Microbe forensics: Oxygen and hydrogen stable isotope ratios in Bacillus subtilis cells and spores

Abstract

Bacillus subtilis, a Gram-positive, endospore-forming soil bacterium, was grown in media made with water of varying oxygen (␦ 18 O) and hydrogen (␦D) stable isotope ratios. Logarithmically growing cells and spores were each harvested from the cultures and their ␦ 18 O and ␦D values determined. Oxygen and hydrogen stable isotope ratios of organic matter were linearly related with those of the media water. We used the relationships determined in these experiments to calculate the effective whole-cell fractionation factors between water and organic matter for B. O) that form the organic matter of those organisms. For example, isotope ratios have been used to trace the origins of migratory butterflies (1), birds (2, 3), and elephants (4, 5). Point-of-origin information can span several spatial scales. As an example, the physiological differences between C 3 and C 4 photosynthetic pathways, which result in large 13 C͞ 12 C differences, allow one to trace the flow of this organic carbon as differential dietary inputs to animals (6), the transport of carbon across ecosystems (7), and, ultimately, the movement of region-specific carbon back into the atmosphere (8-10). This sourcing aspect of stable isotopes has also been applied to forensic issues, such as determining the pointof-origin of illicit drugs (11, 12) and adulteration of foods and alcoholic beverages (13-15).Continentality, storm-track trajectories, and moisture origins result in substantial geographic gradients in the ␦ 18O and ␦D values of precipitation and, therefore, of local waters (16). Several maps have been produced that show the extent of isotopic variations in waters at the continental and global scales (17, 18). Again, organisms often record the isotopic composition of these source waters in their organic compounds. In trees, for example, the isotopic composition of cellulose is correlated with that of source water (19,20). Similar examples are found in animals where oxygen isotope ratios of bone and blood are closely related to that of the local water (21).We hypothesized that microorganisms, too, should show a record of their growth environment in their cellular components. If relationships between growth environment and cellular isotope composition could be elucidated, it might become possible to draw conclusions about the waters in which the microbes had been grown, particularly for genetically identical organisms cultured in different geographic regions. We used the growth of Bacillus subtilis in laboratory culture as a model system. B. subtilis is a nonpathogenic, endospore-forming, Gram-positive soil bacterium. Because B. subtilis can grow on a variety of nutrients, we focused first on the relationship between the ␦ 18O and ␦D values in the microbial products (cells or spores) and those ratios in the water used to make the culture media. We then tested the values predicted by laboratory model with cultures grown in different geographical regions across the United States. MethodsOur experimental organism was B. subtilis strain 6051 (American ...

Plant Pathogen Forensics: Capabilities, Needs, and Recommendations

“…Some lack of inherency may be useful, however, when the property reflects a feature of the microbe's original environment. For example, a microbe's 18 O and 2 H isotope content can suggest in what water source it was produced (95). A diagnostic marker set discriminates among closely related pathogen strains, allowing confident exclusion of nonmatching suspects.…”

Hydrogen and oxygen isotope ratios in human hair are related to geography

“…T he carbon (␦ 13 C), nitrogen (␦ 15 N), and sulfur (␦ 34 S) isotope ratios of humans, other animals, and microbes are strongly correlated with the isotope ratios of their dietary inputs (1)(2)(3)(4)(5). The adage ''You are what you eat'' reflects the observation that there are limited differences (Յ1‰) between heterotrophic organisms and their diet in either the ␦ 13 C or ␦ 34 S values (6)(7)(8).…”

“…The ␦ 2 H values of bird feathers and butterfly wings (both are largely keratin) and water in the region in which the tissue was produced are highly correlated (14,15). Kreuzer-Martin et al (1) showed that Ϸ70% of the oxygen and Ϸ30% of the hydrogen atoms in microbial spores (Ϸ50% proteinaceous) were derived from the water in the growth medium, whereas the remainder was derived from the organic compounds supplied as substrate (16).…”

“…The value of ␣ ow is unknown, but could be as high as 1.027, the value for fully equilibrated carbonyl-oxygen isotope fractionation between acetone and water (29). Kreuzer et al (1) calculated ␣ ow to be 1.0164 for microbial spore cell walls during non-log-phase growth.…”

Ecological Implications Of The ẟ13c Values of Plant Species Growing in Natural Environment, Greenhouse and Plant Respired Carbon Dioxide (Captured as Carbonate)

“…Stable carbon isotopes ratios (ẟ 13 C) of terrestrial plants are employed across a diverse range of applications in environmental and plant sciences; however, the kind of information that is desired from the ẟ 13 C signals often differs [9]. Further, ẟ 13 C values have been used to solve scientific problems, for example, to trace the flow of carbon as differential dietary inputs (Hobson, 1999[12]), follow the transport of carbon across ecosystems (Conte and Weber, 2002[13]; Ehleringer, Cerling and Dearing, 2002 [14]), address forensic problems such as determining the origin of illicit drugs (Carter, Titterton, Murray and Sleeman 2002 [15]), trace the origin of infectious microbial source (Kreuzer-Martin, Lott, Dorigan and Ehleringer 2003 [16]), Kreuzer-Martin, Chesson, Lott, Dorigan and Ehleringer2004 [17]), fingerprints of biological agents as forensic tool (Horita and Vass, 2003[18]) and differentiating between C 3 and C 4 dicot species of the Centrospermeae families and understanding climatic factors affecting their distribution along altitudinal gradient in Kenya [3] [8]. Thus, at the extremes, the ẟ 13 C value applications ranges between diverse and dynamic field of studies in the society and not only pure environmental and biological criteria [9].…”