Insights & Perspectives Hypotheses How bacterial cell division might cheat turgor pressure – a unified mechanism of septal division in Gram-positive and Gram-negative bacteria Harold P. Erickson An important question for bacterial cell division is how the invaginating septum can periplasm, which is a major focus of the overcome the turgor force generated by the high osmolarity of the cytoplasm. I present analysis. To achieve division all suggest that it may not need to. Several studies in Gram-negative bacteria have layers of the cell envelope must invagi- nate into the division site, although not shown that the periplasm is isoosmolar with the cytoplasm. Indirect evidence at the same time, and eventually fully suggests that this is also true for Gram-positive bacteria. In this case the invagination cover the daughter cells. of the septum takes place within the uniformly high osmotic pressure environment, In 2008, Osawa et al. [6] demon- and does not have to fight turgor pressure. A related question is how the V-shaped strated that FtsZ-mts could assemble Z constriction of Gram-negative bacteria relates to the plate-like septum of Gram- rings when reconstituted in tubular liposomes. The mts (membrane-target- positive bacteria. I collected evidence that Gram-negative bacteria have a latent ing sequence) is an amphipathic helix capability of forming plate-like septa, and present a model in which septal division is that can tether FtsZ directly to mem- the basic mechanism in both Gram-positive and Gram-negative bacteria. brane. These reconstituted Z rings could constrict the thick walls of Keywords: multilamellar liposomes. Both Z-ring .bacteria; cell division; cytokinesis; FtsZ; peptidoglycan; tubulin; turgor assembly and constriction were achieved by FtsZ-mts alone À no other division protein was needed. This Introduction cycle. Near the end of the cell cycle the Z discovery elevated FtsZ as the prime ring, including all the downstream candidate for generating the constric- In bacterial cytokinesis FtsZ provides proteins, constricts over a period of tion force in bacterial cell division. the cytoskeletal framework for the Z minutes to pinch the cell in two. The How could FtsZ constrict mem- ring, which serves as the docking site for mechanisms of FtsZ have been investi- branes? Previous work had demon- up to two dozen other division proteins. gated intensively since the discovery strated that FtsZ protofilaments can The Z ring assembles early in the cell 25 years ago that FtsZ is a homolog of switch conformation from straight to cycle and remains in place without any eukaryotic tubulin, and that it is local- curved, and it was suggested that the obvious constriction for most of the ized at the site of constriction [1–5]. curved protofilaments could generate a Bacteria are surrounded by a cell bending force on the membrane and DOI 10.1002/bies.201700045 envelope. In Gram-positive bacteria the pull it inward [7, 8]. Additional experi- cell envelope consists of an inner lipid ments with “inside-out Z rings” sup- Department of Cell Biology, Duke University membrane (IM) and a rigid cell wall ported this mechanism [9–12]. Medical School, Durham, NC 27710, USA composed primarily of peptidoglycan Recently this Z-centric hypothesis (the abbreviation PG will refer to the cell has been questioned. Coltharp and Corresponding author: wall, implicitly including all other Xiao [13, 14] suggested that the force Harold P. Erickson E-mail: [email protected] components). Gram-negative bacteria from bending FtsZ protofilaments have an additional outer lipid mem- would be insufficient to constrict the Abbreviations: brane (OM), which is closely attached to IM against the turgor pressure gener- EM, electron microscopy; IM, inner membrane; MDO, membrane-derived oligosaccharide; OM, the PG. The PG layer is separated from ated by the high osmolarity of the outer membrane; PG, peptidoglycan. the IM by a variable space termed the bacterial cytoplasm. They suggested Bioessays 39: 1700045, ß 2017 WILEY Periodicals, Inc. www.bioessays-journal.com 1700045 (1 of 10) H. P. Erickson Insights & Perspectives..... that the major force for constriction is and relies on the natural contrast of the likely generated by inward growth of the membranes and protein structures with PG wall, pushing the IM from the surrounding water. Most of the results outside. In this mechanism the role of discussed below (Figs. 1–3) were FtsZ is primarily to serve as a docking obtained from high pressure freezing site for the PG synthetic enzymes, and to followed by cryosectioning. An alter- regulate assembly of the PG. native to cryosectioning is cryoEM Here, I am not arguing for one tomography, where the whole bacte- constriction mechanism or the other. I rium is imaged at multiple tilts and a 3- present the hypothesis that the essen- D image is reconstructed (Fig. 4). The tial steps of cell division, constricting thin samples for tomography are typi- Hypotheses the IM, and building new PG to form a cally prepared by plunge freezing Figure 1. CryoEM (high pressure freezing E. septum, likely take place completely without the need for high pressure. and cryosection) of the cell envelope of coli K-12. The black arrows point to the IM within the high osmolarity environ- Matias, Beveridge, and colleagues and OM. The PG is the lightly stained ment, and never have to resist the applied the technique of high pressure narrow band indicated by the white arrow. turgor pressure. This would mean freezing and cryosectioning to bacteria The periplasm is the lighter stained zone that both potential mechanisms, FtsZ in a series of seminal papers from 2003 between the PG and IM. Bar is 50 nm. bending and cell wall ingrowth, would to 2008. Figure 1 shows the cell enve- Reprinted with permission from [16]. need only modest forces to achieve lope of E. coli K12 [16]. The IM and OM division. are each seen as a single, thin, dark staining layer. The PG appears as a line medium density “outer wall zone” that of slightly enhanced density close to the can be identified as the PG layer. The cell envelope of OM. The periplasm is the lighter stain- Several other Gram-positive bacteria Gram-positive and Gram- ing space between the IM and the PG. have been imaged bythe same technique, Matias et al. [16] noted two important anddimensionsof theircellenvelopes are negative bacteria features of the periplasmic space: its given in Table 1. For most of these the visualized by cryoEM width varied by 10% over non- periplasm is 20 nm thick versus 10 nm compressed sections of the cell enve- for E. coli, and the PG is much thicker, The structural relationships of the mem- lope; and it could be significantly 20–30 nm versus 6.4 nm for E. coli.An branes and PG wall have been determined compressed by the sectioning knife. exception is Mycobacterium bovis, whose by thin section electron microscopy (EM). They suggested that the periplasmic periplasm and PG are closer to the In the early days of EM, bacteria were space has inherent compressibility or dimensions of E. coli. Interestingly, M. chemically fixed, dehydrated and infil- flexibility that allows the IM to move bovis and related Corynebacterineae were trated with plastic, and thin sections were closer or farther away from the PG. It is shown to have an outer lipid bilayer, cut. Aldehydes and OsO4 were the most not a rigid gel. chemically different but structurally sim- popular fixation agents. There was, how- The term “periplasm” has had two ilar to the OM of E. coli [19]. ever, always concern about changes in the different meanings in the literature. A structure that might occur during the time number of authors use the term to refer to for the fixation to work and during the entire space between the IM and OM, The periplasm of Gram- subsequent processing. with the PG layer being contained within negative bacteria is Rapid freezing offered the promise this periplasm. However, we will use the of instantaneous fixation, but ice terms periplasm or periplasmic space to isoosmotic with the crystal formation disrupted tissue refer specifically to thespacebetweenthe cytoplasm structures more than a few mmfrom PG and IM. This designation was used in the freezing surface. Cryoprotectants the original study of osmolarity of the An important concern for bacterial divi- could reduce ice crystals, but might compartments [17], and it is also consis- sionishowitdealswiththeturgorforceof alter the cellular structures. A major tent with the definition of periplasm in the bacterial cytoplasm. To understand advance was the development of high Gram-positive bacteria. this we need to know which elements of pressure freezing; see McDonald for a Early studies had suggested that in the cell envelope generate and which review of the history and applica- Gram-positive bacteria the IM was elements contain the turgor force. tions [15]. Briefly, the sample is sub- pressed in tight apposition to the In Gram-negative bacteria the OM is jected to 2,000 bar pressure relatively thick PG cell wall [18]. In tightly coupled to the PG by the very simultaneously with rapid freezing, 2005, Matias and Beveridge [18] over- abundant lipoprotein Lpp (other attach- both being achieved in 30 micro- turned this model when they applied ments exist but Lpp is the most seconds. The high pressure prevents cryosectioning to image the envelope of abundant). Lpp is a triple helical rod, formation of ice crystals, and the rapid Bacillus subtilis. Figure 2 reproduces 8.3 nm long with three fatty acid chains freezing preserves the organic struc- their definitive images. The cell enve- on its N-terminus that insert into the tures. The frozen specimen can then be lope comprises the IM, resolved here as OM [20].