Physical basis for the adaptive flexibility of Bacillus spore coats

Abstract

Bacillus spores are highly resistant dormant cells formed in response to starvation. The spore is surrounded by a structurally complex protein shell, the coat, which protects the genetic material. In spite of its dormancy, once nutrient is available (or an appropriate physical stimulus is provided) the spore is able to resume metabolic activity and return to vegetative growth, a process requiring the coat to be shed. Spores dynamically expand and contract in response to humidity, demanding that the coat be flexible. Despite the coat's critical biological functions, essentially nothing is known about the design principles that allow the coat to be tough but also flexible and, when metabolic activity resumes, to be efficiently shed. Here, we investigated the hypothesis that these apparently incompatible characteristics derive from an adaptive mechanical response of the coat. We generated a mechanical model predicting the emergence and dynamics of the folding patterns uniformly seen in Bacillus spore coats. According to this model, spores carefully harness mechanical instabilities to fold into a wrinkled pattern during sporulation. Owing to the inherent nonlinearity in their formation, these wrinkles persist during dormancy and allow the spore to accommodate changes in volume without compromising structural and biochemical integrity. This characteristic of the spore and its coat may inspire design of adaptive materials.

Figure 1.

B. subtilis spore morphology. (a) Wild-type (strain PY79) spores were analysed by SEM, (b,c) TEM [2] (by fixation in glutaraldehyde and osmium, dehydration, embedment in Spurr's resin, and then thin-section transmission electron microscopy) and (d) with AFM height profiles. Separate height profiles obtained on sterne strain of B. anthracis are also given in (e). (c) Is a magnification part of (b). Cortex (Cx), inner coat (IC), outer coat (OC) and a ruck (R) are indicated in (b) and/or (c). The size bars indicate (a) 250, (b) 500 and (c) 100 nm. (The outer most layer of the coat, the crust [3] is not seen because of the fixation method). The spore has an ovoid shape with a long and a short axis [4]. (d) Height profiles measured along the short axis of a spore are recorded at low (35%, solid line) and high (95%, dashed line) relative humidity depict partial unfolding of the wrinkles at a high relative humidity. (e) Height profiles measured for B. anthracis sterne also exhibit partial unfolding of the wrinkles at a high relative humidity. To plot the two curves as closely as possible, an offset is added to the height profiles at a low relative humidity because the overall height of the spore increases with relative humidity. Note that the widths of the spores are larger than the widths of the curves in (d,e), which are plotted across the ridges on top of the spore.

Figure 2.

Model of formation of folds in the spore coat and their response to spore shrinking. (a–c) Simulation of the ruck formation as the radius of spore interior (Rin) shrinks during sporulation. Rin values are given as percentages of the initial value, 300 nm. Rout is the average outer diameter. Using bending and stretching modulus values estimated from thickness and mechanical measurements of the coat, the model predicts the emergence of rucks that are comparable in width, height and number to previous reports [26]. (d) Upon spore expansion, rucks formed during sporulation do not reattach readily, but rather decrease their height and increase their width. Details of simulation results are given in electronic supplementary material, movie S1. (Online version in colour.)

Figure 3.

(a) Height measurements on wild-type and (b) cotE gerE mutant of B. subtilis, and (c) sterne strain of B. anthracis at low (35%) and high humidity (95%). (a–c) Scale bars, 1 µm. Heights of spores marked on the AFM images are listed on the right for a low and a high relative humidity, together with the percentage of the differences and average values. The mutant B. subtilis spore lacks most of its coat; yet its expansion is comparable to wild-type spores. (Online version in colour.)