Selenium (Se) toxicity is thought to be due to nonspecific incorporation of selenocysteine (Se-Cys) into proteins, replacing Cys. In an attempt to direct Se flow away from incorporation into proteins, a mouse (Mus musculus) Se-Cys lyase (SL) was expressed in the cytosol or chloroplasts of Arabidopsis. This enzyme specifically catalyzes the decomposition of Se-Cys into elemental Se and alanine. The resulting SL transgenics were shown to express the mouse enzyme in the expected intracellular location, and to have SL activities up to 2-fold (cytosolic lines) or 6-fold (chloroplastic lines) higher than wild-type plants. Se incorporation into proteins was reduced 2-fold in both types of SL transgenics, indicating that the approach successfully redirected Se flow in the plant. Both the cytosolic and chloroplastic SL plants showed enhanced shoot Se concentrations, up to 1.5-fold compared with wild type. The cytosolic SL plants showed enhanced tolerance to Se, presumably because of their reduced protein Se levels. Surprisingly, the chloroplastic SL transgenics were less tolerant to Se, indicating that (over) production of elemental Se in the chloroplast is toxic. Expression of SL in the cytosol may be a useful approach for the creation of plants with enhanced Se phytoremediation capacity. Selenium (Se) is an essential element for many organisms, but it is also toxic at higher concentrations. Se is essential because seleno-Cys (Se-Cys) is in the active site of certain selenoproteins, several of which are involved in oxidative stress resistance (Stadtman, 1996). Se becomes toxic at higher levels due to incorporation of Se into sulfur (S)-containing molecules, especially the nonspecific replacement of Cys by Se-Cys in proteins (Ohlendorf et al., 1986; Anderson, 1993). This replacement of S by Se in molecules is due to the chemical similarity of these two elements; most enzymes involved in S metabolism can catalyze the analogous reaction with the corresponding Se substrates with similar affinity for both substrates (Stadtman, 1990; Anderson, 1993). On the other hand, Se-specific enzymes tend to have a much higher affinity for the Se substrate than for the S analog (Mihara et al., 2000).The specific incorporation of Se into selenoproteins involves the translation of UGA opal (stop) codons in specific mRNAs encoding Se-Cys-containing proteins (Bö ck et al., 1991). Se-Cys-tRNA is formed from Ser-tRNA using selenophosphate as a Se substrate. Selenophosphate is formed by selenophosphate synthetase, using elemental Se (Se 0 ) as a substrate (Lacourciere and Stadtman, 1998; Lacourciere et al., 2000). Se 0 is released from Se-Cys by Se-Cys lyase (SL; a pyridoxal phosphate-dependent enzyme of the NifS family), also producing Ala. Se-Cys is produced from selenate by the sulfate assimilation pathway, both in Se-requiring and non-requiring organisms (Stadtman, 1990; Anderson, 1993;Pilon-Smits et al., 1999). In plants, this pathway is localized mainly in the chloroplast (Leustek and Saito, 1999).At this point, it is not clear whether ...

Selenium is an essential nutrient for many organisms, as part of certain selenoproteins. However, selenium is toxic at high levels, which is thought to be due to non-specific replacement of cysteine by selenocysteine leading to disruption of protein function. In an attempt to prevent non-specific incorporation of selenocysteine into proteins and to possibly enhance plant selenium tolerance and accumulation, a mouse selenocysteine lyase was expressed in Brassica juncea (Indian mustard) chloroplasts, the site of selenocysteine synthesis. This selenocysteine lyase specifically breaks down selenocysteine into elemental selenium and alanine. The transgenic cpSL plants showed normal growth under standard conditions. Selenocysteine lyase activity in the cpSL transgenics was up to 6-fold higher than in wild-type plants. The cpSL transgenics contained up to 40% less selenium in protein compared to wild-type plants, indicating that Se flow in the plant was successfully redirected. Surprisingly, the selenium tolerance of the transgenic cpSL plants was reduced, perhaps due to interference of produced elemental selenium with chloroplastic sulphur metabolism. Shoot selenium levels were enhanced up to 50% in the cpSL transgenics, but only during the seedling stage.

Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.