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.
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...
Three phylogenetically distinct groups within the a-proteobacteria which differ in trifolitoxin sensitivity are described. Trifolitoxin sensitivity was found in strains of Agrobacterium, BruceUa, Mycoplana, Ochrobactrum, PhyUobacterium, Rihodobacter, Rhodopseudomonas, RhodospiriUum, and Rhizobium. Strains of Agrobacterium, BruceUla, PhyUobacterium, Rhizobium, and Rhodospirillum were capable of producing trifolitoxin upon conjugal transfer of afABCDEFG.
The effect of temperature on the ability of five species of Trichoderma to decompose cellulose was evaluated in three types of sterilized soils. The loss in tensile strength of cotton strips was used as an index of cellulose decomposition. The influence of levels of nitrogen on cotton degradation was also evaluated using silica sand as a substitute for soil. A greater loss in cotton tensile strength was obtained with T. virens and T. viride than with T. hamatum, T. polysporum, or T. koningii. All species responded similarly to nitrogen levels and temperature, with decreased activity at low nitrogen and low temperature. The soil also had a large influence on the rate of decomposition of the strips. The soil from a maple forest gave rise to greater losses in tensile strength than did soil from a white pine or Norway spruce plantation. These results emphasize the importance of the soil chemistry in determining fungal activities in the field, and the difficulties of extrapolating results of cellulose utilization studies in the laboratory to the field.Key words: Trichoderma, cellulolysis, soil, nitrogen, temperature.
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