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Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation.

Brown AR, Correa E, Xu Y, AlMasoud N, Pimblott SM, Goodacre R, Lloyd JR - PLoS ONE (2015)

Bottom Line: FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines.This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response.Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear.

View Article: PubMed Central - PubMed

Affiliation: Williamson Research Centre for Molecular Environmental Science, University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom; Research Centre for Radwaste and Decommissioning, University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom; School of Earth, Atmospheric and Environmental Sciences, University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom.

ABSTRACT
Biogeochemical processes mediated by Fe(III)-reducing bacteria such as Shewanella oneidensis have the potential to influence the post-closure evolution of a geological disposal facility for radioactive wastes and to affect the solubility of some radionuclides. Furthermore, their potential to reduce both Fe(III) and radionuclides can be harnessed for the bioremediation of radionuclide-contaminated land. As some such sites are likely to have significant radiation fluxes, there is a need to characterise the impact of radiation stress on such microorganisms. There have, however, been few global cell analyses on the impact of ionizing radiation on subsurface bacteria, so here we address the metabolic response of S. oneidensis MR-1 to acute doses of X-radiation. UV/Vis spectroscopy and CFU counts showed that although X-radiation decreased initial viability and extended the lag phase of batch cultures, final biomass yields remained unchanged. FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines. MALDI-TOF-MS detected an increase in total protein expression in cultures exposed to 12 Gy. At 95 Gy, a decrease in total protein levels was generally observed, although an increase in a putative cold shock protein was observed, which may be related to the radiation stress response of this organism. Multivariate statistical analyses applied to these FT-IR and MALDI-TOF-MS spectral data suggested that an irradiated phenotype developed throughout subsequent generations. This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response. Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear.

No MeSH data available.


Related in: MedlinePlus

Fe(III) reduction by irradiated S. oneidensis MR-1.Cultures of S. oneidensis (MR-1) were grown aerobically in tryptic soy broth (30°C; 130 rpm) to late log–early stationary phase. Biomass was harvested and washed twice in sterile 30 mM sodium bicarbonate buffer prior to irradiation with 50 Gy X-radiation (rad). Immediately after irradiation, cell suspensions were driven anoxic with an 80:20 gas mix of N2:CO2 prior to inoculation into an anoxic medium containing 20 mM lactate as electron donor, 50 mM Fe(III) as poorly crystalline insoluble Fe(III) oxide and 30 mM sodium bicarbonate. 10 μM riboflavin (Rf) was added to media post-irradiation as an electron shuttle where necessary. Fe(II) concentrations were determined by ferrozine assay after extraction with 0.5 N HCl. Error bars depict standard error of the mean of triplicate experiments.
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pone.0131249.g006: Fe(III) reduction by irradiated S. oneidensis MR-1.Cultures of S. oneidensis (MR-1) were grown aerobically in tryptic soy broth (30°C; 130 rpm) to late log–early stationary phase. Biomass was harvested and washed twice in sterile 30 mM sodium bicarbonate buffer prior to irradiation with 50 Gy X-radiation (rad). Immediately after irradiation, cell suspensions were driven anoxic with an 80:20 gas mix of N2:CO2 prior to inoculation into an anoxic medium containing 20 mM lactate as electron donor, 50 mM Fe(III) as poorly crystalline insoluble Fe(III) oxide and 30 mM sodium bicarbonate. 10 μM riboflavin (Rf) was added to media post-irradiation as an electron shuttle where necessary. Fe(II) concentrations were determined by ferrozine assay after extraction with 0.5 N HCl. Error bars depict standard error of the mean of triplicate experiments.

Mentions: The data described above suggest that ionizing radiation alters protein and lipid levels. As these molecules are important components of biological membranes, radiation induced changes to these molecules could alter the integrity and function of S. oneidensis membranes, where many of the respiratory chain components and cytochromes required to respire alternative electron acceptors, such as Fe(III), are located. Hence, the ability of the irradiated phenotype to reduce poorly crystalline insoluble Fe(III) oxide was assessed. Cell cultures were irradiated with 50 Gy X-radiation, selected as an approximate median dose from the doses used in the metabolic experiments described above. Systems containing irradiated biomass displayed more than double the levels of Fe(III) reduction (Fig 6) despite biomass displaying only 2.3% viability with respect to non-irradiated controls (determined, as before, by serial dilution in PBS and CFU counts on solid minimal medium agar plates). It is evident that much of the Fe(III) in the poorly crystalline insoluble Fe(III) oxide used in this experimental system was not directly accessible for enzymatic reduction. Indeed, the extent of Fe(II) generation in the systems containing irradiated S. oneidensis was only increased with the addition of riboflavin, indicating that any radiation induced metabolic changes required the presence of an electron shuttle to facilitate reduction of Fe(III). The reason for this is unclear, although it could be related to a general up-regulation of metabolism post-irradiation, or physical damage to the cell structure facilitating extracellular electron transfer. However, the fact that this phenomenon is only observed in the presence of an electron shuttle suggests that this effect is likely not the result of the release of soluble reducing equivalents from lysed cells killed by radiation. The precise mechanism underpinning this result clearly warrants further work, ideally focusing on anaerobically grown cultures adapted to Fe(III) reducing conditions, rather than the aerobically grown cultures that were used in this experiment to facilitate comparisons with the earlier experiments described above. In addition, future experiments may be expanded to determine whether this phenomenon is limited to insoluble Fe(III) or is also applicable to chelated soluble Fe(III) and other inorganic anaerobic electron acceptors.


Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation.

Brown AR, Correa E, Xu Y, AlMasoud N, Pimblott SM, Goodacre R, Lloyd JR - PLoS ONE (2015)

Fe(III) reduction by irradiated S. oneidensis MR-1.Cultures of S. oneidensis (MR-1) were grown aerobically in tryptic soy broth (30°C; 130 rpm) to late log–early stationary phase. Biomass was harvested and washed twice in sterile 30 mM sodium bicarbonate buffer prior to irradiation with 50 Gy X-radiation (rad). Immediately after irradiation, cell suspensions were driven anoxic with an 80:20 gas mix of N2:CO2 prior to inoculation into an anoxic medium containing 20 mM lactate as electron donor, 50 mM Fe(III) as poorly crystalline insoluble Fe(III) oxide and 30 mM sodium bicarbonate. 10 μM riboflavin (Rf) was added to media post-irradiation as an electron shuttle where necessary. Fe(II) concentrations were determined by ferrozine assay after extraction with 0.5 N HCl. Error bars depict standard error of the mean of triplicate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476702&req=5

pone.0131249.g006: Fe(III) reduction by irradiated S. oneidensis MR-1.Cultures of S. oneidensis (MR-1) were grown aerobically in tryptic soy broth (30°C; 130 rpm) to late log–early stationary phase. Biomass was harvested and washed twice in sterile 30 mM sodium bicarbonate buffer prior to irradiation with 50 Gy X-radiation (rad). Immediately after irradiation, cell suspensions were driven anoxic with an 80:20 gas mix of N2:CO2 prior to inoculation into an anoxic medium containing 20 mM lactate as electron donor, 50 mM Fe(III) as poorly crystalline insoluble Fe(III) oxide and 30 mM sodium bicarbonate. 10 μM riboflavin (Rf) was added to media post-irradiation as an electron shuttle where necessary. Fe(II) concentrations were determined by ferrozine assay after extraction with 0.5 N HCl. Error bars depict standard error of the mean of triplicate experiments.
Mentions: The data described above suggest that ionizing radiation alters protein and lipid levels. As these molecules are important components of biological membranes, radiation induced changes to these molecules could alter the integrity and function of S. oneidensis membranes, where many of the respiratory chain components and cytochromes required to respire alternative electron acceptors, such as Fe(III), are located. Hence, the ability of the irradiated phenotype to reduce poorly crystalline insoluble Fe(III) oxide was assessed. Cell cultures were irradiated with 50 Gy X-radiation, selected as an approximate median dose from the doses used in the metabolic experiments described above. Systems containing irradiated biomass displayed more than double the levels of Fe(III) reduction (Fig 6) despite biomass displaying only 2.3% viability with respect to non-irradiated controls (determined, as before, by serial dilution in PBS and CFU counts on solid minimal medium agar plates). It is evident that much of the Fe(III) in the poorly crystalline insoluble Fe(III) oxide used in this experimental system was not directly accessible for enzymatic reduction. Indeed, the extent of Fe(II) generation in the systems containing irradiated S. oneidensis was only increased with the addition of riboflavin, indicating that any radiation induced metabolic changes required the presence of an electron shuttle to facilitate reduction of Fe(III). The reason for this is unclear, although it could be related to a general up-regulation of metabolism post-irradiation, or physical damage to the cell structure facilitating extracellular electron transfer. However, the fact that this phenomenon is only observed in the presence of an electron shuttle suggests that this effect is likely not the result of the release of soluble reducing equivalents from lysed cells killed by radiation. The precise mechanism underpinning this result clearly warrants further work, ideally focusing on anaerobically grown cultures adapted to Fe(III) reducing conditions, rather than the aerobically grown cultures that were used in this experiment to facilitate comparisons with the earlier experiments described above. In addition, future experiments may be expanded to determine whether this phenomenon is limited to insoluble Fe(III) or is also applicable to chelated soluble Fe(III) and other inorganic anaerobic electron acceptors.

Bottom Line: FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines.This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response.Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear.

View Article: PubMed Central - PubMed

Affiliation: Williamson Research Centre for Molecular Environmental Science, University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom; Research Centre for Radwaste and Decommissioning, University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom; School of Earth, Atmospheric and Environmental Sciences, University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom.

ABSTRACT
Biogeochemical processes mediated by Fe(III)-reducing bacteria such as Shewanella oneidensis have the potential to influence the post-closure evolution of a geological disposal facility for radioactive wastes and to affect the solubility of some radionuclides. Furthermore, their potential to reduce both Fe(III) and radionuclides can be harnessed for the bioremediation of radionuclide-contaminated land. As some such sites are likely to have significant radiation fluxes, there is a need to characterise the impact of radiation stress on such microorganisms. There have, however, been few global cell analyses on the impact of ionizing radiation on subsurface bacteria, so here we address the metabolic response of S. oneidensis MR-1 to acute doses of X-radiation. UV/Vis spectroscopy and CFU counts showed that although X-radiation decreased initial viability and extended the lag phase of batch cultures, final biomass yields remained unchanged. FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines. MALDI-TOF-MS detected an increase in total protein expression in cultures exposed to 12 Gy. At 95 Gy, a decrease in total protein levels was generally observed, although an increase in a putative cold shock protein was observed, which may be related to the radiation stress response of this organism. Multivariate statistical analyses applied to these FT-IR and MALDI-TOF-MS spectral data suggested that an irradiated phenotype developed throughout subsequent generations. This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response. Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear.

No MeSH data available.


Related in: MedlinePlus