Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity

Abstract

Using an automated scanning microscope, we report the surprising result that individual dormant spores of Bacillus thuringiensis grow and shrink in response to increasing and decreasing relative humidity. We simultaneously monitored the size of inorganic calibration particles. We found that the spores consistently swell in response to increased relative humidity, and shrink to near their original size on reexposure to dry air. Although the dispersion of swelling amplitudes within an ensemble of spores is wide (Ϸ30% of the average amplitude), amplitudes for individual spores are highly correlated between different swelling episodes, suggesting that individual spores respond consistently to changes in humidity. We find evidence for two distinct time scales for swelling: one with a time scale of no more than Ϸ50 s, and another with a time scale of Ϸ8 min. We speculate that these two mechanisms may be due to rapid diffusion of water into the spore coat ؉ cortex, followed by slower diffusion of water into the spore core, respectively. Humidity-dependent swelling may account for the greater kill effectiveness of spores by gas-phase chlorine dioxide, formaldehyde, and ethylene oxide at very high relative humidity. In the aftermath of the anthrax attacks in Fall 2001, we looked into measurable physical properties of bacterial endospores that might distinguish Bacillus anthracis from other species. This led to two striking discoveries: The first, which we report in this paper, is that dormant spores are not entirely static but rapidly swell and shrink in response to increases and decreases in relative humidity (RH), with interesting biological implications. The second, which we report in a subsequent paper, is that, when RH and temperature are held constant, accurate measurements of sizes of Bacillus spores can discriminate among species.It has long been known from the high refractive index of spores that the core has a low water content. Neihof et al.(1) measured water sorption and desorption of both freeze-dried and crushed Bacillus subtilis spores as a function of RH. From the sigmoid shape, hysteresis, and similar behavior of normal and crushed spores, they inferred that swelling accompanies sorption and that an anhydrous spore core is not maintained by a water permeability barrier.Studies of the inverse correlation between spore heat resistance and water content have led to the present view that an osmotic pressure within the spore is responsible for maintaining the partly dry core (2, 3). The requisite tension and pressure are thought to be generated in the cortex by peptidoglycan, initially polymerized in an ordered tightly packed conformation, fixed by cross-linking of the glycan chains (3). From experiments with deuterated water, Marshall and Murrell (4) concluded that at least 97% of the water in spores was able to exchange with the environment. In experiments with tritiated water, they found that water exchange in a population of spores as well as of vegetative cells was almost complete in 2-3 min at 0°C. In m...

The physical state of water in bacterial spores

“…Consistent with this view, our data indicate fast water exchange across the coat on the 2 H and 17 O relaxation time scales. Scanning microscope observation of B. thuringiensis spores exposed to variable humidity identified two distinct time scales, 50 and 500 s, for spore swelling, tentatively identified with water influx into the coat ϩ cortex and into the core, respectively (44). These time scales refer to swelling of dried spores and they are much longer than the time scales found here for water exchange within fully hydrated spores.…”

Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile

“…The spore core volume was determined as described above, but substituting 5 M [ 14 C]sorbitol for methylamine. Water has been reported to penetrate the entire spore, whereas sorbitol does not (4)(5)(6)(7). The amount of methylamine in the spore core and the core pH were calculated as described (4, 7).…”

“…One factor important in dormant spore resistance to such chemicals appears to be their slow permeation into the spore core or protoplast. A variety of data indicate that it is the spore's inner membrane that is the major permeability barrier restricting the passage of small molecules into the spore core (3)(4)(5)(6)(7), although the lipid composition of the inner membrane exhibits no anomalies that might explain its unusual properties (8)(9)(10)(11)(12). In addition, the dormant spore's inner membrane has the potential to expand significantly, because electron microscopy indicates that the volume this membrane encompasses (the spore core): (i) decreases as much as 2-fold late in sporulation (13); and (ii) increases up to 2-fold in the first minute of spore germination, when the spore's large peptidoglycan cortex is degraded and the germ cell wall expands (13)(14)(15).…”

Coherent X-ray diffractive imaging on the single-cell-level of microbial samples

“…For instance, a socalled honeycomb crystalline layer with a period of about 9 nm and ∼ 5−6 nm holes or pits seems to be present in the coat of many Bacillus species [231,232]. Finally, it became only recently understood that the endospore morphology changes drastically due to an increase or decrease of relative humidity of its environment [68,325].…”

“…In particular, it was shown by Westphal et al [325] that endospores undergo swelling when exposed to an increase in relative humidity. They could identify two di erent swelling time scales (t 1 < 50 s and t 2 ≈ 8 min), that may be related to di usion times of water into coat & cortex and core, respectively.…”