A newly discovered gene, tfuA, involved in the production of the ribosomally synthesized peptide antibiotic trifolitoxin.
Trifolitoxin (TFX) is a gene-encoded, posttranslationally modified peptide antibiotic. Previously, we have shown that tfxABCDEFG from Rhizobium leguminosarum bv. trifolii T24 is sufficient to confer TFX production and resistance to nonproducing strains within a distinct taxonomic group of the alpha-proteobacteria (E. W. Triplett, B. T. Breil, and G. A. Splitter, Appl. Environ. Microbiol. 60:4163-4166, 1994). Here we describe strain Tn5-2, a Tn5 mutant of T24 defective in the production of TFX, whose insertion maps outside of the tfx cluster. It is not altered in growth compared with T24, nor does it inactivate TFX in its proximity. The wild-type analog of the mutated region of Tn5-2 was cloned. Sequencing, transcriptional fusion mutagenesis, and subcloning were used to identify tfuA, a gene involved in TFX production. On the basis of computer analysis, the putative TfuA protein has a mass of 72.9 kDa and includes a peroxidase motif but no transmembrane domains. TFX production studies show that extra copies of the tfxABCDEFG fragment increase TFX production in a T24 background while additional copies of tfuA do not. Lysate ribonuclease protection assays suggest that tfuA does not regulate transcription of tfxA. Upstream of tfuA are two open reading frames (ORFs). The putative product of ORF1 shows high similarity to the LysR family of transcriptional regulators. The putative product of ORF2 shows high similarity to the cytosine deaminase (CodA) of Escherichia coli.
Breil
B
B
Cellular and Molecular Biology Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Borneman
J
J
Triplett
E W
EW
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D043525:Cytosine Deaminase; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D009700:Nucleoside Deaminases / Q000235:genetics; D009842:Oligopeptides / Q000096:biosynthesis*; D010455:Peptides; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D016961:Rhizobium leguminosarum / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009842:Oligopeptides; D010455:Peptides; C100966:TfuA protein, Rhizobium leguminosarum; D014157:Transcription Factors; C081619:trifolitoxin; C073470:LysR protein, Bacteria; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; C468827:codA protein, E coli