Is there evidence for such forces and for roles for the SUMIs in the modern descendants of non-equilibrium hyperstructures?

The non-equilibrium type of hyperstructure is exemplified by the nucleolus in eukaryotic cells and, by “nucleolus-like” microcompartments in prokaryotic cells where rRNA genes are transcribed and where ribosomal proteins assemble onto the nascent rRNA, so bringing together genes encoding rRNA and rproteins, nascent RNA, as well as nascent ribosomal proteins and their genes [119]. A non-equilibrium structure requires a flow of energy and this requirement would correspond to the appearance of the putative ribosomal hyperstructure in E. coli under rapid growth conditions [26],[120]. The non-equilibrium composomal ancestor of the ribosomal hyperstructure may also have been the ancestor of the EF-Tu hyperstructure. EF-Tu is a GTPase that delivers amino-acylated tRNAs to the ribosome during the elongation step of translation and that, in bacteria, forms filaments and networks [121],[122] which may have a role in the sensing of metabolic activity [123]. Coupling transcription and translation to insertion into the membrane (transertion) or to assembly into ribosomes can expand or condense, respectively, the bacterial nucleoid [120],[124]. Such transertion can generate forces sufficient to resist turgor pressure [125]; significantly, therefore, truncating EF-Tu results in cell lysis [126].

In the scenario of the prebiotic ecology, the organization of SUMIs into equilibrium composomes, such as acidocalcisomes, and into non-equilibrium composomes, such as an “EF-Tu/ribosomal” composome (or indeed composomal precursors of any of the modern coupled transcription-translation hyperstructures), may have involved a form of peptide synthesis that preceded polynucleotide-directed template synthesis. In modern cells, spermine stabilizes the conformation of the yeast Tyr-tRNA and Phe-tRNA, stimulates their binding to the A and P sites of the E. coli ribosomes [127], whilst in other studies polyamines have been shown to act at both the initiation and elongation steps in translation, and to affect the levels of proteins encoded by the polyamine modulon such as the RNA polymerase sigma subunit, RpoS, and Fis, which itself increases the level of rRNA and tRNA [20]. Covalent modification by PHB would have facilitated the interactions between peptides, membranes and nucleic acids within the composomal ancestor of the ribosomal and EF-Tu hyperstructures given that in E. coli, ribosomal/EF-Tu hyperstructure constituents that undergo modification by PHB include the elongation factors EF-Tu and EF-Ts themselves, ribosomal proteins RplL and S1, and RNA polymerase subunit, RpoA [17]. PolyP has been shown to catalyze peptide synthesis in a variety of aqueous prebiotic conditions [128]-[130] aided by magnesium as a catalyst [131]. More importantly, phosphate binding on proteins often involves a very simple, highly conserved motif of three to six residues [132],[133], suggesting that PolyP might have provided a template for the synthesis of the peptides to which it binds. This has led to the suggestion that a PolyP-peptide synergy linking phosphate metabolism with peptide synthesis was one of the key steps in the emergence of life [134]. One of the results of this linkage could have been the PolyP-template-directed synthesis of the peptide precursors of the modern translational machinery and the various kinases, phosphatases and ion channels that are incorporated into acidocalcisomes. A similar mechanism linking calcium-directed templating of PolyP-stimulated peptide synthesis could have produced the highly conserved modules, such as EF hands, made up of the dozen amino acids that form most calcium binding sites in modern proteins [135]. In this way, a localized primitive metabolism could have evolved in the absence of cellular life and provided one of the key components incorporated into it.

Reviewer 1 (Doron Lancet)

“This paper is revealing and important, but requires a major overhaul of the presentation flow.

The major idea, as far as I reckon, is that that there are substances, playing key roles in present-day living cells, which are more likely to have been early “jump-starters” for life as compared to the usually implicated “mainstream” compounds – DNA, RNA and proteins. This idea is appealing, because in the eyes of many, the latter three complex, sequence-centered biopolymers are rather unlikely to have emerged abiotically under early prebiotic conditions.

Three of the potentially prebiotic compounds innovatively implicated by the authors are polymers/oligomers. The first is polyphosphate, chains of diester-bonded orthophosphate groups (PolyP), the second is a homopolymeric polyester, polyhydroxybutyrate (PHB), and the third is a class of relatively short oligomers – polyamines. A fourth implicated component is lipids and a fifth is divalent calcium ions. The authors coin the acronym SUMIs for such compounds – simple universal molecules and ions. The name is not very appropriate, as there is not much that is universal about such compounds, and there is little in common between the polymers lipids and ions, so it would be good to change the acronym. I do admit that PolyP, PHB and polyamines (heretofore called PPP) are indeed simple, in being homopolymeric (nearly so for polyamines), and not portraying the combinatorial complexity of sequence-based heteropolymers. Lipids are a class of their own in this respect, being essentially monomeric, but share the attribute of lacking sequence-related combinatorics.

Two special attributes of PPP emerge from the admirable literature search presented in this paper: 1) They are ubiquitously seen in living cells (with special emphasis on bacteria), and found to participate in numerous contemporary cell functions; 2) They show a curious capacity to interact non-covalently (sometime covalently) with each other and with other components (e.g. proteins) and form supramolecular assemblies. Consequently, the most fundamental hypothesis of the paper, in my view, is that compounds such as PPP and lipids, with the mediation of metal ions (first and foremost calcium), may form readily under prebiotic/abiotic conditions. They are endowed with a capacity, evidenced in contemporary organisms, to form noncovalent complexes that have biochemical functions surprisingly similar to those of their much more elaborate, sequence-based descendants – proteins and RNA. They thus are natural candidates for a transition from a “primordial soup” to full-fledged living cells. I strongly suggest that this clear line of argument become the spine of a revised paper.

Reviewer 2 (Kepa Ruiz-Mirazo)

Response to Doron Lancet

The authors coin the acronym SUMIs for such compounds – simple universal molecules and ions. The name is not very appropriate, as there is not much that is universal about such compounds, and there is little in common between the polymers lipids and ions, so it would be good to change the acronym.

We are not sure why the reviewer thinks that “there is not much universal” about lipids and these polymers. Lipids, PHB and PolyP are, to our knowledge, present in all species whilst calcium is everywhere (the problem is often keeping it out). That said, polyamines have been reported as absent in Staphlococcus aureus and we now cite this paper. The suggestion for a different acronym is a good one but we would prefer to stick with the original one (which has a meaning in Japanese – a sumi is a stick of ink used by painters – that is somewhat appropriate).

1) The abstract requires a major revision. Its first paragraph begins with the most obscure sentence in the entire paper: “The solutions to these problems are likely to be found with the organic and inorganic molecules and inorganic ions that constituted the first cells …”. Which problems? In what way are the mentioned substances a solution? Similarly, the last sentence of the first paragraph: “Here, we explore the possibility that the direct interactions between PHB, PolyP, polyamines and lipids - modulated by calcium - played a central role in solving the fundamental problems faced by early and modern cells”. Which fundamental problems?

Unfortunately, a cut-and-paste error led to the absence of the required information in what should have been the background. We have now inserted this: “Fundamental problems faced by the precurors of cells (protocells) and by their modern descendants include how to go from one phenotypic state to another, how to escape from a basin of attraction in the space of phenotypes, how to reconcile conflicting growth and survival strategies (and thereby live on ‘the scales of equilibria’) and, more generally still, how to create a coherent, reproducible phenotype at all from a multitude of constituents”.

The second paragraph does not make the readers’ life easier: “We reason that since these SUMIs are important for modern cells and were probably abundant and available for the first cells, there may be evidence for their interactions in modern cells.” If the authors are trying to decipher protocellular function, why does the punchline address modern cells?

We have rewritten this section and have incorporated much of the paragraph proposed by the reviewer: “We review the evidence that the SUMIs (1) were probably abundant and available for the first cells, (2) are widespread in modern cells, (3) interact with one another and with other constituents in modern cells to create new structures with new functions that are surprisingly similar to those of proteins and RNA, and (4) are essential to helping modern bacteria create coherent phenotypes. We reason therefore that the SUMIs are natural candidates for both reducing the immensity of phenotype space and making the transition from a “primordial soup” to full-fledged living cells”.

The last abstract paragraph is just as confusing: terms like “prebiotic ecology” and “initial phenotype space” are not too revealing and should be better left out.

We have rewritten this paragraph, we define composomes and now say in this paragraph: “prebiotic ecology of co-evolving populations of composomes” and “initial phenotype space of composomes”. Later in the text, we try to make it clear why these concepts are essential to our hypothesis (see below).

“(3) these interactions constituted a system of global regulation that limited phenotype space to the two large attractors of growth and survival and to the transitions between them”. These obscure terms and seemingly formal arguments are never defined or supported in the paper, and should better be left out completely.

We have put these problems upfront in our paper and, rather than delete them, we have tried to explain them better. We say in the Background: “The first of these fundamental problems is how to generate reproducible, coherent phenotypes from a large number of effectively different molecules. In modern cells, this number runs into thousands - if not millions –if genes and other nucleic acid sequences are considered as separate entities, if post-translational modifications are taken into account and if the small molecules of metabolism are included. The combination of the activities of these molecules generates the phenotype on which natural selection acts. Since there would appear to be an almost unlimited number of such combinations, there should be an almost unlimited number of phenotypes. … The upshot of all this is that probably less than a hundred different hyperstructures, created in part by molecular complementarity, determine the phenotype of the bacterium. This means that the number of hyperstructures is orders of magnitude less than the number of macromolecules so the number of phenotypes resulting from combinations of hyperstructures is much smaller than the number resulting from combinations of macromolecules. In other words, phenotype space is dramatically reduced but remains huge. … A second, fundamental problem is how to generate phenotypes that can satisfy the incompatible requirements of survival and growth. This is the problem of ‘Life on the scales’ whereby cells are damned if they simply grow (since they risk being destroyed if conditions turn bad) and damned if they eschew growth, for example, to sporulate (since they risk being out-competed by other, growing, cells if conditions remain good) {Norris, 2012 #5109}. To simplify it, at one extreme, survival requires a cell that approaches an equilibrium state in which it is relatively static and robust but does not grow (with interactions between cellular constituents are strong and stable) whilst, at the other extreme, growth requires a cell in a non-equilibrium state in which it is highly dynamic and metabolically active but risks death (with interactions between cellular constituents that are weak and unstable). Moreover, cells must be able to go between these states. One of the solutions adopted by modern bacteria is to have a cell cycle that gives daughter cells with different phenotypes as evidenced by the division of Caulobacter crescentus into stalked and swarmer cells each of which can generate the other {Bowman, 2013 #5758}. In the hyperstructure hypothesis, these different phenotypes are conferred by different combinations of equilibrium (technically, quasi-equilibrium) and non-equilibrium hyperstructures {Norris, 2007 #2798}”.

I suggest to lump all background information … before presenting the hypothesis. The hypothesis section should then be devoted purely to presenting all the authors’ conjectures, based on all the information displayed beforehand. To exemplify this point, in the current hypothesis section does not include a hypothesis point shown 2 paragraphs earlier in the Background section - …source and a reservoir of carbon, phosphorus and nitrogen…; In turn the current hypothesis section has a point not obviously belonging there “roles…can still be discerned in modern cells”.

In the limit, there are two ways to present a hypothesis. One is the way proposed by the reviewer. The disadvantage is that the reason for the choice of facts that precede and build up to the hypothesis is unclear until the reader reaches the hypothesis. Of course, this is fine for a review in which the hypothesis is only of minor importance. The other way, which we prefer and which is recommended by the journal, is to spell out the hypothesis first. This might seem to have the disadvantage that at least some background information still has to be given first. However, we subscribe to the view that this background should be about the nature of “what is the problem?” rather than about the properties of PolyP, PHB, polyamines, lipids and calcium, so we would prefer to retain our original structure. That said, the reviewer’s suggestion has several merits and we have therefore included much more information about the SUMIs in the Background section.

We have rewritten the presentation of the hypothesis and have added to it the missing bit about the SUMIs being a reservoir of carbon, phosphorus and nitrogen. The reviewer says that the statement that “roles … (of the SUMIs)…can still be discerned in modern cells” is out of place in the presentation of the hypothesis. As we see it, this statement is an assumption (i.e., a hypothesis in its own right) that justifies our gathering information in support of our principal hypothesis and its best place is therefore in the presentation section. We have, however, modified the phrase by adding “in their universal utilization of ribosome microcompartments and acidocalcisomes to regulate key cellular processes”.

Non-covalent assemblies are often referred to in the paper as “composomes”. Indeed composomes (REF 5) have been invoked as non-covalent heterogeneous molecular assemblies present at the early stages of Life’s emergence. But the most crucial aspect of composomes is that they are dynamic structures that manifest rudimentary information transfer from one growth-split generation to another. It would be good to address this latter property in reference to the molecular non-covalent complexes invoked in the present paper.

This is particularly important in relation two of the hypothesis points: “the interactions between SUMIs determined the behavior of composomes”. It is really necessary to back such a statement with specific relationships.

The problem here is that the reviewer and his group have coined an excellent term which we should not and cannot avoid! Composomes, in our version of the concept, fall into two classes, non-equilibrium and (quasi-)equilibrium, the former being much more dynamic than the latter (which we previously termed ‘protocells’). This is important because the behaviours of the two classes of composomes would have been very different: one class is equipped for growth and the other for survival; moreover, we believe that the phenotype of modern cells is governed by non-equilibrium and equilibrium classes of the modern counterparts of composomes (that is, hyperstructures). There is sufficient evidence out there to implicate the SUMIs in growth, survival and the transitions between these behaviours. We therefore now say in the Background:

Although the current scenario of the prebiotic ecology offers solutions to fundamental problems, these solutions are incomplete. How was phenotype space sufficiently constrained by composomes to have enabled natural selection to act effectively? How did composomes or collections of composomes go from an equilibrium state to a non-equilibrium one and back again? How exactly was energy generated and catalysis achieved?Addressing these questions by invoking peptides and oligonucleotides would be understandable. There are, however, other molecules that merit consideration. Not so long ago, the late Arthur Kornberg chided the scientific community for dismissing polyphosphate (PolyP) and its metabolism as a mere “molecular fossil” {Kornberg, 2008 #5480}. In modern cells, PolyP is implicated in quorum sensing, biofilm formation, motility, virulence, sporulation phosphate storage and energy metabolism {Rao, 2009 #3546}. PolyP is not alone in receiving insufficient attention. Short chain poly-(R)-3-hydroxybutyrate (PHB) can form ion and DNA uptake channels (with PolyP) and pumps {Das, 1997 #983} {Reusch, 1997 #2028} and may directly modulate interactions between proteins, nucleic acids and membranes {Huang, 1996 #807} {Reusch, 2002 #1615}; moreover, PHB can act as a carbon store {Wahl, 2012 #5157}. Polyamines bind to nucleic acids and proteins, decrease membrane permeability, may help cells survive abiotic stresses {Igarashi, 2006 #2726} {Groppa, 2008 #2724}; polyamines may also act as nitrogen stores.

We also now say in the section “From composome to hyperstructure (and back again)”:

At a very early stage of the evolution of the prebiotic ecology, equilibrium composomes could have exploited the free energy associated with the attraction of peptides to the boundaries of lipid domains {Netz, 1996 #818} to drive polymerization. Such boundaries can concentrate, align and orientate monomers, reduce hydrolysis and increase condensation. Indeed, a model of abiotic catalysis shows the free energy change from the redistribution of peptides within domains is sufficient to drive the formation of the peptide bond; the resulting polymerization is significant and the equilibrium distribution of polymer lengths is shifted towards longer peptides. This model is supported by the observation of shifts in equilibria in reactions in organic solvents {Deschrevel, 1992 #1595}{Hitz, 2000 #6148} and by the 1011 increase in reaction rates due to orientation effects {Milstien, 1970 #1594}. etc.

Prebiotic non-covalent structures, composomes, hyperstructures, present day organelles (e.g. acidocalcisome) and conjectural assembles such as EF-Tu/ribosomal structures are all intermixed in the paper’s argumentation, particularly in the From composome to hyperstructure section, in ways that require considerable further clarification. What happened first? What was the evolutionary progression? What were the driving forces for such? In fact the abovementioned section contains many statements that should be part of a well-balanced hypothesis section (see comment 3). The final sentence of the second paragraph in this section addresses energy considerations not at all mentioned in the hypothesis section – needs to be corrected. The text within this very sentence “…via integrative interactions between hyperstructures, convert this energy into biologically useful forms” is truly obscure and unless supported by more formal arguments needs to be eliminated.

We have rewritten the “From composome to hyperstructure” section to make it clear what happened first:

“In our hypothesis, boundaries or interfaces between combinations of SUMIs and other molecules abounded in the equilibrium composomes which dominated early prebiotic evolution. Catalysis on these interfaces resulted from the dynamic effects on the composomes of changes in the physical and chemical nature of their environment, and division to generate a network on interacting polymers. This process was guided by the molecular complementarity between the SUMIs and between the monomers and polymers with the SUMIs”.

We have transferred hypotheses to the hypothesis section:

“The equivalents of the non-equilibrium types of composome in which macromolecules are synthesized are epitomized by the nucleolus in eukaryotic cells and by the “nucleolus-like” hyperstructures in prokaryotic cells as well as by the other prokaryotic hyperstructures in which transcription and translation are coupled. The equivalents of the equilibrium types of hyperstructures are epitomized by acidocalcisomes which help regulate osmolarity and pH and which are found in every major branch of life”.

We have changed “The resulting composomes were able to accumulate phosphates while regulating calcium and other ion concentrations and fluxes in order to store the resulting energy and, via integrative interactions between hyperstructures, convert this energy into biologically useful forms” into “Could the molecular complementarity between SUMIs involved in acidocalcisome metabolism have resulted in the formation of self-organizing composomes in which integrative interactions between SUMIs (in the appropriate conditions) converted energy into biologically useful forms such as polymerization? Universality and conservation are criteria that may help answer these questions. etc.”

In the Testing the hypothesis section, the idea of adding to compounds highlighted by this paper to a Miller-Urey type experiment makes little sense. The experiment addresses the production of organic compounds from inorganic gases. It produces a plethora of small and large molecules that were never fully analyzed. The experiment has no delineation of a progression from the organic compounds to the next steps in prebiotic evolution. So even if the authors meant adding compounds such as PolyP and PHB to the organic residue of the Miller Urey experiment, this involves no testable predictions. On the other hand, it is quite clear that experiments should be suggested to investigate the abiogenesis of PPP.

We have modified the Testing section to clarify the experiment we mean and to respond to the point about abiogenesis of the SUMIs: “Second, adding SUMIs such as PolyP and calcium to Urey-Miller type experiments {Miller, 1959 #5276} should have a dramatic effect on the nature of the resulting products. The addition of one SUMI should influence the appearance of the SUMIs with which it interacts. Seeding the starting mix with the SUMIs should lead to the formation of self-aggregating complexes and produce specific peptide sequences that can interact functionally with phosphates, PHB, polyamines, etc. In such modified reruns of the Urey-Miller experiment, it would be important to add a step to counter-select those molecules that do not interact with one another and that therefore might be preferentially eliminated by exposure to uv radiation; we predict that, by going through many cycles, the wide variety of molecules generated in the original Urey-Miller experiment would be narrowed onto the limited number of those that interact due to molecular complementary”.

The authors strive to portray events that preceded true cellular life, but often inappropriately use the term “first cells”, likely intending to say something like “protocells”.

Yes. We have now replaced “first cells” and “early cells” with “protocells”.

I do not see the validity of the statement in the Interactions with Calcium section: “The first cells would have had to cope with huge amounts of extracellular calcium and it is therefore not surprising that calcium is involved in many different processes…”. There were large amounts of many ions, and a role for an ion is likely brought about by evolutionary emergence of molecular mechanisms and not by need “to cope”.

Calcium would initially have been a problem for protocells. We now say: “Protocells would have had to cope with problems such as the solubility of phosphate posed by huge amounts of extracellular calcium and it may be that, in overcoming these problems, calcium became involved in many different processes as evidenced in modern bacteria”

Response to Kepa Ruiz-Mirazo