Spatially resolved characterization of water and ion incorporation in Bacillus spores

Abstract

We present the first direct visualization and quantification of water and ion uptake into the core of individual dormant Bacillus thuringiensis subsp. israelensis (B. thuringiensis subsp. israelensis) endospores. Isotopic and elemental gradients in the B. thuringiensis subsp. israelensis spores show the permeation and incorporation of deuterium in deuterated water (D(2)O) and solvated ions throughout individual spores, including the spore core. Under hydrated conditions, incorporation into a spore occurs on a time scale of minutes, with subsequent uptake of the permeating species continuing over a period of days. The distribution of available adsorption sites is shown to vary with the permeating species. Adsorption sites for Li(+), Cs(+), and Cl(-) are more abundant within the spore outer structures (exosporium, coat, and cortex) relative to the core, while F(-) adsorption sites are more abundant in the core. The results presented here demonstrate that elemental abundance and distribution in dormant spores are influenced by the ambient environment. As such, this study highlights the importance of understanding how microbial elemental and isotopic signatures can be altered postproduction, including during sample preparation for analysis, and therefore, this study is immediately relevant to the use of elemental and isotopic markers in environmental microbiology and microbial forensics.

FIG. 1.

Representative distribution of C, F, P, and Cl in a single B. thuringiensis subsp. israelensis endospore, as determined by NanoSIMS imaging depth profile analysis using a ¹³³Cs⁺ primary ion beam. (A) The ion images obtained from serial scans of the spore by the Cs beam represent the summed ion counts for each species. Lighter colors represent higher counts. Scale bar = 500 nm. (B) The depth profile data are extracted from serial images of the spore as it is eroded away by the Cs beam. The data are extracted from the ∼200-nm-diameter circle, which is centered on the P image. Depth is estimated from the lateral dimension of the spore and the sputter rate of similar samples.

FIG. 2.

Deuterium uptake with depth and time data obtained from B. thuringiensis subsp. israelensis spores exposed to D₂O. (A) Distribution of deuterium (D) and hydrogen (H) as a function of depth in a single spore exposed to D₂O. The depth profile data are normalized to those of C⁻. Error bars represent 2 standard errors and are not visible where smaller than data points. (B) Summed H⁻ and D⁻ images of a single spore. Color scales are linear and indicate counts per pixel. Scale bar = 500 nm. (C) Uptake and exchange of D₂O by B. thuringiensis subsp. israelensis spores as a function of time of exposure to D₂O vapor and when the analysis took place relative to the end of the exposure experiment (day 1 and 3 months, respectively). D⁻/H⁻ ratios are whole-spore averages. Open symbols represent data obtained with Parr bomb treatment (2 days at 80°C). Untreated spores yielded the natural abundance D/H ratio of ∼0.00016.

FIG. 3.

Li uptake into B. thuringiensis subsp. israelensis spores from 10 mM LiF solution. (A) Depth profiles of ⁷Li⁺ in control spores (no exposure) and spores exposed to LiF solution for 3 days, showing uptake of Li from solution throughout the spore, with greater uptake in the outer structures. Li content in the treated spores (measured as a ⁷Li⁺/¹²C⁺ ion ratio) increased by greater than 4 orders of magnitude relative to the control sample. (B, C) Summed ⁴⁴Ca⁺ and ⁷Li⁺ NanoSIMS ion images of spores with no exposure (B) and 3 days of exposure to 10 mM LiF solution (C). The Ca images show the locations of the spores, and the Li images show the low background in the control sample (B) and that Li is localized in the spores in the exposed samples (C).

FIG. 4.

Average Li uptake by B. thuringiensis subsp. israelensis spores. (A) ⁷Li⁺/¹²C⁺ ion ratios in the spore outer structures and core as a function of exposure time to 0.4 mM LiF solution. The rate of Li incorporation into the spore core is modeled by fitting the core Li⁺/C⁺ ionic ratio as a function of exposure time to the rate equation, R(t) = R₀ + ΔR[1 − e^−(t/Γ)]. Here, [⁷Li⁺/¹²C⁺] = 1.6 − 1.5e^t/−17.9. (B) Average ⁷Li⁺ depth profiles for spores exposed to 0.4 mM LiF for different exposure times. The spores in the depth profiles with exposure times of 7 h and 3 days are not fully consumed. The numbers of spore analyses performed for each parameters in panels A and B are 21 for 5 min, 12 for 15 min, 12 for 7 h, 13 for 1 day, and 13 for 3 days.

FIG. 5.

(A to C) Average F content (measured as ¹⁹F⁻/¹²C⁻) in the spore outer structures and core as a function of exposure time to 0.4 mM LiF solution (A), as a function of LiF solution concentration (B), and as a function of exposure time to deionized water (C). The data shown in panel B correspond to 3 days of exposure for each concentration. Open symbols correspond to the control samples. The control used for the 0.4 mM samples was stored hydrated, and the control used for the other treatments was stored dry. The number of spore analyses performed for each parameter is 27 for the control, 10 for 7 h, 11 for 1 day, and 33 for 3 days (A); 6 for the control, 33 for 0.4 mM, 5 for 10 mM, and 8 for 40 mM LiF (B); and 6 for the control, 5 for 10 min, and 5 for 3 days (C).

FIG. 6.

(A to C) Change in spore core and outer structure Cl content (measured as ³⁵Cl⁻/¹²C⁻) with exposure time to 0.4 mM LiF solution (A), LiF concentration (B), and exposure time to deionized water (C). Open symbols indicate data for the control samples. The control used for the 0.4 mM samples was stored hydrated, and the control used for the other treatments was stored dry. The number of spore analyses performed for each parameter is 27 for the control, 10 for 7 h, 11 for 1 day, and 33 for 3 days (A); 6 for the control, 5 for 10 mM, and 8 for 40 mM LiF (B); and 6 for the control, 5 for 10 min, and 5 for 3 days (C).