2007 NSTI Nanotechnology Conference and Trade Show - Nanotech 2007 - 10th Annual

Protons’ binding within nanometer scaled compartments of natural ionic reservoirs: Bacillus subtilis spores

S. Kazakov, E. Bonvouloir and I. Gazaryan
Pace University, US

natural ionic reservoirs, Bacillus spores, nanometer scaled compartments, proton binding, kinetics

Dormant spores of bacteria of Bacillus genus are intriguing micrometer-scaled objects for a physico-chemical study. The most paradoxical feature of dormant spores is that, being metabolically inactive and resistant to environmental insults, they actually monitor the external environment continuously in order to sense a specific germinant, i.e., life of a spore is determined by the balance between stabilization factors and germination effectors. One may suggest that this dynamic alert system is linked to the spore’s multi-layered structure which can change with time. The structure of Bacillus spores and mechanisms of spore resistance and dormancy support the hypothesis on the spore’s ability to act as a natural ionic reservoir accumulating/releasing ions in response to environmental stimuli. At least two of the spore integuments, cortex and core, can be considered as interacting ion-sensitive polymer networks with different mobility of small ions. Since the physical dimensions of spore compartments are of nanometer-scale, the kinetics of proton exchange between spores and environment can be examined by time-resolved micro-potentiometry, the method we recently developed for micro- and nanogels (hydrogel spheres of nanometer diameter) [Kazakov et al. J. Phys. Chem. B 2006, 110, 15107]. In the present work, the method was applied for the spore suspensions of different concentrations. A Scatchard-like approach to extract quantitative characteristics of the spore ability to accumulate or release protons (number of binding sites per spore, N, and apparent binding constant, K) has been developed on the basis of proton equilibrium binding to spore ionizable groups. The spore has been shown to behave like an almost infinite reservoir capable of accumulating billions of protons (N ~ 1010 per each spore). Two types of “binding sites” have been discovered: one, with K1 constant in the process of spore protonation, and second, with K2 dependent on the acidity of an aqueous titrant. One may speculate that the second constant actually reflects remodeling of the spore surface/structure making available a different combination of protonation sites. Proton uptake kinetics by dormant Bacillus subtilis spores corresponds to a multi-step process: proton accumulation can be modeled by (i) occupation of binding sites on the surface of spores, followed by (ii) penetration inside the bulk and binding to the ionizable groups within. The first process is too fast to be numerically analyzed, nevertheless, the data allow us to suggest the existence of at least two steps with characteristic time constants of less than 10 sec. The second process is also a two-step one, but its characteristic time constants are greater than 100 sec. The multi-step kinetic scheme for the proton uptake has to be attributed to the multi-layered spore structure, which includes exosporium, coats, cortex, inner membrane, and core. The result is the first step toward understanding the spore’s alert sensory mechanism based on monitoring the environmental conditions favoring germination. The future studies are of great potential for designing novel nature-inspired bionanosensors.

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