Intracellular copper accumulation enhances the growth of Kineococcus radiotolerans during chronic irradiation

Abstract

The actinobacterium Kineococcus radiotolerans is highly resistant to ionizing radiation, desiccation, and oxidative stress, though the underlying biochemical mechanisms are unknown. The purpose of this study was to explore a possible linkage between the uptake of transition metals and extreme resistance to ionizing radiation and oxidative stress. The effects of six different divalent cationic metals on growth were examined in the absence of ionizing radiation. None of the metals tested were stimulatory, though cobalt was inhibitory to growth. In contrast, copper supplementation dramatically increased colony formation during chronic irradiation. K. radiotolerans exhibited specific uptake and intracellular accumulation of copper, compared to only a weak response to both iron and manganese supplementation. Copper accumulation sensitized cells to hydrogen peroxide. Acute-irradiation-induced DNA damage levels were similar in the copper-loaded culture and the age-synchronized no-copper control culture, though low-molecular-weight DNA was more persistent during postirradiation recovery in the Cu-loaded culture. Still, the estimated times for genome restoration differed by only 2 h between treatments. While we cannot discount the possibility that copper fulfills an unexpectedly important biochemical role in a low-radioactivity environment, K. radiotolerans has a high capacity for intracellular copper sequestration and presumably efficiently coordinated oxidative stress defenses and detoxification systems, which confers cross-protection from the damaging effects of ionizing radiation.

FIG. 1.

Flow diagram illustrating the experiments conducted to determine the effect of transition metals on the growth of K. radiotolerans during gamma irradiation.

FIG. 2.

Effects of different transition metals on the growth of K. radiotolerans during chronic irradiation. Black bars indicate the growth of the no-irradiation control cultures with and without metals. Dark gray bars indicate cultures that were grown to exponential phase in TGY medium and then streak plated onto metal-supplemented TGY plates. White bars indicate cultures that were pregrown to exponential phase in metal-supplemented liquid medium and then streak plated onto non-metal-containing TGY plates. The horizontal baseline (light gray panel) of 75 ± 25 CFU was established from the no-metal, irradiated control cultures. All plates were irradiated for 4 days at 60 Gy/h at a constant temperature of 30°C.

FIG. 3.

Recovery response of K. radiotolerans following H₂O₂ exposure. Cultures (n = 3) were grown to exponential phase in TGY or TGY amended with a divalent cationic metal (100 μM), incubated in H₂O₂ (4%) for 10 min, and then allowed to recover in fresh TGY medium at 28°C and 150 rpm. ▴, TGY (control); ▪, TGY plus Mn²⁺; ♦, TGY plus Cu²⁺; •, TGY plus Fe²⁺; ○, TGY plus Zn²⁺; □, TGY plus Mo²⁺; and ▵, TGY plus Co²⁺.

FIG. 4.

Intracellular accumulation of transition metals in early-stationary-phase K. radiotolerans cultures. Bars indicate normalized metal contents of Fe²⁺ (solid), Mn²⁺ (gray), and Cu²⁺ (white) in no-metal control and metal-supplemented cultures (x axis).

FIG. 5.

Electron micrographs of K. radiotolerans cell clusters. (A) Scanning electron micrograph. Scale bar = 20 μm. (B) Transmission electron micrograph of a stained thin section. Scale bar = 0.5 μm. (C) Transmission electron micrograph of an unstained thin section. Elemental spectra were collected along a transect (indicated by spots A to E) spanning a single cell within a cluster. Scale bar = 0.5 μm.

FIG. 6.

X-ray elemental spectra from a thin K. radiotolerans cell section. Each of the EDS panels corresponds to a specific sampling location from the thin section shown in Fig. 5C. (A) EDS spectrum of the embedding resin. (B) EDS spectrum of the extracellular milieu. (C and D) EDS spectra from two different locations within the cytoplasm. (E) EDS spectrum from the cell membrane and cell wall and the interstitial space between dividing cells within the cluster.

FIG. 7.

DNA damage repair in K. radiotolerans cultures following acute irradiation. (A) DNA damage as a function of radiation dose. (B and C) Timing of radiation-induced DNA damage repair for control and Cu-grown cultures. MW, molecular weight.