A newly discovered gene, tfuA, involved in the production of the ribosomally synthesized peptide antibiotic trifolitoxin

Abstract

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 ␣-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.Trifolitoxin (TFX) is a ribosomally synthesized, posttranslationally modified peptide antibiotic produced by Rhizobium leguminosarum bv. trifolii T24 (4, 29). A number of features make TFX an interesting antibiotic for study. Its spectrum of activity is quite narrow but includes bacteria that are plant symbionts and plant and animal pathogens (29,34,35). TFX has already been shown to limit nodules formed by TFXsensitive strains (32-34) and so may provide a solution to the Rhizobium competition problem (9, 36). This problem arises when inoculant strains are not competitive for legume root nodulation against strains indigenous to soil. Its ribosomal synthesis makes it an easy substrate for drug modification (12,16,17,27). Though the structure of TFX has not been completely elucidated, it is known to contain a thiazoline ring and another cyclic chromophore (23). The latter appears to be a novel, pyrimidine-like structure (unpublished results). It may be possible to use the enzymes that modify TFX to modify different substrates, thereby creating new molecules. EpiD from the epidermin system has been used to catalyze the oxidative decarboxylation of heptapeptides (21), thereby showing the very real possibility of using antibiotic posttranslational modification enzymes to catalyze reactions on molecules other than their natural preantibiotic target. Given the TFX system's agricultural, medical, and biochemical potential, elucidation of the genetics of TFX production is important.A number of genes have been shown to be required for TFX production and resistance. T...

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Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis

“…TvaB, TvaM, and TvaN are categorized as regulatory proteins based on sequence similarities. TvaI is also assignable as a regulator, because a homologous protein, TfuA, can regulate the production of trifolitoxin, a RiPP family antibiotic (19).…”