Bradyrhizobium japonicum rhizobitoxine genes and putative enzyme functions: expression requires a translational frameshift.
Some strains of Bradyrhizobium japonicum produce rhizobitoxine, a phytotoxin that causes foliar chlorosis on susceptible host plants. We have previously obtained Tn5-induced rhizobitoxine null mutants of B. japonicum. DNA sequence analysis of the region surrounding two Tn5 insertions identifies two overlapping open reading frames. The first open reading frame (rtxA) predicts a 54-kDa protein for which the N-terminal 280 residues have sequence similarity to serine: pyruvate aminotransferase. The sequence homology to aminotransferase is consistent with the involvement of this gene in serinol production, a likely intermediate in rhizobitoxine biosynthesis. Previously, a mutant in this open reading frame was shown not to make serinol. The predicted amino acid sequence of the second open reading frame (rtxB) has similarity to yeast O-acetylhomoserine sulfhydrolase. This enzyme function is similar to that required for dihydrorhizobitoxine synthase. The DNA sequence shows that the rtxB open reading frame overlaps rtxA, suggesting that expression of rtxB requires a -1 translational frameshift. Protein expression experiments demonstrate production of an RtxAB fusion protein. The ability of the overlapping rtxA and rtxB sequences to promote a translational frameshift was confirmed in a heterologous expression system. In Escherichia coli, this frameshift appears to be unusually efficient, occurring at a frequency of 80-90%.
Ruan
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X
Ohio State Biotechnology Center, Ohio State University, Columbus.
Zhang
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C
Peters
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NK
D000595:Amino Acid Sequence; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D003547:Cysteine Synthase; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D016368:Frameshift Mutation; D005798:Genes, Bacterial; D008190:Lyases / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D010957:Plasmids; D011412:Propanolamines / Q000378:metabolism*; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012230:Rhizobiaceae / Q000201:enzymology* / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D000637:Transaminases / Q000235:genetics
D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011412:Propanolamines; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D003547:Cysteine Synthase; C103381:MET17 protein, S cerevisiae; C018725:O-acetylhomoserine (thiol)-lyase; D000637:Transaminases; C022638:serine-pyruvate aminotransferase; D008190:Lyases; D019757:Carbon-Oxygen Lyases; C041618:rhizobitoxine