DNA-SEQUENCE AND MUTATIONAL ANALYSIS OF GENES INVOLVED IN THE PRODUCTION AND RESISTANCE OF THE ANTIBIOTIC PEPTIDE TRIFOLITOXIN

The 7.1-kb fragment of Rhizobium leguminosarum bv. trifolii T24 DNA wh ich confers trifolitoxin production and resistance to nonproducing, se nsitive Rhizobium strains (E. W. Triplett, M. J. Schink, and K. L. Noe ldner, Mol. Plant-Microbe Interact. 2:202-208, 1989) was subcloned, se quenced, and mutagenized with a transcriptional fusion cassette. The s equence of this fragment revealed seven complete open reading frames, tfxABCDEFG, all transcribed in the same direction. TfxA has an 11-amin o-acid carboxy terminus identical to the known amino acid sequence of the trifolitoxin backbone, DIGGSRXGCVA, where X is an UV-absorbing chr omophore. This is evidence that trifolitoxin is synthesized ribosomall y as a prepeptide that is posttranslationally modified to yield the ac tive peptide. TfxB shows 27.6% identity with McbC, a protein required for the production of the ribosomally synthesized antibiotic microcin B17. Tn3GUS transcriptional fusion insertions in tfxA, tfxB, tfxD, or tfxF caused a nonproducing, trifolitoxin-resistant phenotype and confi rmed the direction of transcription of these frames. No insertion muta tions were found in tfxE or tfxG. Sequence analysis along with inserti on and deletion mutation analysis suggest that (i) trifolitoxin is syn thesized ribosomally from tfx4; (ii) tfxA, tfxE, and tfxG have their o wn promoters; (iii) TfxG is required for immunity; (iv) TfxB, TfxD, an d TfxF are required for trifolitoxin production; and (v) the UV-absorb ing chromophore is derived from glutamine.