Resolution of distinct selenium-containing formate dehydrogenases from Escherichia coli

Cox, John C.; Edwards, E S; DeMoss, John A.

doi:10.1128/jb.145.3.1317-1324.1981

Cited by 89 publications

(20 citation statements)

References 23 publications

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“…Selenium labelling was performed as described by Cox et al (1981). Cells were grown aerobically for 8 h in TP medium (Heider et al, 1992) supplemented with 0.4% glycerol and 50 μg/ml cysteine.…”

Section: In Vivo Labelling With [ 75 Se]selenitementioning

confidence: 99%

Dynamics and efficiency invivo of UGA-directed selenocysteine insertion at the ribosome

Suppmann¹,

Persson

Böck³

1999

The EMBO Journal

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The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo. A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10%. This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS. When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced. When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression. The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present. The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs.

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Section: In Vivo Labelling With [ 75 Se]selenitementioning

confidence: 99%

Dynamics and efficiency invivo of UGA-directed selenocysteine insertion at the ribosome

Suppmann¹,

Persson

Böck³

1999

The EMBO Journal

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“…Incorporation of the radioactive isotope [75Se] into FDHH and FDHN and their resolution on SDS-polyacrylamide gels were carried out as described by Cox et aL (1981).…”

Section: Incorporation Of [75se] Into Fdhsmentioning

confidence: 99%

tRNA genes and pathogenicity islands: influence on virulence and metabolic properties of uropathogenic Escherichia coli

Ritter¹,

Blum²,

Emödy

et al. 1995

Molecular Microbiology

109

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The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two unstable DNA regions on its chromosome which were termed pathogenicity islands (Pais). Both pathogenicity islands, Pai I and Pai II, are incorporated into tRNA specific loci: Pai I is located in the tRNA gene for selenocysteine (selC), and Pai II is integrated in the leucine-specific tRNA locus leuX. Mutant strain 536-21 has lost the two pathogenicity islands together with the intact tRNA genes. While 536 is a virulent strain, 536-21 has lost a number of properties, including in vivo virulence. In previous publications we reported that the genes coding for two haemolysins (hly I, hly II) and P-related fimbria (prf) are located on the Pais. In this paper, we demonstrate that the expression of other gene products influencing metabolic properties in addition to in vivo virulence are strongly dependent on the intact tRNA loci selC and leuX. In order to determine the influence of the two tRNAs on the expression of these properties, the genes selC and leuX were cloned from the genome of strain 536 and then introduced into the mutant 536-21. Our results clearly show that the seleno-cysteine-specific tRNA (tRNA(Sec)) directly influences the ability of the bacteria to grow under anaerobic conditions, because selenocysteine is part of the enzyme formate dehydrogenase (FDH) which is involved in mixed acid fermentation. The rare leucine-specific tRNA5(Leu), encoded by leuX, influences a number if properties including type 1 fimbria production, flagellation and motility, production of enterobactin and serum resistance, and is also necessary for full in vivo virulence. While the tRNA(Sec) is directly involved in the production of FDHs, the leuX specific tRNA5(Leu) appears to influence the expression of various factors through specific transcriptional or translational control mechanisms.

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“…Both FDH-1 and FDH-2 are selenoproteins (18) but differ in their molecular weights. Cox et al have observed that FDH-1 and FDH-2 had molecular weights of 110,000 and 80,000 respectively (8). The two enzymes also failed to cross-react immunologically (11).…”

Section: Materials and Methoqsmentioning

confidence: 99%

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli

Sankar¹,

Lee²,

Shanmugam³

1985

J Bacteriol

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Escherichja coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-Ill carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also'eptablished the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobasepair DNA (pSE-130) or fragments derived from this DNA in different orientations inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, straip K-10, but only during an early induction period. These results, based on complementation analysjs with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of-active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, ffl4 andfdv are also njeded, All four genes map between 58 and 59 min in the E. coli chromosome. SE-5, SE-6, SE-7, and SE-6}, respectively. Table 1 provides the pedigree and hyd genotype of the hydrogenase-defective mutant strains of E. coli used in this study, and these mutant strains were isolated by E$V selection (22).Media and growth conditions. Luria broth, glucose minimal medium, and HF have been described previously (22).Ampicillin and tetracycliqp, when present, were added to the medium after autoclaving to a final concentration of 100 and 15 p.g/ml, respectively. Cultures for enzyme assays were 353 on July 31, 2020 by guest

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Resolution of distinct selenium-containing formate dehydrogenases from Escherichia coli

Cited by 89 publications

References 23 publications

Dynamics and efficiency invivo of UGA-directed selenocysteine insertion at the ribosome

Dynamics and efficiency invivo of UGA-directed selenocysteine insertion at the ribosome

tRNA genes and pathogenicity islands: influence on virulence and metabolic properties of uropathogenic Escherichia coli

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli

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