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Water behavior in bacterial spores by deuterium NMR spectroscopy.

Friedline AW, Zachariah MM, Johnson K, Thomas KJ, Middaugh AN, Garimella R, Powell DR, Vaishampayan PA, Rice CV - J Phys Chem B (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma , 101 Stephenson Parkway, Norman, Oklahoma 73019, United States.

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.

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Postexchange B. subtilis 1A578 spore spectrum(Figure 4) overlaid on the same spectrum asthe five-day lyophilized deuterated BSA (Figure 5B). BSA’s spectrum fits well to the broad peak feature in1A578 but less so to the mobile water peak feature. The differencein line shape between this and Figure 10 isattributed to structural changes in B. subtilis 1A578resulting in higher protein conformational homogeneity and may beresponsible for the differences in line shape overall between B. subtilis 1A578 and B. subtilis ATCC6051.
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fig11: Postexchange B. subtilis 1A578 spore spectrum(Figure 4) overlaid on the same spectrum asthe five-day lyophilized deuterated BSA (Figure 5B). BSA’s spectrum fits well to the broad peak feature in1A578 but less so to the mobile water peak feature. The differencein line shape between this and Figure 10 isattributed to structural changes in B. subtilis 1A578resulting in higher protein conformational homogeneity and may beresponsible for the differences in line shape overall between B. subtilis 1A578 and B. subtilis ATCC6051.

Mentions: If feature III of the spore deuterium NMR spectrumin Figure 1A was indicative of labile deuterons,asserted byKaieda et al.,28 the spectrum observedin Figure 3 should look identical (or nearlyso) to that seen in Figure 1A. Instead, Figure 3 shows a feature II that is practically indistinguishablefrom the central mobile deuterium peak and broad shoulders insteadof the sharp Pake doublet seen in feature III of Figure 1A. Likewise, if feature III were the result of O–D···O=Chydrogen bonds susceptible to exchange, the H-to-D experiment shouldproduce a sharp Pake doublet. Instead, we speculate that the broadsignals in Figures 3 and 4 are from labile deuterons of proteins. When the spectra in Figures 3 and 4 are compared to aspectrum of BSA, the result is a nearly identical overlap for thewild-type 6051 spores (Figure 10). However,with its large fraction of mobile water, the resistant 1A578 spores(Figure 11) do not overlap with the BSA spectrumunless the protein spectrum is reduced in height. While the scalingfactor was 3.3, this number is somewhat meaningless as we are notconvinced that labile deuterons in the coat proteins of 1A578 sporeswould be exchanged to a similar degree as labile deuterons in a homogeneoussolution of BSA.


Water behavior in bacterial spores by deuterium NMR spectroscopy.

Friedline AW, Zachariah MM, Johnson K, Thomas KJ, Middaugh AN, Garimella R, Powell DR, Vaishampayan PA, Rice CV - J Phys Chem B (2014)

Postexchange B. subtilis 1A578 spore spectrum(Figure 4) overlaid on the same spectrum asthe five-day lyophilized deuterated BSA (Figure 5B). BSA’s spectrum fits well to the broad peak feature in1A578 but less so to the mobile water peak feature. The differencein line shape between this and Figure 10 isattributed to structural changes in B. subtilis 1A578resulting in higher protein conformational homogeneity and may beresponsible for the differences in line shape overall between B. subtilis 1A578 and B. subtilis ATCC6051.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216197&req=5

fig11: Postexchange B. subtilis 1A578 spore spectrum(Figure 4) overlaid on the same spectrum asthe five-day lyophilized deuterated BSA (Figure 5B). BSA’s spectrum fits well to the broad peak feature in1A578 but less so to the mobile water peak feature. The differencein line shape between this and Figure 10 isattributed to structural changes in B. subtilis 1A578resulting in higher protein conformational homogeneity and may beresponsible for the differences in line shape overall between B. subtilis 1A578 and B. subtilis ATCC6051.
Mentions: If feature III of the spore deuterium NMR spectrumin Figure 1A was indicative of labile deuterons,asserted byKaieda et al.,28 the spectrum observedin Figure 3 should look identical (or nearlyso) to that seen in Figure 1A. Instead, Figure 3 shows a feature II that is practically indistinguishablefrom the central mobile deuterium peak and broad shoulders insteadof the sharp Pake doublet seen in feature III of Figure 1A. Likewise, if feature III were the result of O–D···O=Chydrogen bonds susceptible to exchange, the H-to-D experiment shouldproduce a sharp Pake doublet. Instead, we speculate that the broadsignals in Figures 3 and 4 are from labile deuterons of proteins. When the spectra in Figures 3 and 4 are compared to aspectrum of BSA, the result is a nearly identical overlap for thewild-type 6051 spores (Figure 10). However,with its large fraction of mobile water, the resistant 1A578 spores(Figure 11) do not overlap with the BSA spectrumunless the protein spectrum is reduced in height. While the scalingfactor was 3.3, this number is somewhat meaningless as we are notconvinced that labile deuterons in the coat proteins of 1A578 sporeswould be exchanged to a similar degree as labile deuterons in a homogeneoussolution of BSA.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma , 101 Stephenson Parkway, Norman, Oklahoma 73019, United States.

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.

Show MeSH
Related in: MedlinePlus