Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier that defines cell shape and allows the cell to sustain large mechanical loads such as turgor pressure. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper-and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless "stored" growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan.bacterial morphogenesis | cell mechanics C ell growth is the result of a complex system of biochemical processes and mechanical forces. For bacterial, plant, and fungal cells, growth requires both the synthesis of cytoplasmic components and the expansion of the cell wall, a stiff polymeric network that encloses these cells. It is well established that plant cells use turgor pressure, the outward normal force exerted by the cytoplasm on the cell wall, to drive mechanical expansion of the cell wall during growth (1, 2). In contrast, our understanding of the physical mechanisms of cell-wall expansion in bacteria is limited. Furthermore, the bacterial cell wall is distinct from its eukaryotic counterparts in both ultrastructure and chemical composition. In particular, the peptidoglycan cell wall of Gram-negative bacteria is extremely thin, comprising perhaps a molecular monolayer (3). This raises the question of whether these organisms require turgor pressure for cell-wall expansion, or whether they use a different strategy than organisms with thicker walls.Turgor pressure is established within cells according to the Morse equation, P = RTðC in − C out Þ, where C in is the osmolarity of the cytoplasm, C out is the osmolarity of the extracellular medium, R is the gas constant, and T is the temperature. In the Gram-negative bacterium Escherichia coli, P has been estimate...

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Morphogenesis of plant cells is tantamount to the shaping of the stiff cell wall that surrounds them. To this end, these cells integrate two concomitant processes: 1), deposition of new material into the existing wall, and 2), mechanical deformation of this material by the turgor pressure. However, due to uncertainty regarding the mechanisms that coordinate these processes, existing models typically adopt a limiting case in which either one or the other dictates morphogenesis. In this report, we formulate a simple mechanism in pollen tubes by which deposition causes turnover of cell wall cross-links, thereby facilitating mechanical deformation. Accordingly, deposition and mechanics are coupled and are both integral aspects of the morphogenetic process. Among the key experimental qualifications of this model are: its ability to precisely reproduce the morphologies of pollen tubes; its prediction of the growth oscillations exhibited by rapidly growing pollen tubes; and its prediction of the observed phase relationships between variables such as wall thickness, cell morphology, and growth rate within oscillatory cells. In short, the model captures the rich phenomenology of pollen tube morphogenesis and has implications for other plant cell types.

When Staphylococcus aureus undergoes cytokinesis, it builds a septum generating two hemispherical daughters whose cell walls are only connected via a narrow peripheral ring. We found that resolution of this ring occurred within milliseconds (“popping”), without detectable changes in cell volume. The likelihood of popping depended on cell wall stress, and the separating cells split open asymmetrically leaving the daughters connected by a hinge. An elastostatic model of the wall indicated high circumferential stress in the peripheral ring before popping. Finally, we observed small perforations in the peripheral ring that are likely initial points of mechanical failure. Thus, the ultrafast daughter cell separation in S. aureus appears to be driven by accumulation of stress in the peripheral ring, and exhibits hallmarks of mechanical crack propagation.

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Bacterial multi‐drug resistance is a burgeoning crisis whose origin remains mechanistically opaque. Through altered permeability or efflux activity, the membrane prevents many antibiotics from reaching high enough intracellular concentrations to exert a therapeutic effect. We show here that protein synthesis of membrane‐associated genes involves translation of proline CC[C/U] codons, which require N1‐methylation of guanosine 37 (m1G37) on the 3′‐side of the tRNA anticodon. TrmD is the bacterial methyl transferase that catalyzes m1G37‐methylation. Removal of m1G37 by trmD inactivation reduces biosynthesis of membrane proteins, impairs membrane structure and mechanics, and sensitizes Gram‐negative bacteria to multiple classes of antibiotics and suppresses development of resistance and persistence. Codon engineering in the major efflux gene tolC removes the dependence on trmD for biosynthesis of membrane proteins. TrmD activity requires the conserved G37 in tRNAPro; replacement of G37 with C37 increases membrane permeability and accelerates cell death. These findings demonstrate the potential to diminish multi‐drug resistance by inaction of TrmD and its m1G37‐tRNA product. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Feedback mechanisms are required to coordinate balanced synthesis of subcellular components during cell growth. However, these coordination mechanisms are not apparent at steady state. Here, we elucidate the interdependence of cell growth, membrane tension, and cell-wall synthesis by observing their rapid re-coordination after osmotic shocks in Gram-positive bacteria. Single-cell experiments and mathematical modeling demonstrate that mechanical forces dually regulate cell growth: while turgor pressure produces mechanical stress within the cell wall that promotes its expansion through wall synthesis, membrane tension induces growth arrest by inhibiting wall synthesis. Tension inhibition occurs concurrently with membrane depolarization, and depolarization arrested growth independently of shock, indicating that electrical signals implement the negative feedback characteristic of homeostasis. Thus, competing influences of membrane tension and cell-wall mechanical stress on growth allow cells to rapidly correct for mismatches between membrane and wall synthesis rates, ensuring balanced growth.