We have developed a radiation resistant bacterium for the treatment of mixed radioactive wastes containing ionic mercury. The high cost of remediating radioactive waste sites from nuclear weapons production has stimulated the development of bioremediation strategies using Deinococcus radiodurans, the most radiation resistant organism known. As a frequent constituent of these sites is the highly toxic ionic mercury (Hg) (II), we have generated several D. radiodurans strains expressing the cloned Hg (II) resistance gene (merA) from Escherichia coli strain BL308. We designed four different expression vectors for this purpose, and compared the relative advantages of each. The strains were shown to grow in the presence of both radiation and ionic mercury at concentrations well above those found in radioactive waste sites, and to effectively reduce Hg (II) to the less toxic volatile elemental mercury. We also demonstrated that different gene clusters could be used to engineer D. radiodurans for treatment of mixed radioactive wastes by developing a strain to detoxify both mercury and toluene. These expression systems could provide models to guide future D. radiodurans engineering efforts aimed at integrating several remediation functions into a single host.

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 radiationresistant 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.

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The interaction of the adenyosylcobalamin-dependent ribonucleoside diphosphate reductase of Cornyebacterium nephridii with 2'-C-methyladenosine 5'-diphosphate (2'-MeADP) and 2'-C-methyluridine 5'-diphosphate (2'-MeUDP) has been investigated. The nucleotide analogs are converted to adenine and uracil, respectively, suggesting that they may be mechanism-based inhibitors. In addition, both analogs generate nucleotides with properties expected for the 2'-deoxy-2'-C-methylnucleotides. The nucleoside obtained after enzymatic dephosphorylation of the product formed from 2'-MeADP has been identified as 2'-deoxy-2'-C-methyladenosine by 1H NMR and mass spectroscopies. Adenine is the major product derived from 2'-MeADP, indicating that the degradation pathway predominates. During the reaction, the carbon-cobalt bond of the coenzyme is cleaved irreversibly to yield 5'-deoxyadenosine and cob(II)alamin. 2'-MeADP is a potent competitive inhibitor of the reduction of the purine nucleotides ADP and GDP, while 2'-MeUDP competitively inhibits the reduction of the pyrimidine nucleotides UDP and CDP. 2'-MeADP is a very effective promoter of the tritium exchange reaction between [5'-3H2]adenosylcobalamin and the solvent, indicating that the exchange reaction is an integral part of the overall reduction. All these observations are consistent with the reaction mechanism proposed by Stubbe and co-workers [Harris, G., Ashley, G. W., Robins, M. J., Tolman, R. L., & Stubbe, J. (1987) Biochemistry 26, 1895-1902 (1987); Stubbe, J. (1990) J. Biol. Chem. 265, 5329-5332] in which they suggest that the partitioning between reduction and inactivation occurs at the level of the 2'-deoxy-3'-ketoribonucleotide intermediate.

A protein catalyzing the tritium exchange of [5-3H]deoxyuridine monophosphate for solvent protons and the dehalogenation of 5-bromo-deoxyuridine monophosphate (Br-dUMP) has been isolated from the methanogenic archaea Methanobacterium thermoautotrophicum. These two activities are well-established side reactions of thymidylate synthase and do not require cofactors. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the purified enzyme showed a single band with a molecular mass of 27 kDa. The suggested molecular mass of the native protein calculated from sedimentation equilibrium experiments was 33.5 kDa, indicating that the enzyme is a monomer. The pH optima were 9.0 and 7.0 for the exchange reaction and the dehalogenation, respectively. The effects of temperature, salt, reducing agent and inhibitors were determined. The apparent K,,, for the tritium exchange from [5-3H]dUMP was 7 pM and for the dehalogenation of Br-dUMP was 14 pM. However, thus far, the conditions for dTMP synthesis from dUMP have not yet been established. Incubation of the enzyme with dUMP, tetrahydromethanopterin, a folate analog present in methanogens, and formaldehyde did not yield dTMP. The first 30 amino acids of the amino terminus have been sequenced. However, there is no similarity with any of the thymidylate synthases. Surprisingly, the protein from M. thermoautotrophicum appears to be related to chitin synthases from several organisms.Thymidylate synthase catalyzes the conversion of dUMP to dTMP using 5,lO-methylenetetrahydrofolate as the onecarbon donor and reductant. The enzyme also promotes the specific exchange of [5-'H]dUMP for protons of water in the presence or absence of a cofactor as well as the dehalogenation of S-bromo-2'-deoxyuridine monophosphate (Br-dUMP) to dUMP (Santi and Brewer, 1973;Garrett et al., 1979;Pogolotti et al., 1979). Thymidylate synthase has been isolated from several eukaryotes and eubacteria (Perry et al., 1990;Kim et al., 1992). Sequencing studies have revealed that it is the most conserved enzyme known to date. In general, it is a dimer of identical subunits of about 35 kDa each. However, a much larger bifunctional protein with both thymidylate synthase and dihydrofolate reductase activities was isolated from protozoa (reviewed by Santi and Danenberg, 1984).Thymidylate synthase has not yet been purified from archaea, which are distinct from eubacteria in their unique

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 ( J . Biol. Chem. 256,[9174][9175][9176][9177][9178][9179][9180][9181][9182], has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUCl3 and transformants were identified by complementation of a thioredoxin negative (trxA -) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biof. Clzem. 262, 12 114-12 1191. In this paper we present the purification and characterization of one of these thioredoxins.The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactoharillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP') and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. twphridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys-at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.Thioredoxins are small, acidic, ubiquitous proteins with two redox-active half-cystine residues in an exposed active center having the amino acid sequence : -Cys-Gly-Pro-Cys-. They exist in the reduced [thioredoxin-(SH),] or the oxidized form (thioredoxin-S2) where the half-cystine residues form a 14-membered intramolecular ring. During the last 25 years, thioredoxins have been isolated from many sources and these small redox proteins have been found to participate in a variety of biological reactions [l -31. They are hydrogen donors for various reductive enzymes, protein disulfide oxidoreductases, photosynthetic regulatory factors, one subunit of a virus DNA polymerase, essential components for the assembly of small viruses, and possibly protein disulfide isomerases. The protein was originally purified in Escherichia coli as the hydrogen donor for ribonucleotide reductase [4].

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