Induction of a Futile Embden-Meyerhof-Parnas Pathway in Deinococcus radiodurans by Mn: Possible Role of the Pentose Phosphate Pathway in Cell Survival

Abstract

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO 2 . Mntreated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.The genus Deinococcus is composed of a group of bacteria characterized by their extreme resistance to ionizing radiation (1,7,9,(17)(18)(19). Most experts agree that Deinococcus radiodurans has a very effective system to repair its DNA, but the molecular basis of this effective system is not clear (14,17,18). It appears that D. radiodurans achieves its resistance to radiation by changing the operating conditions of many "ordinary" enzymes (3). Most researchers use the tryptone-glucose-yeast extract (TGY) medium to grow Deinococcus (American Type Culture Collection catalogue. 1996. American Type Culture Collection, Manassas, Va.) (19), even though these organisms are generally considered inactive to sugars (21). Chou and Tan (5) showed that the addition of Mn to a stationary-phase culture of D. radiodurans could induce new rounds of cell division. They termed this mode of growth Mn-induced cell division (MnCD) and suggested that MnCD is a form of starvation growth (23). The authors also showed that the addition of Mn to the culture also induced higher superoxide dismutase (SOD) and catalase activity in the culture. Interestingly, D. radiodurans also became more sensitive to radiation when Mn was included in the culture. Mn is required in many metabolic enzymes. In D. radiodurans, this element serves as a cofactor of SOD (12). The binding of Mn to the chromosomes of the intact cells may contribute to its resistance to radiation (14). Evans and Moseley (9) suggested that Mn served as a cofac...

Oxidative Stress Resistance inDeinococcus radiodurans

“…radiodurans derives its energy mainly from proteolysis (i.e., amino acids and peptides). The biomass formation during growth in TGY medium is supported largely by tryptone and yeast extract (i.e., amino acids), with only a marginal contribution from glucose in the later stages of growth (246,683). D. radiodurans displays a high level of proteolytic activity when grown on plates with skimmed milk, forming large halo-shaped areas of clearing around colonies (207).…”

“…Although D. radiodurans is largely proteolytic, glucose metabolism is important for recovery from DNA-damaging agents (683). In D. radiodurans, carbohydrates are stored in granules (615), appearing as light areas in electron micrographs, which give a positive reaction with periodic acid-Schiff (PAS) stain (Murray and Robinow, presented at the Seventh International Congress of Microbiology, 1958) and with Thiery reagent (574) (Fig.…”

“…RADIODURANS OXIDATIVE STRESS RESISTANCE 137 phosphate pathway (PPP), the tricarboxylic acid cycle, and glyoxylate bypass (381) ( Table 2). The PPP is highly active in D. radiodurans (502), with the activity of glucose-6-phosphate dehydrogenase (G6PDH) (the marker enzyme for PPP) being 4-fold higher than that in Escherichia coli (683). G6PDH is upregulated in gamma-irradiated cells (677), and mutants defective in G6PDH are sensitive to UV light, hydrogen peroxide (H 2 O 2 ), and MMC (367,682).…”

“…Glutathione and thioredoxin are antioxidants, which reduce hydrogen peroxide and oxidized cysteines. By providing substrates for DNA repair and for protection against ROS toxicity, glucose enhances resistance to UV radiation (683), whereas glucose depletion results in an increased susceptibility to the lethal effects of UV and ionizing radiation, MMC, and N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) (96,362). D. radiodurans normally incorporates 8% of the transported 14 C from the glucose into its DNA, which increases to 18% after UV irradiation (683).…”

“…By providing substrates for DNA repair and for protection against ROS toxicity, glucose enhances resistance to UV radiation (683), whereas glucose depletion results in an increased susceptibility to the lethal effects of UV and ionizing radiation, MMC, and N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) (96,362). D. radiodurans normally incorporates 8% of the transported 14 C from the glucose into its DNA, which increases to 18% after UV irradiation (683). When glucose is depleted from the growth medium by the addition of a high concentration of manganese (100 M MnCl 2 ), the incorporation of 14 C from glucose into DNA is reduced to 4% and 6% before and after UV irradiation, respectively (683).…”