Physiologic determinants of radiation resistance in Deinococcus radiodurans

Abstract

Immense volumes of radioactive wastes, which were generated during nuclear weapons production, were disposed of directly in the ground during the Cold War, a period when national security priorities often surmounted concerns over the environment. The bacterium Deinococcus radiodurans is the most radiation-resistant organism known and is currently being engineered for remediation of the toxic metal and organic components of these environmental wastes. Understanding the biotic potential of D. radiodurans and its global physiological integrity in nutritionally restricted radioactive environments is important in development of this organism for in situ bioremediation. We have previously shown that D. radiodurans can grow on rich medium in the presence of continuous radiation (6,000 rads/h) without lethality. In this study we developed a chemically defined minimal medium that can be used to analyze growth of this organism in the presence and in the absence of continuous radiation; whereas cell growth was not affected in the absence of radiation, cells did not grow and were killed in the presence of continuous radiation. Under nutrient-limiting conditions, DNA repair was found to be limited by the metabolic capabilities of D. radiodurans and not by any nutritionally induced defect in genetic repair. The results of our growth studies and analysis of the complete D. radiodurans genomic sequence support the hypothesis that there are several defects in D. radiodurans global metabolic regulation that limit carbon, nitrogen, and DNA metabolism. We identified key nutritional constituents that restore growth of D. radiodurans in nutritionally limiting radioactive environments.

FIG. 1

Growth of D. radiodurans in liquid minimal media (Table 1, composition I) containing different carbon sources at a concentration of 2 mg/ml. An optical density at 600 nm (OD₆₀₀) of 1.0 was equivalent to ∼1 × 10⁸ CFU/ml. Cells were pregrown on solid minimal (fructose) medium before they were inoculated into liquid minimal medium.

FIG. 2

Relationship between amino acid concentration and growth of D. radiodurans in liquid minimal medium. The nutrient conditions were the conditions described in Table 1 except for the amino acid composition. Fructose was the carbon source. The amino acid composition was as follows: glutamine, 25%; cysteine, 18%; and a mixture containing tyrosine, tryptophan, and phenylalanine and buffered with 5% glycine, 10%. Cultures were inoculated with 5 × 10⁶ CFU/ml by using cells that were pregrown on solid minimal (fructose) medium (Table 1). Optical densities at 600 nm (OD₆₀₀) were determined 96 h after inoculation.

FIG. 3

Effect of nutrient conditions on the viability and DNA content of D. radiodurans exposed to chronic gamma irradiation in liquid culture. Cells were irradiated at a rate of 6,000 rads/h (144,000 rads/day) at 23°C. Both irradiated and control cultures were diluted to a concentration 5 × 10⁶ CFU/ml at the start of the experiment. (A) Survival curves. Symbols: ■, control, TGY, no irradiation; □, TGY, gamma irradiation; ●, control, minimal (fructose) medium (Table 1, composition I), no irradiation; ○, minimal (fructose) medium (Table 1, composition I), gamma irradiation. (B) Total DNA was prepared from cells obtained at each of the time points shown in panel A. Each lane contained DNA from ∼3 × 10⁶ cells, as determined by hemocytometer counting (7). TGY+γ, cells that were grown in TGY and received gamma irradiation; Min+γ, cells that were grown in minimal (fructose) medium (Table 1, composition I) and received gamma irradiation; TGY and Min, controls incubated in the absence of irradiation. Lanes λ/H contained lambda phage DNA cut with HindIII. DNA sizes (in kilobases) are indicated on the left. The gel migration positions of DNA and rRNA are indicated on the right. Gel electrophoresis was performed with a 0.66% agarose gel for 17 h at 45 V.

FIG. 4

Effect of growth substrate and recovery substrate on survival of D. radiodurans following acute gamma irradiation. Cells were grown to the early stationary phase and irradiated on ice at a rate of 1.33 Mrads/h. Symbols: □, cells pregrown in liquid TGY, irradiated, and plated onto solid TGY; ▴, cells pregrown in liquid TGY, irradiated, and plated onto solid minimal (fructose) medium (Table 1, composition I); ○, cells pregrown in liquid minimal (fructose) medium, irradiated, and plated onto solid TGY; ⧫, cells pregrown in liquid minimal (fructose) medium, irradiated, and plated onto solid minimal (fructose) medium.

FIG. 5

(A) Production of pp(p)Gpp in D. radiodurans and E. coli. ³²P-labeled ppGpp and ppGpp were detected by polyethyleneimine-cellulose chromatography (see Materials and Methods). Stationary-phase cells grown in either TGY or minimal medium (Table 1) were suspended in PFLM (see Materials and Methods) with or without the amino acid analogue serine hydroxamate. Lanes 1 and 2, D. radiodurans cells obtained from minimal (fructose) medium (Table 1, composition I) and incubated in PFLM containing serine hydroxamate (equivalent to lanes 4 and 5 in panel B); lanes 3 and 4, D. radiodurans cells obtained from minimal (fructose) medium (Table 1) and incubated in PFLM containing the 16 amino acids listed in Table 1 (final amino acid concentration, 50 μg/ml); lane 5, D. radiodurans cells obtained from TGY and incubated in PFLM containing serine hydroxamate; lane 6, E. coli (relA deleted) incubated in PFLM containing serine hydroxamate (control); lane 7, E. coli wild type incubated in PFLM containing serine hydroxamate (control). (B) Formation of ppGpp and pppGpp in D. radiodurans. Cells were treated as described above in the presence of serine hydroxamate. Lane 1, control, no cells; lanes 2 and 3, cells grown in TGY; lanes 4 and 5, cells grown in minimal (fructose) medium (Table 1). (C) Formation of ppGpp and pppGpp in D. radiodurans grown in minimal medium. Cells were treated as described above. Lane 1, cells treated in the absence of serine hydroxamate; lane 2, cells treated in the presence of serine hydroxamate.

FIG. 6

CSLM. Bacterial cells were stained with acridine orange. Acridine orange-stained single-stranded nucleic acids result in complexes that fluoresce red and were used to localize RNA. Acridine orange-stained double-stranded nucleic acid complexes fluoresce green and were used to localize DNA. Note that when DNA and RNA were both present, the cells were yellow. (a) Minimal (fructose) medium (Table 1, composition I). (b) TGY. Bars = 5 μm.