A NEWLY DISCOVERED GENE, TFUA, INVOLVED IN THE PRODUCTION OF THE RIBOSOMALLY SYNTHESIZED PEPTIDE ANTIBIOTIC TRIFOLITOXIN

Trifolitoxin (TFX) is a gene-encoded, posttranslationally modified pep tide antibiotic. Previously, we have shown that tfxABCDEFG from Rhizob ium leguminosarum bv. trifolii T24 is sufficient to confer TFX product ion 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 al tered in growth compared with T24, nor does it inactivate TFX in its p roximity. The wild-type analog of the mutated region of Tn5-2 was clon ed. Sequencing, transcriptional fusion mutagenesis, and subcloning wer e used to identify tfuA, a gene involved in TFX production. On the bas is 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 tfuA do not. Lysate ribon uclease protection assays suggest that tfuA does not regulate transcri ption of tfxA. Upstream of tfuA are two open reading frames (ORFs). Th e putative product of ORF1 shows high similarity to the LysR family of transcriptional regulators. The putative product of ORF2 shows high s imilarity to the cytosine deaminase (CodA) of Escherichia coli.