Water Behavior in Bacterial Spores by Deuterium NMR Spectroscopy

Abstract

Dormant bacterial spores are able to survive long periods of time without nutrients, withstand harsh environmental conditions, and germinate into metabolically active bacteria when conditions are favorable. Numerous factors influence this hardiness, including the spore structure and the presence of compounds to protect DNA from damage. It is known that the water content of the spore core plays a role in resistance to degradation, but the exact state of water inside the core is a subject of discussion. Two main theories present themselves: either the water in the spore core is mostly immobile and the core and its components are in a glassy state, or the core is a gel with mobile water around components which themselves have limited mobility. Using deuterium solid-state NMR experiments, we examine the nature of the water in the spore core. Our data show the presence of unbound water, bound water, and deuterated biomolecules that also contain labile deuterons. Deuterium–hydrogen exchange experiments show that most of these deuterons are inaccessible by external water. We believe that these unreachable deuterons are in a chemical bonding state that prevents exchange. Variable-temperature NMR results suggest that the spore core is more rigid than would be expected for a gel-like state. However, our rigid core interpretation may only apply to dried spores whereas a gel core may exist in aqueous suspension. Nonetheless, the gel core, if present, is inaccessible to external water.

Sterilization Resistance of Bacterial Spores Explained with Water Chemistry

“…As described below and in previous work, 29 Feature I is attributed to mobile water and it is possible its intensity is a function of varying drying conditions. Nevertheless, the relative intensity of Features II and III are quite 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Variable-temperature deuterium NMR was also performed on spores of B. subtilis 1A578 to compare its behavior to that of B. pumilus SAFR-032.…”

“…Through NMR spectroscopy, we have been able to identify different chemical environments of water within the spore, reveal which internal water molecules can be exchanged with external water molecules, and show that rigid core water resists melting at elevated temperatures. 29 As described below, these properties are more profound in spores that are resistant to sterilization with hydrogen peroxide and UV light. Thus, NMR spectroscopy provides atomic level information of water chemistry that lends insight into the resistance mechanisms of bacterial spores.…”

“…These spectra are different than that shown for B. subtilis ATCC 6051 ( Figure 7C) and deuterated bovine serum albumin ( Figure 7B). 29 Difference spectra were also generated in order to determine how much deuterium signal was lost upon heating of spores during variable-temperature experiments and attribute that loss to Feature I, II, or III. Figure 8 shows a comparison of two such difference spectra, made by subtracting the spectrum obtained post-100 °C heating from that obtained post-50 °C heating for both B. subtilis 1A578 and B. pumilus SAFR-032.…”

“…26 Other papers published recently by Kong et al using single-spore Raman imaging reported that coreassociated CaDPA was found with water, though in a state closer to an amorphous solid or a glassy core than a pure powder, and that D 2 O in the spore is both unevenly distributed and in a weakly hydrogen-bonding mode. [27][28] Recent data from our laboratory support the core water residing in a rigid, glassy, state 29 in marked contrast to the gel-like core of Sunde et al 25 and Kaieda et al 26 With this understanding, we were motivated to determine if the spore water chemical environment can help explain spore sterilization resistance traits. A variety of isolates of Bacillus pumilus were obtained in 2004 from a clean room at the Jet Propulsion Laboratory's Spacecraft Assembly Facility in Pasadena, CA, and studied for UV resistance characteristics.…”

“…The variable-temperature NMR experiments indicate a rigid glassy core similar to our earlier work with B. subtilis. 29 Variable-temperature deuterium NMR was also performed with B. subtilis 1A578 for comparison to B. pumilus SAFR-032. The SAFR-032 spores demonstrated greater core rigidity than spores of 1A578, which would explain the higher UV resistance of that B. pumilus SAFR-032.…”