Bacterial Cell Division and Peptidoglycan Synthesis: An Evolutionary Enigma
The video above shows the process by which bacterial cells reproduce themselves. Looks simple, doesn't it? It's only a colony of cells elongating before splitting in two. Don't be fooled -- appearances can be deceiving. As is so common throughout biology, the apparent simplicity at the macro level masks remarkable complexity at the micro or molecular level.
In eukaryotes, cell division occurs by either meiosis (sex cells) or mitosis (somatic cells). Bacteria, however, undergo neither of those processes (they are asexual and contain no membrane-enclosed organelles or nuclei). Bacterial cell division occurs by a process known as binary fission. Rod-shaped bacteria (e.g. Escherichia coli or Salmonella typhimurium) elongate to twice their original length. This is followed by invagination of the cell membrane, and the formation of a septal ring in the middle (Vicente et al., 2006; Weiss, 2004). The elongated bacterial cell splits down the middle, forming two daughter cells. Some bacteria exhibit variations on this mechanism. For example, in Caulobacter, no septum is formed (Poindexter and Hagenzieker, 1981) and its division is asymmetrical (Judd et al., 2003).
FtsZ Ring Assembly
A family of proteins called Fts proteins are necessary for proper cell division. One of these proteins, FtsZ, is key to cell division and is found ubiquitously in almost all known prokaryotes (Erickson et al., 2010; Margolin, 2005; Romberg and Levin, 2003; Erickson, 1997). There are a few notable exceptions to this generalization, including certain species of the genus Mycoplasma, (Lluch-Senar et al., 2010; Alarcon et al., 2007), Ureaplasma urealyticum (Vaughan et al., 2004; Brown and Rockey 2000; Glass et al., 2000), and the gammaproteobacterial clam symbiont Calyptogena okutanii (Kuwahara et al., 2007).
Cells with mutated FtsZ proteins are unable to divide; instead, they yield long filamentous cells (Addinall et al., 1996; Pla et al., 1991). In fact, for this reason FtsZ is a target for antibiotics (Schaffner-Barbero et al., 2012; Ma and Ma, 2012; Margalit et al., 2004). Togther, the Fts proteins build an apparatus for cell division called a divisome (Lutkenhaus et al., 2012; Gamba et al., 2009; Aarsman et al., 2005).
FtsZ is believed to be homologous to tubulin, but this is based largely on structural similarity, since the sequence identity is relatively weak (less than 20% overall). The sequence identity is strongest over the N-terminal GTP-binding domains but is almost completely absent over the C-terminal domains.
The first stage in formation of the divisome is the polymerization of thousands of FtsZ molecules to form a ring, the so-called FtsZ ring (Fu et al., 2010; Adams and Errington, 2009; Stricker et al., 2002). Two additional proteins that are recruited independently of each other, called ZipA and FtsA, function to tether the FtsZ ring to the inner membrane (Huang et al., 2013; Pichoff and Lutkenhaus, 2002; Erickson, 2001). FtsZ ring assembly occurs provided that at least one of those two proteins is present (Pichoff and Lutkenhaus, 2005). ZipA homologues are not found outside the γ-proteobacterial family, but "the requirement for ZipA can be bypassed completely by a single alteration in a conserved residue of FtsA" (Geissler et al., 2003). There is also evidence to suggest that elevated concentrations of FtsA (which is far more widely distributed) can compensate for a lack of ZipA. For example, FtsA is present in significantly higher concentrations in the Firmicute bacterium Bacillus subtilis than in Escherichia coli (Feucht et al., 2001). Although a particular anchor protein (such as ZipA) is not necessarily required for successful cell division, at least some anchor protein is certainly required. I would argue that here we have a case of irreducible complexity, since, unless FtsZ and a tether are present together, the system is useless. Both of these proteins also play a role in the recruitment of other division proteins (Pichoff and Lutkenhaus, 2002).
The FtsZ ring assembles following replication of the bacterial DNA (the nucleoids block FtsZ ring assembly prior to segregation). Location of the cell's center occurs by a very clever mechanism: Proteins called MinC and MinD oscillate from pole to pole, inhibiting assembly of the FtsZ ring (Dajkovic et al., 2008; Shih et al., 2003; Johnson et al., 2002; Cordell and Lowe, 2001 Shapiro and Losick, 2000; de Boer et al., 1992; de Boer et al., 1989; de Boer et al., 1988). Since the oscillation cycle of MinC and MinD entails that they spend more time at the poles of the cell than the middle, the cell's center has, on average, a lower concentration of these proteins than elsewhere. Midcell suppression of MinC and MinD is facilitated by an additional protein called MinE (Shen and Lutkenhaus, 2011; Sullivan and Maddock, 2000; Raskin and de Boer, 1997). Consequently, the cell's mid-point is most conducive to FtsZ ring assembly. As might be expected given its asymmetrical division, there appear to be no homologues of MinC or MinD in Caulobacter (Nierman et al., 2001).
Cell Shape Determination
In addition to those proteins that orchestrate cell division, there are also proteins that determine cell shape. Four significant shape-determining proteins in prokaryotes are MreB, MreC, MreD, and Mbl. Despite low sequence-identity, these proteins are thought to be homologous to actin based on structural similarities (Carballido-Lopez, 2006). For more than a decade, it has been thought that these proteins polymerize into spiral-shaped filaments in the cytoplasm, thereby forming a simple cytoskeleton (White et al., 2010; Divakaruni et al., 2007; Figge et al., 2004; Shih et al., 2003; Jones et al., 2001). This understanding was based on images obtained through fluorescence microscopy. In December of last year, however, an interesting study was published in the Journal of Bacteriology, which called this hypothesis into question (Swulius and Jensen, 2012). Using a technique known as electron cryotomography, the paper's authors reported that "MreB does in fact form extended helices and ﬁlaments in Escherichia coli when yellow ﬂuorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not," concluding that "the helices are therefore an artifact of the placement of the ﬂuorescent protein tag," and that "the many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered." For further discussion, see Margolin (2012): "The Price of Tags in Protein Localization Studies."
Although the mechanism is not entirely clear, there is definite evidence that these proteins are involved in cellular shape-determination. Inactivating mutations in the genes encoding them result in coccoid-shaped bacteria (Kawai et al., 2009; Bendezu and Boer, 2008; Figge et al., 2004; Doi et al., 1988; Wachi et al., 1987). In fact, naturally coccus-shaped bacteria do not possess a homologue of the mreB gene (suggesting that coccus-shaped bacteria is the default), although it has been reported that MreC and MreD proteins have been characterized and localized in some ovococcus species such as Streptococcus pneumoniae (Land and Winkler, 2011). The rod-shaped bacterium Caulobacter crescentus produces an additional shape-determining protein called crescentin, which is responsible for its characteristic curved morphology (Esue et al., 2010; Kim and Sun, 2009; Ausmees, 2006; Møller-Jensen and Lowe, 2005).
Bacteria are divided into two categories based on their response to gram staining, a chemical technique that differentiates bacteria according to the nature of their cell wall. So-called gram-positive bacteria possess a thick cell wall, whereas gram-negative bacteria possess a thinner cell wall. Gram-negative bacteria also characteristically possess a double membrane, whereas gram-positive bacteria possess only a single membrane. Almost all bacteria possess a peptidoglycan cell wall. In gram-negative bacteria, the region between the cell membrane and the cell wall is called the periplasmic space. Gram-positive bacteria possess a smaller periplasmic space between the cell wall and cytoplasmic membrane. Peptidoglycan is composed of N-acetylmuramic acid and N-acetylglucosamine, which alternate to form a polymer. These polymers are cross-linked by either a pentapeptide bridge (most gram-positive bacteria) or direct covalent bonding (most gram-negative bacteria) (Schleifer and Kandler, 1972). For a detailed review of peptidoglycan structure and architecture, I refer readers to an excellent review by Vollmer et al. (2008).
Bacterial cell division by binary fission requires the growth of the peptidoglycan cell wall as the bacterium elongates (Amir and Nelson, 2012). In rod-shaped bacteria, the cell wall grows at multiple locations along the cell. In the majority of cocci bacteria, the cell wall grows outward from the FtsZ ring in opposite directions. This process occurs by the severing of the peptidoglycan backbone and the synthesis of new cell wall material. Enzymes called autolysins hydrolyze the β-1,4 glycosidic bonds that link N-acetylglucosamine and N-acetylmuramic acid, and the gaps are filled in with additional cell wall material (Zoll et al., 2010; Smith et al., 2000). Since these enzymes can result in programmed cell death, careful regulation is required (Rice and Bayles, 2003; Calamita and Doyle, 2002).
The first stage of re-synthesis of the cell wall is the formation of the peptidoglycan precursors. A chain of five amino acids (a pentapeptide) is added to N-acetylmuramic acid. N-acetylglucosamine is subsequently attached to the end of the N-acetylmuramic acid. The result is a peptidoglycan precursor.
An extremely hydrophobic molecule called bactoprenol bonds to cell wall peptidoglycan precursors, and transports them across the cytoplasmic membrane by making them hydrophobic enough to pass through the membrane. In the periplasmic space, bactoprenol subsequently interacts with transglycosylase enzymes that are responsible for integrating the cell wall precursors into the cell wall and catalyzing the formation of the glycosidic bonds.
The final step of the process is called transpeptidation, the stage of cross-linking. In the case of gram-negative bacteria, this involves the formation of peptide cross-links between diaminopimelic acid and D-alanine on adjacent peptides. In gram-positive bacteria, cross-links typically occur from an L-lysine to a D-alanine of adjacent peptides. At the end of the peptidoglycan precursor, there exists initially two D-alanine residues, but one is removed during the reaction leaving one in the final molecule. A transpeptidase enzyme is responsible for the cross-linking of peptidoglycan. In E. coli a specialized penicillin-binding protein called FtsI is the key player in transpeptidation at the septum (Wissel and Weiss, 2004; Wang et al., 1998; Weiss et al., 1997). Localization of FtsI to the septum itself requires an intact N-terminal membrane anchor in addition to the division proteins FtsZ, FtsA, FtsQ, and FtsL (Weiss et al., 1999).
For more detailed reviews of the process of peptidoglycan biosynthesis, I refer readers to Lovering et al. (2012); Barreteau et al. (2008); Hett and Rubin (2008); Heijenoort (2001); and Heijenoort (1998). Be sure to also check out this video animation.
An Evolutionary Enigma
Re-synthesis of peptidoglycan is absolutely essential for viable cell division in virtually all bacteria. How do we know this? The inhibition of peptidoglycan cross-linking is what makes beta-lactam antibiotics -- which include penicillins (e.g. ampicillin), cephalosporins and monobactams -- so potent as antibacterial agents (Blundell and Perkins, 1981). Penicillin, for example, binds to penicillin-binding proteins, causing them to lose their enzymatic activity. The cell wall is thereby so weakened by the activity of the autolysins that the cell bursts. Some other, non beta-lactam, antibiotics (e.g. lactivicins) have a similar mechanism of action (Macheboeuf et al., 2007).
Cell wall precursors are also targets for antibiotics. For example, the antibiotic nisin associates with cell wall precursor lipid II, and effectively inhibits the peptidoglycan synthesis cycle by locking the cell wall precursor in a stable complex (Muller et al., 2012; Scherer et al., 2011).
Now, here's the conundrum. Consider the following two observations: (1) Critical to the elongation process is the severing of the peptidoglycan cell wall by the autolysins. (2) Critical to cell viability is the re-synthesis of the peptidoglycan cell wall. This has to be done in a coordinated fashion. Breaking of the cell wall can serve no adaptive utility until a mechanism has arisen for the simultaneous integration of peptidoglycan precursors. Indeed, without the latter mechanism, the cell is rendered non-viable. This is a classic example of what one might describe as an irreducibly complex system.
Observant readers will notice that I cautiously stated that peptidoglycan synthesis is essential for viable cell division in "virtually all bacteria". As those familiar with the field of microbiology will know only too well, where there is a rule, there are exceptions -- and this case is no different. One notable exception to the above generalization is species belonging to the genus Mycoplasma, which lack a peptidoglycan cell wall (Kornspan and Rottem, 2012; Hasselbring et al., 2006; Waites and Talkington, 2004; Razin et al., 1998). This fact, however, has little relevance to explaining the origins of the cell division machinery (including the mechanisms for severing and re-synthesizing peptidoglycan) in bacteria species that do possess a cell wall. In any case, Mycoplasma bacteria, although requiring a minimal genome and no cell wall, are obligate parasites. Mycoplasma also live in osmotically protected habitats such as an organism's body. They also typically possess sterols in their cytoplasmic membrane, imparting to them greater rigidity and strength.
Having considered the complex molecular machinery needed for successful cell division and the biosynthesis of peptidoglycan, ask yourself this question: Is this kind of system amenable to a step-wise Darwinian pathway of incremental modification? The problem outlined above strikes me as a fiendishly difficult challenge to modern evolutionary theory. Add this to the continuously expanding list of macromolecular machines that defy explanation by neo-Darwinian evolution.