Escherichia coli genes whose products are involved in selenium metabolism

Abstract

Mutants of Escherichia coli were isolated which were affected in the formation of both formate dehydrogenase N (phenazine methosulfate reducing) (FDHN) and formate dehydrogenase H (benzylviologen reducing) (FDHH). They were analyzed, together with previously characterized pleiotropicfdh mutants (fdhA, fdhB, and fdhC), for their ability to incorporate selenium into the selenopolypeptide subunits of FDHN and FDHH. Eight of the isolated strains, along with the fdhA and fdhC mutants, maintained the ability to selenylate tRNA, but were unable to insert selenocysteine into the two selenopolypeptides. ThefdhB mutant tested had lost the ability to incorporate selenium into both protein and tRNA. fdhF, which is the gene coding for the 80-kilodalton selenopolypeptide of FDHH, was expressed from the T7 promoter-polymerase system in the pleiotropic fdh mutants. A truncated polypeptide of 15 kilodaltons was formed; but no full-length (80-kilodalton) gene product was detected, indicating that translation terminates at the UGA codon directing the insertion of selenocysteine. A mutant fdhF gene in which the UGA was changed to UCA expressed the 80-kilodalton gene product exclusively. This strongly supports the notion that the pleiotropicfdh mutants analyzed possess a lesion in the gene(s) encoding the biosynthesis or the incorporation of selenocysteine. The gene complementing the defect in one of the isolated mutants was cloned from a cosmid library. Subclones were tested for complementation of other pleiotropic fdh mutants. The results revealed that the mutations in the eight isolates fell into two complementation groups, one of them containing the fdhA mutation. fdhB, fdhC, and two of the new fdh isolates do not belong to these complementation groups. A new nomenclature (set) is proposed for pleiotropic fdh mutations affecting selenium metabolism. Four genes have been identified so far: selA and selB (at thefdhA locus), selC (previously fdhC), and selD (previously fdhB).Several enzymes from procaryotic and eucaryotic organisms and a number of tRNA species contain selenium in a covalently bound form (21). For bacteria, the first indication for a biological role of this trace element dates back to 1954, when Pinsent detected that gas production by anaerobic cultures of Escherichia coli depended upon the presence of selenium in the medium (20); enzymological studies revealed that it was the formate dehydrogenase component of the formate-hydrogen-lyase complex which specifically required selenium for activity (14,20). By using [75Se]selenite, it was subsequently demonstrated that the isotope was incorporated into just two proteins in E. coli: an 80-kilodalton polypeptide, which is a constituent component of formate dehydrogenase H (FDHH) and is involved in gas formation, and a 110-kilodalton polypeptide, which is part of formate dehydrogenase N (FDHN) and delivers the electrons from formate to nitrate reductase (8,19). FDHH is formed under anaerobic conditions in the absence of external electron acceptors, whereas synthesis of FDH...

TheiscSgene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

“…2). These results show that the iscS mutant is unable to incorporate 75 Se into the tRNA nucleosides and in this respect resembles the E. coli selD mutant strain MB08 (35). The selD Ϫ strain cannot produce FDH H or selenium-containing tRNAs because it is defective in the formation of monoselenophosphate.…”

“…Aliquots of supernatants containing 6-8 g of protein were analyzed for 75 Se-containing tRNA by SDS͞PAGE (12%) and PhosphorImager detection of radioactivity. 35 S-labeled tRNA was isolated as described (11,16). Nuclease digestion of tRNA was performed as described by Gehrke et al (33) with minor modifications.…”

“…Elution positions of the four major nucleosides are indicated by arrows. tRNA labeled with 35 S was digested to nucleosides and analyzed by HPLC at a flow rate of 0.5 ml͞min as described.…”

Selenium in Biology: Facts and Medical Perspectives

“…Recoding of TGA as a selenocysteine codon requires an mRNA secondary structure called SECIS (for selenocysteine-insertion sequence), which, in bacteria, is localized immediately downstream of the UGA codon. This secondary structure is recognized by a special translation factor, SelB (Baron et al, 1993), which directs a special tRNA (Ser)Sec , encoded by the SelC gene, to the ribosome, where the latter enables the incorporation of selenocysteine by means of a UGA anticodon (Leinfelder et al, 1988;Forchhammer et al, 1991). The selenocystyl-loaded tRNA (Ser)Sec is synthesized from a seryl-loaded tRNA (Ser)Sec by means of selenophosSelenium in Biology 853 Figure 2 illustrates the conformational aspects of thyroid hormones and the bulky iodine atoms (black) of the non-planar diphenylether aromatic ring system (modified from Cody et al, 1986).…”

On the road to selenocysteine

“…9). In fact, genetic analyses of bacterial mutants unable to synthesize Sec-containing formate dehydrogenases resulted in the identification of the components of the prokaryotic selenoprotein biosynthesis machinery (10).…”