Isolation and characterization of Escherichia coli mutants affected in aerobic respiration: the cloning and nucleotide sequence of ubiG. Identification of an S-adenosylmethionine-binding motif in protein, RNA, and small-molecule methyltransferases.
We report the isolation and characterization of a mutant of Escherichia coli unable to grow aerobically on non-fermentable substrates, except for very slow growth on glycerol. The mutant contains cytochrome oxidases o and d, and grows anaerobically with alternative electron acceptors. Oxygen consumption rates of cell-free extracts were low relative to activities in an isogenic control strain, but were restored in vitro by adding ubiquinone-1 to cell-free extracts. Transformation with a cloned 2.8 kb ClaI-EcoRV fragment of chromosomal DNA restored the ability of this mutant (AN2571) to grow on succinate and also restored cellular quinone levels in this strain. The plasmid also complemented a previously isolated ubiG mutant (AN151) for aerobic growth on succinate. The nucleotide sequence revealed a 0.7 kb portion of gyrA. Unidirectional nested deletions from this fragment and complementation analysis identified an open reading frame encoding a protein with a predicted molecular mass of 26.5 kDa. This gene (ubiG) encodes the enzyme 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase, which catalyses the terminal step in the biosynthesis of ubiquinone. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by three palindromic unit sequences. Comparison of the inferred amino acid sequence of UbiG with the sequence of other S-adenosylmethionine (AdoMet)-dependent methyltransferases reveals a highly conserved AdoMet-binding region. The cloned 2.8 kb fragment also contains a sequence encoding the C-terminus of a protein with 42-44% identity to fungal acetyl-CoA synthetases.
Wu
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G
Microbial Physiology Research Group, King's College London, UK.
Williams
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HD
Zamanian
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M
Gibson
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Poole
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RK
D000332:Aerobiosis; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003580:Cytochromes / Q000032:analysis; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008780:Methyltransferases / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010101:Oxygen Consumption / Q000235:genetics*; D011809:Quinones / Q000032:analysis; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014451:Ubiquinone / Q000096:biosynthesis
D003580:Cytochromes; D029968:Escherichia coli Proteins; D011809:Quinones; D014451:Ubiquinone; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C019616:UbiG protein, E coli