Limits...
Protein oxidation implicated as the primary determinant of bacterial radioresistance.

Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, Leapman RD, Lai B, Ravel B, Li SM, Kemner KM, Fredrickson JK - PLoS Biol. (2007)

Bottom Line: For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups.X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells.For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America. mdaly@usuhs.mil

ABSTRACT
In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.

Show MeSH

Related in: MedlinePlus

Additional Oxidative Protein Damage Assays(A) In vitro IR-induced oxidative protein damage. Western blot (W) immunoassay of protein-bound carbonyl groups in D. radiodurans (non-irradiated) cell extract adjusted to 500 μM FeCl2 and irradiated to the indicated doses. A total of 20 μg of protein extract loaded per lane.(B) Mn-depleted, radiosensitive D. radiodurans cells [1] are highly susceptible to oxidative protein damage during irradiation. D. radiodurans was grown in defined rich medium without Mn supplementation (no-Mn DRM) [1] to OD600 0.8 and exposed aerobically to 10 kGy. A total of 20 μg of protein extract loaded per lane.(C) Decreased survival of D. radiodurans irradiated at pH 10.5 correlates with oxidative protein damage. D. radiodurans was grown to OD600 approximately 0.9 in TGY (pH 7), adjusted to pH 10.5, and exposed aerobically to the indicated doses. A total of 20 μg of protein extract loaded per lane.(D) P. putida proteins are similarly susceptible to oxidative protein damage when cells are irradiated anaerobically (+Ar) or aerobically (+O2). P. putida was grown to OD600 approximately 0.9 in TGY, purged with ultra-high purity Ar, and irradiated in sealed tubes to 4 kGy.Values for intracellular Mn/Fe concentration ratios and D10 at the bottom of (B), (C), and (D), as reported previously [1]. A total of 20 μg of protein extract loaded per lane.C, Coomassie-stained polyacrylamide denaturing gel; M, mixture of artificial IgG-binding protein standards; O, oxidized protein standards; S, wide-range protein standards; +, DNPH treated;−, DNPH untreated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1828145&req=5

pbio-0050092-g004: Additional Oxidative Protein Damage Assays(A) In vitro IR-induced oxidative protein damage. Western blot (W) immunoassay of protein-bound carbonyl groups in D. radiodurans (non-irradiated) cell extract adjusted to 500 μM FeCl2 and irradiated to the indicated doses. A total of 20 μg of protein extract loaded per lane.(B) Mn-depleted, radiosensitive D. radiodurans cells [1] are highly susceptible to oxidative protein damage during irradiation. D. radiodurans was grown in defined rich medium without Mn supplementation (no-Mn DRM) [1] to OD600 0.8 and exposed aerobically to 10 kGy. A total of 20 μg of protein extract loaded per lane.(C) Decreased survival of D. radiodurans irradiated at pH 10.5 correlates with oxidative protein damage. D. radiodurans was grown to OD600 approximately 0.9 in TGY (pH 7), adjusted to pH 10.5, and exposed aerobically to the indicated doses. A total of 20 μg of protein extract loaded per lane.(D) P. putida proteins are similarly susceptible to oxidative protein damage when cells are irradiated anaerobically (+Ar) or aerobically (+O2). P. putida was grown to OD600 approximately 0.9 in TGY, purged with ultra-high purity Ar, and irradiated in sealed tubes to 4 kGy.Values for intracellular Mn/Fe concentration ratios and D10 at the bottom of (B), (C), and (D), as reported previously [1]. A total of 20 μg of protein extract loaded per lane.C, Coomassie-stained polyacrylamide denaturing gel; M, mixture of artificial IgG-binding protein standards; O, oxidized protein standards; S, wide-range protein standards; +, DNPH treated;−, DNPH untreated.

Mentions: To demonstrate a mechanistic link between solution-phase radiochemistry of Mn ions (Figure 2) and their physiological targets in vivo, we examined IR-induced protein damage in IR-sensitive and IR-resistant bacteria (Figure 3). Cellular proteins in the most-sensitive bacteria were substantially more vulnerable to IR-induced oxidation than proteins in the most-resistant bacteria (Figure 3); and from the pattern of oxidized bands, we infer that not all proteins in sensitive bacteria are equally susceptible to carbonylation. At 4 kGy, high levels of protein oxidation occurred in cells with the lowest intracellular Mn/Fe concentration ratios, whereas no protein oxidation was detected in cells with the highest Mn/Fe ratios (see bottom of Figure 3 for bacterial IR survival values and Mn/Fe concentration ratios). In vitro, proteins from resistant bacteria were readily carbonylated when exposed to IR in the presence of Fe (Figure 4A), which confirmed that proteins in resistant bacteria are not inherently resistant to oxidation. Furthermore, we previously reported that D. radiodurans cells grown in defined rich medium without Mn supplementation (No-Mn DRM) were depleted in Mn and, at 10 kGy, displayed a 1,000-fold reduction in survival compared to cells with normal Mn concentrations [1]. D. radiodurans cells grown in DRM without Mn were highly susceptible to protein oxidation during irradiation (Figure 4B). In comparison, D. radiodurans cells with normal intracellular Mn concentrations were sensitized to IR and protein oxidation when irradiated at pH 10.5 (Figure 4C). Pseudomonas putida cells irradiated anaerobically were equally sensitive to IR and as susceptible to IR-induced protein carbonylation as cells irradiated aerobically (Figure 4D). Thus, high levels of IR-induced protein oxidation in bacteria correlated with IR sensitivity in the presence or absence of atmospheric O2, and the IR resistance and level of protein oxidation in D. radiodurans cells with normal intracellular Mn concentrations could be controlled exogenously.


Protein oxidation implicated as the primary determinant of bacterial radioresistance.

Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, Leapman RD, Lai B, Ravel B, Li SM, Kemner KM, Fredrickson JK - PLoS Biol. (2007)

Additional Oxidative Protein Damage Assays(A) In vitro IR-induced oxidative protein damage. Western blot (W) immunoassay of protein-bound carbonyl groups in D. radiodurans (non-irradiated) cell extract adjusted to 500 μM FeCl2 and irradiated to the indicated doses. A total of 20 μg of protein extract loaded per lane.(B) Mn-depleted, radiosensitive D. radiodurans cells [1] are highly susceptible to oxidative protein damage during irradiation. D. radiodurans was grown in defined rich medium without Mn supplementation (no-Mn DRM) [1] to OD600 0.8 and exposed aerobically to 10 kGy. A total of 20 μg of protein extract loaded per lane.(C) Decreased survival of D. radiodurans irradiated at pH 10.5 correlates with oxidative protein damage. D. radiodurans was grown to OD600 approximately 0.9 in TGY (pH 7), adjusted to pH 10.5, and exposed aerobically to the indicated doses. A total of 20 μg of protein extract loaded per lane.(D) P. putida proteins are similarly susceptible to oxidative protein damage when cells are irradiated anaerobically (+Ar) or aerobically (+O2). P. putida was grown to OD600 approximately 0.9 in TGY, purged with ultra-high purity Ar, and irradiated in sealed tubes to 4 kGy.Values for intracellular Mn/Fe concentration ratios and D10 at the bottom of (B), (C), and (D), as reported previously [1]. A total of 20 μg of protein extract loaded per lane.C, Coomassie-stained polyacrylamide denaturing gel; M, mixture of artificial IgG-binding protein standards; O, oxidized protein standards; S, wide-range protein standards; +, DNPH treated;−, DNPH untreated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1828145&req=5

pbio-0050092-g004: Additional Oxidative Protein Damage Assays(A) In vitro IR-induced oxidative protein damage. Western blot (W) immunoassay of protein-bound carbonyl groups in D. radiodurans (non-irradiated) cell extract adjusted to 500 μM FeCl2 and irradiated to the indicated doses. A total of 20 μg of protein extract loaded per lane.(B) Mn-depleted, radiosensitive D. radiodurans cells [1] are highly susceptible to oxidative protein damage during irradiation. D. radiodurans was grown in defined rich medium without Mn supplementation (no-Mn DRM) [1] to OD600 0.8 and exposed aerobically to 10 kGy. A total of 20 μg of protein extract loaded per lane.(C) Decreased survival of D. radiodurans irradiated at pH 10.5 correlates with oxidative protein damage. D. radiodurans was grown to OD600 approximately 0.9 in TGY (pH 7), adjusted to pH 10.5, and exposed aerobically to the indicated doses. A total of 20 μg of protein extract loaded per lane.(D) P. putida proteins are similarly susceptible to oxidative protein damage when cells are irradiated anaerobically (+Ar) or aerobically (+O2). P. putida was grown to OD600 approximately 0.9 in TGY, purged with ultra-high purity Ar, and irradiated in sealed tubes to 4 kGy.Values for intracellular Mn/Fe concentration ratios and D10 at the bottom of (B), (C), and (D), as reported previously [1]. A total of 20 μg of protein extract loaded per lane.C, Coomassie-stained polyacrylamide denaturing gel; M, mixture of artificial IgG-binding protein standards; O, oxidized protein standards; S, wide-range protein standards; +, DNPH treated;−, DNPH untreated.
Mentions: To demonstrate a mechanistic link between solution-phase radiochemistry of Mn ions (Figure 2) and their physiological targets in vivo, we examined IR-induced protein damage in IR-sensitive and IR-resistant bacteria (Figure 3). Cellular proteins in the most-sensitive bacteria were substantially more vulnerable to IR-induced oxidation than proteins in the most-resistant bacteria (Figure 3); and from the pattern of oxidized bands, we infer that not all proteins in sensitive bacteria are equally susceptible to carbonylation. At 4 kGy, high levels of protein oxidation occurred in cells with the lowest intracellular Mn/Fe concentration ratios, whereas no protein oxidation was detected in cells with the highest Mn/Fe ratios (see bottom of Figure 3 for bacterial IR survival values and Mn/Fe concentration ratios). In vitro, proteins from resistant bacteria were readily carbonylated when exposed to IR in the presence of Fe (Figure 4A), which confirmed that proteins in resistant bacteria are not inherently resistant to oxidation. Furthermore, we previously reported that D. radiodurans cells grown in defined rich medium without Mn supplementation (No-Mn DRM) were depleted in Mn and, at 10 kGy, displayed a 1,000-fold reduction in survival compared to cells with normal Mn concentrations [1]. D. radiodurans cells grown in DRM without Mn were highly susceptible to protein oxidation during irradiation (Figure 4B). In comparison, D. radiodurans cells with normal intracellular Mn concentrations were sensitized to IR and protein oxidation when irradiated at pH 10.5 (Figure 4C). Pseudomonas putida cells irradiated anaerobically were equally sensitive to IR and as susceptible to IR-induced protein carbonylation as cells irradiated aerobically (Figure 4D). Thus, high levels of IR-induced protein oxidation in bacteria correlated with IR sensitivity in the presence or absence of atmospheric O2, and the IR resistance and level of protein oxidation in D. radiodurans cells with normal intracellular Mn concentrations could be controlled exogenously.

Bottom Line: For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups.X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells.For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America. mdaly@usuhs.mil

ABSTRACT
In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.

Show MeSH
Related in: MedlinePlus