Mechanical Consequences of Cell-Wall Turnover in the Elongation of a Gram-Positive Bacterium

Abstract

A common feature of walled organisms is their exposure to osmotic forces that challenge the mechanical integrity of cells while driving elongation. Most bacteria rely on their cell wall to bear osmotic stress and determine cell shape. Wall thickness can vary greatly among species, with Gram-positive bacteria having a thicker wall than Gram-negative bacteria. How wall dimensions and mechanical properties are regulated and how they affect growth have not yet been elucidated. To investigate the regulation of wall thickness in the rod-shaped Gram-positive bacterium Bacillus subtilis, we analyzed exponentially growing cells in different media. Using transmission electron and epifluorescence microscopy, we found that wall thickness and strain were maintained even between media that yielded a threefold change in growth rate. To probe mechanisms of elongation, we developed a biophysical model of the Gram-positive wall that balances the mechanical effects of synthesis of new material and removal of old material through hydrolysis. Our results suggest that cells can vary their growth rate without changing wall thickness or strain by maintaining a constant ratio of synthesis and hydrolysis rates. Our model also indicates that steady growth requires wall turnover on the same timescale as elongation, which can be driven primarily by hydrolysis rather than insertion. This perspective of turnover-driven elongation provides mechanistic insight into previous experiments involving mutants whose growth rate was accelerated by the addition of lysozyme or autolysin. Our approach provides a general framework for deconstructing shape maintenance in cells with thick walls by integrating wall mechanics with the kinetics and regulation of synthesis and turnover.

Cell-Biological Studies of Osmotic Shock Response in Streptomyces spp

“…For immunostaining and Western blot experiments, the osmotic shift was performed in liquid culture. S. coelicolor strain M145 was grown in liquid YEME medium without sucrose for about 12 h. Grown cultures were centrifuged, resuspended in liquid YEME medium with sucrose, and incubated for different time intervals (10,30,60,120,150, and 180 min) before harvesting samples for Western blot analysis or immunostaining. Cultures for immunostaining were grown in medium also containing 0.5% glycine.…”

“…Studies of the regulation of bacterial stress responses are now increasingly being complemented by imaging approaches that enable studies of single cells in situ under osmotic stress. Such investigations have resulted in new insights into turgor pressure, cell growth, cell wall synthesis machineries, cytoskeleton, chromosome topology, and physical properties of the cytoplasm (8)(9)(10)(11).…”

“…Pilizota and Shaevitz also showed that turgor pressure is not responsible for the slow growth of E. coli on high-osmolality media (16). However, in the Gram-positive bacterium Bacillus subtilis, turgor pressure was suggested to be essential for cell growth (10). To understand the fundamental relationships between osmotic stress, growth, and turgor pressure in bacteria, more research is needed on bacteria with contrasting cell wall structures and growth strategies.…”

Getting into shape: How do rod-like bacteria control their geometry?

“…In Gram-positive bacteria the cell wall is much thicker than in Gram-negatives (e.g., in B. subtilis the cell wall approximately 30 nm thick (Beeby et al 2013;Misra et al 2013). Recent electron cryotomography and surface AFM experiments have revealed circumferential furrows in the cell-wall surface (Beeby et al 2013;Andre et al 2010;Hayhurst et al 2008) with a spacing of roughly 50 nm.…”

Polydiglycosylphosphate Transferase PdtA (SCO2578) of Streptomyces coelicolor A3(2) Is Crucial for Proper Sporulation and Apical Tip Extension under Stress Conditions

“…In case of osmotic upshift, bacteria need to increase the internal osmolyte concentration to avoid efflux of water leading to changes in turgor pressure (47). Turgor pressure was recently shown to be essential for the growth of B. subtilis (48). In 6% NaCl, growth of ⌬pdtA mutant liquid cultures was severely impaired, and only 4% of the normal biomass was produced.…”