A selDABC cluster for selenocysteine incorporation in Eubacterium acidaminophilum

The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions. The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA). A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA. SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD. The function of selA, selB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate. selA and selC from E. acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity.     

Escherichia coli genes whose products are involved in selenium metabolism.

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 pleiotropic fdh 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. The fdhB 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 pleiotropic fdh 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 (sel) is proposed for pleiotropic fdh mutations affecting selenium metabolism. Four genes have been identified so far: selA and selB (at the fdhA locus), selC (previously fdhC), and selD (previously fdhB).     

Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs

Mutation of a single gene, referred to as selA1 in Salmonella typhimurium and as selD in Escherichia coli, results in the inability of these organisms to insert selenium specifically into the selenopolypeptides of formate dehydrogenase and into the 2-selenouridine residues of tRNAs. The mutation does not involve transport of selenite into the cell or reduction of selenite to selenide since both mutant strains synthesize selenocysteine and selenomethionine from added selenite and incorporate these selenoamino acids non-specifically into numerous proteins of the bacterial cells. Complementation of the mutation in S. typhimurium with the selD gene from E. coli indicates functional identity of the selA1 and selD genes. Although the selA1 gene maps at approximately 21 min on the S. typhimurium chromosome and the selD gene at approximately 38 min on the E. coli chromosome, only a single gene in wild-type S. typhimurium hybridized to the E. coli selD gene probe. Transformation of the mutant Salmonella strain with a plasmid bearing the E. coli selD gene restored formate dehydrogenase activity, 75Se incorporation into formate dehydrogenase seleno-polypeptides and [75Se]seleno-tRNA synthesis. Transformation with an additional plasmid carrying an E. coli formate dehydrogenase selenopolypeptide-lacZ gene fusion showed that the selD gene allowed readthrough of the UGA codon and synthesis of beta-galactosidase in the Salmonella mutant.     

In vivo 5'' terminus and length of the mRNA for the proton-translocating ATPase (unc) operon of Escherichia coli.

The promoter for the proton-translocating ATPase (unc) operon of Escherichia coli was localized by using a plasmid promoter-screening vector system. S1 nuclease analysis, using the appropriate single-stranded DNA probe from this promoter region and in vivo mRNA, revealed that the 5' end of the in vivo unc mRNA initiates with a guanine residue 73 bases before the start of the proposed gene 1 or 474 bases before uncB. An in vivo unc mRNA species of approximately 7,000 nucleotides in length which initiates in the unc promoter region was shown to exist by RNA-DNA hybridization analysis. This unc mRNA species (based on DNA sequence analysis) is sufficient in length to contain all nine genes, gene 1 and uncBEFHAGDC. That gene 1 is cotranscribed with the unc genes was confirmed by using hybridization probes containing the promoter-proximal (gene 1) or -distal gene (uncC). No strong internal promoters within the unc operon were revealed with either the promoter-screening vector system or the RNA-DNA hybridization analysis. The 5' terminus and the length of the unc mRNA were found to be identical in cells grown either aerobically or anaerobically. The level of unc operon expression, as assayed with the unc promoter plasmid, did not significantly differ when cells bearing the plasmid were grown either aerobically or anaerobically.     

Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli

Summary The ntrA gene product, required for expression of genes involved in nitrogen fixation (nif) and regulation (ntr), was shown to be necessary for the expression of the two enzymes of the anaerobically inducible formate hydrogenlyase (FHL) pathway, formate dehydrogenase (FDH_H) and hydrogenase isoenzyme 3. Consistent with this finding, the gene encoding the selenopolypeptide (fdhF) of FDH_H was shown to have a nif consensus promoter. The levels of six other anaerobically inducible enzymes were examined and found to be ntrA independent. Significantly, these latter six enzymes are dependent upon the fnr gene product for their expression while FDH_H and hydrogenase 3 are fnr independent. These findings indicate that there are at least two classes of anaerobically regulated promoters: one class which is ntrA dependent and fnr independent and a second class which is fnr dependent and ntrA independent.     

Cobalamin (vitamin B(12)) biosynthesis in Rhodobacter capsulatus

In Rhodobacter capsulatus, cobalamin biosynthesis has been shown to occur when the bacteria are grown either aerobically or anaerobically. However, a comparison of the main cobalamin biosynthetic operon found within R. capsulatus would suggest that the encoded proteins belong to the oxygen-dependent pathway for cobalamin biosynthesis, although, significantly, no homologue of the essential mono-oxygenase CobG has yet been detected. Nonetheless, within this main cob operon is found a large open reading frame termed orf663 that is not found in any other cobalamin biosynthetic operon. When overproduced in Escherichia coli, orf663 was found to encode a 90 kDa integral membrane protein. Some of this protein is cleaved within E. coli to give a soluble N-terminal region that can easily be purified and yields a 50 kDa flavoprotein. When expressed in harness with the genes for precorrin-3a synthesis, ORF663 appears to mediate the transformation of precorrin-3a into a new chromophoric compound. Another open reading frame in close proximity to orf663 is termed orf647, and was found to encode a 2Fe-2S ferredoxin-like protein. We suggest that these two proteins may provide an alternative oxygen-independent mechanism for ring contraction within R. capsulatus.     

Sequence and characterization of the Escherichia coli genome between the ndk and gcpE genes

Abstract The region of the chromosome immediately upstream of the Escherichia coli gene gcpE has been cloned and sequenced. This region contains two functional open reading frames, orf 384 and orf 337, encoding proteins of 43082 and 36189 Da, respectively. Sequencing analysis (this paper) and the isolation of a DNA fragment containing a functional promoter (Talukder, A.A., Yanai, S., and Yamada, M. (1994) Biosci. Biotech. Biochem. 58, 117–120) indicate that orf 337 is in an operon with gcpE . The gene orf 384 is immediately downstream of the gene ndk , which encodes nucleoside diphosphate kinase.     

Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.     

Structural Genes for Nitrate-Inducible Formate Dehydrogenase in Escherichia Coli K-12

Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.