Expression of tfx and sensitivity to the rhizobial peptide antibiotic trifolitoxin in a taxonomically distinct group of alpha-proteobacteria including the animal pathogen Brucella abortus.

Three phylogenetically distinct groups within the alpha-proteobacteria which differ in trifolitoxin sensitivity are described. Trifolitoxin sensitivity was found in strains of Agrobacterium, Brucella, Mycoplana, Ochrobactrum, Phyllobacterium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, and Rhizobium. Strains of Agrobacterium, Brucella, Phyllobacterium, Rhizobium, and Rhodospirillum were capable of producing trifolitoxin upon conjugal transfer of tfxABCDEFG.

DNA sequence of the common nodulation genes of Bradyrhizobium elkanii and their phylogenetic relationship to those of other nodulating bacteria.

A 6.6-kb BamHI fragment containing the common nodulation genes of Bradyrhizobium elkanii USDA94 was identified by southern hybridization using the common nod genes of B. japonicum as a probe. This fragment was cloned and sequenced. Analysis of the sequence showed open reading frames highly homologous to nolA, nodD2, nodD1, and nodKABC from other bradyrhizobial sources. The sequence showed higher homology to the common nod genes of Bradyrhizobium sp. (Parasponia) than to those from B. japonicum. The open reading frame identified between nodD1 and nodA in the B. elkanii sequence was far more similar to nodK from Bradyrhizobium sp. (Parasponia) than to nodY from B. japonicum. The molecular phylogeny of nodD and nodAB from many sources was analyzed. The genetic distance between the nod genes is far greater than the distance between the 16S rRNA and nifH genes. The differences between the nod genes among the species of Rhizobium is as great as that between Bradyrhizobium and Rhizobium. The host range of the microsymbiont was found to be a better predictor of the similarities of the common nod genes than the 16S rRNA or nifH genes. We propose two groups of nod genes among the rhizobia and bradyrhizobia, based on molecular phylogenetic analysis: those which nodulate legumes of temperate origin in the tribes Vicieae or Trifolieae and those which nodulate legumes of tropical origin in the tribe Phaseoleae.

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 which confers trifolitoxin production and resistance to nonproducing, sensitive Rhizobium strains (E. W. Triplett, M. J. Schink, and K. L. Noeldner, Mol. Plant-Microbe Interact. 2:202-208, 1989) was subcloned, sequenced, and mutagenized with a transcriptional fusion cassette. The sequence of this fragment revealed seven complete open reading frames, tfxABCDEFG, all transcribed in the same direction. TfxA has an 11-amino-acid carboxy terminus identical to the known amino acid sequence of the trifolitoxin backbone, DIGGSRXGCVA, where X is an UV-absorbing chromophore. This is evidence that trifolitoxin is synthesized ribosomally as a prepeptide that is posttranslationally modified to yield the active 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 confirmed the direction of transcription of these frames. No insertion mutations were found in tfxE or tfxG. Sequence analysis along with insertion and deletion mutation analysis suggest that (i) trifolitoxin is synthesized ribosomally from tfxA; (ii) tfxA, tfxE, and tfxG have their own promoters; (iii) TfxG is required for immunity; (iv) TfxB, TfxD, and TfxF are required for trifolitoxin production; and (v) the UV-absorbing chromophore is derived from glutamine.