166 Opinion TRENDS in Vol.11 No.4 April 2003

Were Gram-positive rods the first ?

Arthur L. Koch

Biology Department, Indiana University, Jordan Hall 142, 1001 East Third Street, Bloomington, IN 47405–6801, USA

At some point in the evolution of life, the Bac- The first cells: Gram-positive or Gram-negative? teria arose from prokaryotic progenitors. The cell that Arguments for Gram-positive rod-shaped cells gave rise to the first bacterium has been given the Arguments from three groups suggest that the first cell to name (among several other names) ‘last universal separate from the monophyletic prokaryotic predecessor of ancestor (LUA)’. This cell had an extensive, well-devel- the bacteria was a Gram-positive, rod-shaped organism. oped suite of biochemical strategies that increased its Seifert and Fox [6] noted that rod-shaped structures ability to grow. The first bacterium is thought to have cluster at the base of the bacterial phylogenetic tree. They acquired a covering, called a sacculus or exoskeleton, compared the morphology of the cells in the various that made it stress-resistant. This protected it from rup- branches of the Woese’s 16S rRNA tree (see Ref. [1] and turing as a result of turgor pressure stress arising from Olsen et al. [7]). Seifert and Fox stated: ‘It [also] seems the success of its metabolic abilities. So what were the likely that the last common ancestor of the domain properties of this cell’s wall? Was it Gram-positive or Bacteria was rod-shaped’. Gram-negative? And was it a coccus or a rod? Tamames et al. [8] drew conclusions from the analysis of the dcw (division and ) clusters present in many Bacteria evolved as a separate domain some time before bacteria. There are 15 genes in the dcw cluster of or Eukarya [1]. They developed at a critical time and around the same number in other that defined the ‘last universal ancestor (LUA)’, and arose species. Tamames et al. compared the coding order of the after the basic groups of physiological processes had genes (i.e. the sequence of genes on the chromosome) in a evolved to a functional point. These major processes variety of species. They found that there was a pattern, must have included DNA replication, ribosome and and that the dcw cluster was more compact and conserved protein synthesis, central intermediate metabolism, cell in bacilli, implying that rod-shaped bacteria came first. In division, DNA repair and responses to fluctuation in the the bacilli, in contrast to the cocci that they studied, there environment. Such properties are possessed by almost all was a more constant order on the chromosome. These modern cells [2] and each would have improved their bacilli encompassed both Gram-positive and Gram- ability to grow compared with the first living cell on this negative forms. planet. The differences among the bacterial, archaeal and Gupta et al. [9–11] analyzed the completed, published eukaryotic cells are slight compared with their similarities sequences of many genomes, both bacterial and archaeal, [2]. One important difference between the immediate and concluded that Gram-positive bacteria arose first, and precursor of the three cell domains and the first bacterium that Gram-negative bacteria arose from Gram-positive was the latter’s development of a functional and stress- bacteria through a sequence of several other groups. resistant wall [2,3], which allowed it to support a higher Gupta’s phylogenetic tree [11] corroborates the standard internal osmotic pressure and remain intact in a low 16S rRNA tree [12]. However, the Woese group has osmotic pressure environment. The issue here is the presented convincing evidence from the 16S rRNA nature of this cell wall. Was the wall thick or thin? Was it a coccus or a rod-shaped (Figs 1 and 2)? In the Rod-shaped Bacillus Coccus Kandler and Schleifer designation system [4,5], what category would it be? That is, what was the chemical structure of the original bacterium’s cell wall? This Gram-positive classification system uses three symbols. Thus, for Escher- ichia coli, it is A1g. The A indicates that the third amino-acid residue is meso-diaminopimelyl; the 1 indicates that there is Gram-negative no intervening peptide that bridges between the two peptides at the tail-to-tail linkage; and the g indicates that the third amino acid is meso and not LL. TRENDS in Microbiology

Fig. 1. Four possibilities for the wall of the first bacterium. These four types rep- resent a majority of organisms. There are other shapes (curved, spiral and tapered) Corresponding author: Arthur L. Koch ([email protected]). but these are probably less likely than the initial bacterial form. http://timi.trends.com 0966-842X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0966-842X(03)00063-5 Opinion TRENDS in Microbiology Vol.11 No.4 April 2003 167

arose only when there were plants, animals and fungi to be Gram-positive parasitized and resisted. As a consequence, their evolution would have been a later event.

Older wall Arguments for Gram-negative cells Woese [1] argued that Bacteria arose some time earlier than the split that led to the separation of Archaea and Eukarya Cytoplasmic membrane from the ‘progenote’ or LUA. The derived phylogeny of Gram-negative Bacteria, which is currently well accepted, does not group Penta-muropeptides the Gram-positive organisms together and does not group the rod-shaped cells separately from the cocci. A major proponent for the idea that Gram-negative cells Up arose first is Cavalier-Smith, who has presented extensive peptide Nona- discussions of the origin and evolution of life [13,14].He muropeptide To other argued first that life started on the outside of a bilayered tessera lipid vesicle that had been produced abiotically [15]. If life Down started on the outside of the lipid membrane, the problem peptide of transport across a lipid bilayer of hydrophilic material is avoided but the theory requires that biomolecules of TRENDS in Microbiology crucial importance remain attached to the outer surface. Fig. 2. Wall-side wall growth mechanisms. In Gram-positive organisms, the wall is Later, when life had developed adequately, this phospho- formed by the continuous laying down of layers of murein just outside of the cyto- lipid vesicle engulfed the living portion. When fusion was plasmic membrane. After they are hydrolysed the layers become highly stretched and peripheral. This mode of the addition of underlying layers that move to the complete, this formed the first cell that was surrounded by outside of the cell wall is called the inside-to-outside mechanism. In Gram-nega- two bilayers, and was thus a Gram-negative organism. tive organisms, the wall grows by the extrusion of penta-muropeptide through the The space between these layers corresponds to the cytoplasmic membrane and their incorporation into a functioning wall structure under tension. This mode is called the nana-muropeptide stress mechanism. periplasmic space. Cavalier-Smith’s suggestion [15] is a modification of one proposed by Blobel [16], who originally suggests that the sequences to show that Archaea and Eukarya separated cell started ‘inside out’. Blobel presumed that life arose on from a prokaryotic precursor and are not derivatives of the a solid rock surface. Cavalier-Smith proposes that this Bacteria [1,12], as Gupta believes [9]. Although the origin location solves the energy problem because of the presence of Archaea or Eukarya is not directly relevant to the of polyphosphates in the rocks, and that only later did present discussion, this conflict is pertinent. invagination take place, he assumes to convert this ‘obcell’ Gupta et al. [9–11] looked for corresponding regions in (obverse cell) to the usual arrangement [15]. Some the available sequenced genomes and for differences descendents of this cell later degenerated to become the characteristic to all members of a taxonomic group and single-membraned, thick-cell-walled Gram-positive cells. that, additionally, were unique to some, but not other, taxonomic groups. Such differences of ‘significance’ they Cell morphology: cocci or rod-shaped called ‘indels’ (insertions/deletions); consequently, these Until the cell developed mechanisms to establish its shape, the default appears to be that the phospholipid- or lipid- indels correlate with a taxonomic group. When these bilayer-enclosed cell formed an ever-increasing sphere as persisted in all members of a taxonomic group, Gupta it grew. Thus, unless the cell had already developed a concluded that the group originated from a founder cell cytoskeleton or a strong wall (that is, unless it had an exo- generated from another phylogenetic group that, by or endo-skeleton), there was no mechanism to make it chance, happened to have this particular indel. The divide to produce new cells or cells of a constant mean size indel was therefore present in all members of the group, or particular shape. If the cell was not attached to any and then was passed on when a founder from this group led object, it must have grown larger and larger without limit. to a newer group. Thus, the indel was common to all This certainly is not a working growth strategy. Attach- members of a group no matter what other lines of ment to surfaces could have helped such a cell to divide, diversification occurred within it later. The important although this would have happened irregularly and conclusion drawn was that major bacterial taxa arose formed daughters of irregular shape. Only one alternative linearly from each other [9,11] and that cells with one to this theory has been suggested in the literature as a membrane, such as Gram-positive organisms, called possible mechanism for division during the time between ‘monoderms’, are the precursors of the cells with both a the first cell and the LUA [17]. This proposal is that the cytoplasmic membrane and an outer membrane. These are lipid constituents of the bilayer were generated within the Gram-negative organisms, such as E. coli. All of these are cell, and that they became inserted into the inner layer of called ‘diderms’ by Gupta’s group. the bilayer and this uneven growth caused the bilayer to A supporting argument for this order of evolution is that invaginate. Eventually, this would lead to cell division. the Gram-negative cells are structurally better able to This strategy might have worked but would have been function as pathogens and to resist . This would irregular and would not have been very effective [17].It be in accordance with the idea that the Gram-negative cell was possibly up to 800 million years later, when a http://timi.trends.com 168 Opinion TRENDS in Microbiology Vol.11 No.4 April 2003 completed the evolution of a stretch-resistant antibiotics [27]). The physical law, derived by LaPlace two ‘fabric’, that an effective alternative arose, which I argue centuries ago, would lead to cylindrical extension [28] was the creation of the domain Bacteria. Similarly, the if the poles were rigid. His law related the radius of a development of pseudomurein could have led to ‘bubble’ of arbitrary shape to the pressure difference the creation of the first Archaea. Of course, later, the acrossthewall,theamountofworkneededtoincrease cytoskeleton arose together with contractile proteins used the surface by a unit amount and constraints upon its by Eukarya. surface (see Ref. [34]). It is not enough in the development of bacteria to The implication of bacterial poles being metabolically develop a means of forming an enclosing strong murein inert is that the first bacterium, like all bacteria (with the sacculus. Several other mechanisms must also have exception of ), formed new poles entirely by arisen. A mechanism that prevents wall growth in the new synthesis and, subsequently, did not enlarge or alter established poles of cells is a key requirement. A them. This implies that the biochemistry and biophysics of mechanism that causes a pole to be metabolically inert bacterial cells must be such that the mature poles are provides a way to maintain the size and shape of cells in blocked from further metabolism or turnover. This succeeding generations. For rod-shaped cells, inert poles prevents the size of completed poles from being further provide support for the elongation of cylindrical growth modified by insertions or being turned over in future [18]. However, this can only function to the degree that the generations. It therefore appears likely that cocci only poles are metabolically inert and rigid [19]. Thus, need to be able to form a septum centrally, and grow the inertness of the poles can also be the basis for the without changing their maximum diameter and the maintenance of the diameter of cylinder-shaped cells in dimensions of a mature pole. They then only needed to balanced growth. Cell mechanisms must also function to allow or aid the septum to split to form two new daughter foster wall growth in an amount consistent with the on- cells of the same diameter. going rate of protoplasmic synthesis. That is, cell This is the way that the coccus hirae and biochemical growth must drive the enlargement of the the rod Bacillus subtillis divide [28–32]. Evidence from cell wall. These aspects are, together, probably the answer the electron microscope is that, at some critical stage of the to the question: why do bacteria not grow larger and larger cell cycle, a septum starts to grow inwards (from the site of and rounder and rounder? the previous septa or in the middle of the cylindrical Experimental evidence for the poles of bacteria started region). As it is formed, it starts to split from the outside in with Cole’s and Hahn’s studies [20] of and the intervening split septal wall bulges outwards, pyogenes, and Doyle’s early unpublished studies of forming two new poles. Consequently, the new pole has the (published in [21]).These studies found same diameter as the old one and, in the next generation, that the established poles of both this Gram-positive these poles are the templates for the new nascent poles. coccus and rod were metabolically inert. There is now This means that the diameter of poles in a culture in definitive evidence for the inertness of the poles of balanced growth is remarkably constant (^5%; Ref. [29]). B. subtilis [22–24] and of E. coli ([25]; A.L. Koch and The growth of cocci is clearly simpler than that of rod- M.A. De Pedro, unpublished). If these findings apply to shaped organisms, which must form the cylindrical walls other bacteria and it is found that the poles of bacteria, by a separate process in addition to septal formation and other than mycoplasma, are rigid, metabolically inert and splitting. This would suggest that that the first bacterium cannot stretch further, then this rigidity is probably the was a coccus [33]. This is not in accordance with the logical defining feature of the domain of bacteria. extension of the ideas of Woese [1], or those of Seifert and The pole metabolisms of B. subtilis and E. coli only have Fox [6], Vicente’s group [8] and Gupta’s group [9]. been studied in sufficient detail to consider the question of Consequently, other roles for rod-shaped cells and their metabolic turnover in the poles. The experimental finding advantages will be considered later in this article to is that the turnover at the poles is negligible [22,24]. support their points of view. However, the sidewalls of rod-shaped cells turn over with a half-life equal to their growth doubling time [24]. The inert Wall growth mechanisms for the first bacterium nature of the poles is surprising from the biochemical point The strategies for wall growth of Gram-positive and Gram- of view because attempts to find a significant chemical negative cells briefly presented here have been studied difference in the murein wall of the poles and of the both theoretically and experimentally. The mechanism for sidewall regions of B. subtilis have failed [26]. Gram-positive cells is well-established [34]. Briefly, new Rigid poles allow the cells to maintain their size during layers of wall are added from the cytoplasmic membrane growth because the poles provide a template for wall surface and autolysis removes the oldest wall. This is enlargement, and this determines the diameters of the called the Gram-positive ‘inside-to-outside’ mechanism. next generation of poles. When initially linked into the By contrast, the mechanism for Gram-negative bacteria wall of stress-bearing organisms, newly polymerized wall requires that new wall units be synthesized, inserted is not extended to its maximum size. Although the murein through the cytoplasmic membrane and covalently is elastic when inserted, it is unstretched. Of course, it will inserted into the stress-bearing wall. The most recent come to be stretched during growth. If there were no proposal depends on stress in the growing wall, altering special controls on insertion and it occurred randomly over the conformation of the new wall. This model is called the the cell surface, the cell would bulge like a soap bubble ‘nona-muropeptide stretch’ mechanism. It can be (as various bacteria do when treated with appropriate appreciated that both sidewall and pole formation for http://timi.trends.com Opinion TRENDS in Microbiology Vol.11 No.4 April 2003 169

Gram-positive organisms are quite simple compared with inserted around the template chain that is then removed the process used by the thin-walled Gram-negative cell. from the wall. This enlarges the wall by doubling the area What are the possibilities for how saccular growth of the template strand. At present this model is the most occurred in the first place? Could either the mechanisms well known model in the literature. However, with either for Gram-positive or Gram-negative cells be those that model it is difficult to imagine from known biochemistry functioned for the progenote LUA cell that had just that the first cell was a Gram-negative cell, as Cavalier- perfected methods to form a cross-linked ‘fabric’ outside Smith would postulate [13–15]. of its cytoplasmic membrane? Being able to form a polymer The coccus is the simplest of possible cell shapes. If outside the cytoplasmic membrane is very complex [34] E. hirae were taken as the model for the first bacterium, and there must have been many problems to overcome. such a cell would only need to form a thick septum and However, in addition for bacterial growth, there must be bisect it and then let the physical forces do the rest. The mechanisms directing the insertion and leading to cell semiconservative process described above for E. hirae division. It is here at the LUA stage that an additional set would not be too difficult to implement simply with a few or of mechanisms had to be generated. Although the early no extracytoplasmic enzymes. mechanisms may not have been as sophisticated as those The reasons why a rod-shaped bacterial organism used in modern bacteria, at the start of the bacterial might have come first have been presented in this Opinion. domain they had to be sufficient, simple and functional. This is the conclusion of three groups based on three quite On the basis of the Gram-positive and Gram-negative different arguments. There are biophysical arguments, possibilities, it would appear plausible that something like based on the kinetics of uptake issues [34], for the a Gram-positive ‘inside-to-outside’ mechanism would be advantages of rod-shaped organisms over coccal-type much simpler than the ‘nona-muropeptide stretch’ mech- cells. There are several different cases for diffusion up to anism for Gram-negative cells. Moreover, it seems self- the cell. In an environment with significant concentrations evident that a coccus instead of a rod-shaped organism is of resources, if every unit of surface area has the same simpler and should have arisen earlier. So the first hunch concentration of uptake sites, the only relevant factor is could be that the original bacterium was a Gram-positive the ratio of surface to volume. Smaller cells have higher coccus. For division, this cell must have been capable of ratios. For a fixed volume, thin rods or flat leaf-like forming a septum or crosswall, possibly with a mechanism structures have higher ratios. By contrast, spheres have similar to that used for crosswall formation in B. subtilis or the lowest ratio. Therefore, a narrow rod would have been . The septal crosswalls of Gram-positive an optimal shape. cells are much thicker than a monolayer of murein and are If different regions of the cell under different environ- around the same thickness as the Gram-positive sidewall mental conditions have a different number of absorbing of B. subtilis. sites, then the actual surface area of the cell is not the only significant quantity, and the number and kind of uptake Conclusions systems per unit of surface area must also be taken into The first bacterial cell appears to have had a sacculus consideration. However, when nutrients are present in [2,3,34]. But other thoughts about the first member of very low concentration, the factor of prime significance is the domain of Bacteria are more varied. It may have been a the diffusion process from the bulk medium to the cell. Now Gram-positive organism, that is, if Gram-positivity only the mathematics for different-shaped objects is quite denotes a thick layer. I argued earlier in this different. But the effect of cell shape is actually minor in article that the Gram-positive strategy for growth is much either low or high substrate concentration. Of course, even simpler than that of Gram-negative cells. Cavalier-Smith if the effect on growth rate is very slight over many argued that a Gram-negative cell appeared first, although generations, it might still be important. The general he considered mostly the membranes and not the murein conclusion from these considerations is that a rod shape is wall and its role in bacterial growth [14]. better than a coccoid shape for a cell of fixed volume. It is probably the case that, during the time between the Are there advantages of rod-shaped growth over coccal- origin of the first cell and the first bacterium, cells had no type growth? Two potential advantages can be suggested. murein layer. Awall was needed after organisms gradually One is that the individual cells in a massed aggregate of became more successful because only then did their turgor cells might have a more effective exposure to the pressure increase. The simplest mode for Gram-negative environment if the aggregate is an assortment of randomly growth suggested so far is the nona-muropeptide stretch oriented rod-shaped cells than if it is a compact mass of model, which is much simpler than earlier models [34].Ifit more spherical particles. This is a possibility that must be functioned at a time when cells had only a partial mathematically explored. functional sacculus and did not involve enzymes passing The second, and more probable, suggestion is that a rod through the cytoplasmic membrane, proteins in holoen- shape is useful when cell division is controlled by cell zyme clusters would not have been required, unlike in growth. At some point in the E. coli cell cycle, the initiation Ho¨ltje’s ‘three-for-one’ model [35].Ho¨ltje’s model postu- of DNA replication takes place and this, in turn, controls lates that penta-muropeptides are secreted through the the timing of the subsequent cell division [36]. This cytoplasmic membrane and are linked by glycan bonds to strategy may have been selected to preclude the possibility form oligopeptides of the same length as a template portion that the cell division event would interfere and break a of the stress-bearing wall. Three of these chains are linked chromosome. Consequently, the rod-shaped mode of cell together by tail-to-tail bonds and the resultant ‘raft’ growth is safer for the genetic integrity of the cell. http://timi.trends.com 170 Opinion TRENDS in Microbiology Vol.11 No.4 April 2003

Contrast this with the behavior of cocci. In E. hirae,it species from completed bacterial genomes, based on conserved indels appears that splitting of the septum is initiated when wall found in various proteins. Int. Microbiol. 4, 187–202 growth cannot occur fast enough for the cell’s needs for 12 Olsen, G.J. (2001) The history of life. Nat. Genet. 28, 197–198 13 Cavalier-Smith, T. (2001) Obcells as proto-organisms: membrane space for its cytoplasm [29–31]. Because of the disparity of heredity, lithophosphorylation, and the origins of the genetic code, the the ratio of volume growth rate to wall surface growth rate first cells, and photosynthesis. J. Mol. Evol. 53, 555–595 throughout the cell cycle, the trigger might be turgor 14 Cavalier-Smith, T. (2002) The neomuran origin of Archaebacteria, the pressure. 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