SummaryDeinococcus radiodurans mutant lacking pyrroloquinoline-quinone (PQQ) synthesis shows sensitivity to g-rays and impairment of DNA double strand break repair. The genome of this bacterium encodes five putative proteins having multiple PQQ binding motifs. The deletion mutants of corresponding genes were generated, and their response to DNA damage was monitored. Only the Ddr2518 mutant exhibited higher sensitivity to DNA damage. Survival of these cells decreased by 3-log cycle both at 6 kGy g-rays and 1200 J m -2 UV (254 nm) radiation, and 2.5-log cycle upon 14 days desiccation at 5% humidity. The Ddr2518 mutant showed complete inhibition of DSB repair until 24 h PIR and disappearance of a few phosphoproteins. The Ddr2518pqqE:cat double mutant showed g-ray sensitivity similar to Ddr2518 indicating functional interaction of these genes in D. radiodurans. DR2518 contains a eukaryotic type Ser/ Thr kinase domain and structural topology suggesting stress responsive transmembrane protein. Its autokinase activity in solution was stimulated by nearly threefold with PQQ and twofold with linear DNA, but not with circular plasmid DNA. More than 15-fold increase in dr2518 transcription and severalfold enhanced in vivo phosphorylation of DR2518 were observed in response to g irradiation. These results suggest that DR2518 as a DNA damageresponsive protein kinase plays an important role in radiation resistance and DNA strand break repair in D. radiodurans.

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Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wildtype bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in ␥ radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of ␥ resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to ␥ radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of 32 P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.Pyrroloquinoline quinone (PQQ) has been shown to be a redox cofactor for periplasmic as well as cytosolic dehydrogenases, contributing to the mineral phosphate solubilization phenotype in bacteria (11). This compound has been reported to act as an antioxidant in vitro (33), in animal systems (13), and in bacterial systems (18) in vivo and as a member of the B group vitamins (16). He and coworkers (13) have shown that the antioxidant nature of PQQ is concentration dependent. Higher concentrations of PQQ induce oxidative stress for mitochondrial activity in rats, which leads to both apoptotic and necrotic cell death. Further studies indicated that the necrotic cell death could be selectively inhibited in the presence of antioxidants, while apoptotic cell death continued by a stillunknown mechanism. Further, a possible role for PQQ as an inducer for proteins kinases involved in distinctly different metabolic and physiological processes has been suggested (20).Deinococcus radiodurans R1, a gram-positive bacterium, exhibits extraordinary tolerance to various abiotic stresses, including radiation, desiccation, and other DNA-damaging factors (3). DNA double strand break repair in D. radiodurans R1 follows biphasic kinetics (8). Phase I is RecA independent and involves an extended synthesis-dependent strand annealing mechanism for reassembly of the fragmented genome (42), while phase II involves RecA-dependent slow crossover events (9). The extreme phenotypes of this bacterium are believed to be due to the presence of an efficient DNA strand break repair mechanism (1, 31) and strong oxidative stress tolerance (27). A comparison of the genome sequence of D. radiodurans R1 (41) with the genome sequenc...

The LexA/RecA-type SOS response is the only characterized mechanism of DNA damage response in bacteria. It regulates cell cycle by attenuating the functions of cell division protein FtsZ and inducing the expression of DNA repair proteins. There are bacteria, including Deinococcus radiodurans, that do not show this classical SOS response. D. radiodurans is known for its extraordinary resistance to gamma radiation, and a DNA damage-responsive Ser/Thr protein kinase (RqkA) has been characterized for its role in radioresistance. RqkA phosphorylates a large number of proteins in solution. The phosphorylation of RecA and PprA by RqkA enhanced their activities. FtsZ phosphorylation is inducible by gamma radiation in wild-type D. radiodurans but not in an rqkA mutant. Phosphorylation affected the interaction of FtsZ and FtsA in this bacterium. This study, therefore, brought forth some findings that might lead to the discovery of a new mechanism regulating the bacterial cell cycle in response to DNA damage.

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