Trifolitoxin Production Research Articles

The novel alkali tolerance function of tfxG in Sinorhizobium meliloti

TfxG, one of the tfxABCDEFG cluster genes that code for trifolitoxin (TFX) production, was initially described in Rhizobium leguminosarum bv. trifolii T24. Although several genes in the tfx family have functions related to TFX production or resistance to TFX, the function of tfxG is largely unknown. Using cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis, we found that expression of the tfxG gene dramatically increased under alkaline culture conditions in Sinorhizobium meliloti CCBAU 81024. This result was confirmed by northern blot analysis. Mutagenesis of tfxG significantly decreased the viability of Sinorhizobium meliloti CCBAU 81024 under alkali stress. Complementation of the tfxG mutant strain using the functional tfxG gene recovered its alkali tolerance to a wild-type level. Genomic analysis of the tfxG gene suggests that choline and homoserine kinase domains may contribute to its alkali tolerance function. This is the first clear evidence that tfxG plays a crucial role in the alkali tolerance of S. meliloti CCBAU 81024, and the finding provides its biological function.

Role of tfxE, but not tfxG, in trifolitoxin resistance.

Eight genes, tfxABCDEFG and tfuA, confer production of trifolitoxin (TFX), a ribosomally synthesized, posttranslationally modified peptide antibiotic, in TFX-sensitive alpha-proteobacteria. An in-frame deletion in tfxE significantly reduced a strain's resistance to TFX in comparison to that of an otherwise identical construct containing wild-type tfxE. The deletion of tfxG had no effect on TFX resistance. Nevertheless, RNase protection assays showed that tfxE and tfxG are transcribed, showing that the tfxDEFG mRNA was produced on the same transcript. Examination of the role of tfxG in TFX production showed that the tfxG mutant expressed slightly less TFX activity and produced only one TFX isomer while four are produced by the wild-type strain. Thus, tfxE plays an important role in TFX resistance while tfxG is important in optimal TFX production through the production of TFX isomers.

Expression of a crown gall biological control phenotype in an avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance genes

BackgroundAgrobacterium vitis is a causal agent of crown-gall disease. Trifolitoxin (TFX) is a peptide antibiotic active only against members of a specific group of α-proteobacteria that includes Agrobacterium and its close relatives. The ability of TFX production by an avirulent strain of Agrobacterium to reduce crown gall disease is examined here.ResultsTFX was shown to be inhibitory in vitro against several A. vitis strains. TFX production, expressed from the stable plasmid pT2TFXK, conferred biological control activity to an avirulent strain of A. vitis. F2/5, against three virulent, TFX-sensitive strains of A. vitis tested on Nicotiana glauca. F2/5(pT2TFXK) is significantly reduces number and size of galls when co-inoculated with tumorigenic strain CG78 at a 10:1 ratio, but is ineffective at 1:1 or 1:10 ratios. F2/5(pT2TFXK) is effective when co-inoculated with tumorigenic strain CG435 at 10:1 and 1:1 ratios, but not at a 1:10 ratio. When F2/5(pT2TFXK) is co-inoculated with CG49 at a 10:1 ratio, the incidence of gall formation does not decline but gall size decreases by more than 70%. A 24 h pre-inoculation with F2/5(pT2TFXK) does not improve biological control at the 1:10 ratio.ConclusionsTFX production by an avirulent strain of Agrobacterium does confer in that strain the ability to control crown gall disease on Nicotiana glauca. This is the first demonstration that the production of a ribosomally synthesized, post-translationally modified peptide antibiotic can confer reduction in plant disease incidence from a bacterial pathogen.

Effects of bacterial antibiotic production on rhizosphere microbial communities from a culture-independent perspective

The effects of antibiotic production on rhizosphere microbial communities of field-grown Phaseolus vulgaris were assessed by using ribosomal intergenic spacer analysis. Inoculum strains of Rhizobium etli CE3 differing only in trifolitoxin production were used. Trifolitoxin production dramatically reduced the diversity of trifolitoxin-sensitive members of the alpha subdivision of the class Proteobacteria with little apparent effect on most microbes.

Trifolitoxin Production Increases Nodulation Competitiveness of Rhizobium etli CE3 under Agricultural Conditions.

A major barrier to the use of nitrogen-fixing inoculum strains for the enhancement of legume productivity is the inability of commercially available strains to compete with indigenous rhizobia for nodule formation. Despite extensive research on nodulation competitiveness, there are no examples of field efficacy studies of strains that have been genetically improved for nodulation competitiveness. We have shown previously that production of the peptide antibiotic trifolitoxin (TFX) by Rhizobium etli results in significantly increased nodule occupancy values in nonsterile soil in growth chamber experiments (E. A. Robleto, A. J. Scupham, and E. W. Triplett, Mol. Plant-Microbe Interact. 10:228-233, 1997). To determine whether TFX production by Rhizobium etli increases nodulation competitiveness in field-grown plants, seeds of Phaseolus vulgaris were inoculated with mixtures of Rhizobium etli strains at different ratios. The three nearly isogenic inoculum strains used included TFX-producing and non-TFX-producing strains, as well as a TFX-sensitive reference strain. Data was obtained over 2 years for nodule occupancy and over 3 years for assessment of the effect of the TFX production phenotype on grain yield. In comparable mixtures in which the test strain accounted for between 5 and 50% of the inoculum, the TFX-producing strain exhibited at least 20% greater nodule occupancy than the non-TFX-producing strain in both years. The TFX production phenotype had no effect on grain yield over 3 years; the average yields reached 2,400 kg/ha. These results show that addition of the TFX production phenotype significantly increases nodule occupancy under field conditions without adverse effects on grain yield. As we used common inoculation methods in this work, there are no practical barriers to the commercial adoption of the TFX system for agriculture.

A Transposable Partitioning Locus Used To Stabilize Plasmid-Borne Hydrogen Oxidation and Trifolitoxin Production Genes in a Sinorhizobium Strain

Improved nitrogen-fixing inoculum strains for leguminous crops must be able to effectively compete with indigenous strains for nodulation, enhance legume productivity compared to the productivity obtained with indigenous strains, and maintain stable expression of any added genes in the absence of selection pressure. We constructed a transposable element containing the tfx region for expression of increased nodulation competitiveness and the par locus for plasmid stability. The transposon was inserted into tetA of pHU52, a broad-host-range plasmid conferring the H2 uptake phenotype. The resulting plasmid, pHUTFXPAR, conferred the plasmid stability, trifolitoxin production, and H2 uptake phenotypes in the broad-host-range organism Sinorhizobium sp. strain ANU280. The broad applications of a transposon conferring plasmid stability are discussed.

Trifolitoxin Production in Rhizobium etli Strain CE3 Increases Competitiveness for Rhizosphere Colonization and Root Nodulation of Phaseolus vulgaris in Soil

The effect of trifolitoxin (TFX) production by Rhizobium etli on rhizosphere colonization and competition for nodulation in soil conditions was determined. TFX is a potent peptide antibiotic made by Rhizobium leguminosarum bv. trifolii T24 that inhibits many α-proteobacteria (E. W. Triplett, B. T. Breil, and G. A. Splitter, Appl. Environ. Microbiol. 60:4163–4166, 1994). Seeds of Phaseolus vulgaris were inoculated with a TFX-sensitive reference strain and either of two isogenic strains that differ only in their ability to produce TFX. The pair of strains were inoculated at different ratios in sterile and nonsterile soil. The representation of the strains in the rhizosphere and nodules was determined at 96 h after inoculation and 3 weeks after planting, respectively. The TFX-producing strain was significantly more competitive for both phenotypes versus the TFX-sensitive strain, compared with the TFX-nonproducing strain versus the TFX-sensitive strain. These results show that nodule occupancy by inoculant strains, often displaced from the nodules by indigenous strains, can be increased by addition of the TFX production phenotype to R. etli in plants grown in either sterile or nonsterile soil. Also, this work shows the efficacy of the TFX system for the first time on a legume host with determinant nodules.

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

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 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 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.

Production of the anti-rhizobial peptide, trifolitoxin, in sterile soils by Rhizobium leguminosarum bv. trifolii T24

Trifolitoxin production by Rhizobium leguminosarum bv. trifolii T24 was observed in three sterile soils. The three soils differed in texture and fertility. Two of these soils were silt loams while the third soil was a silty sand. One of the silt loams was amended with manure and composted. The amount of trifolitoxin in these soils was determined by bioassay after inoculation with T24. The largest amount of trifolitoxin in each soil was observed at 48 h after inoculation; after 48 h, trifolitoxin declined. The amended soil contained 5–10 fold more trifolitoxin than did the unamended soils. The production of trifolitoxin in soil by T24 was verified in two ways: (i) T24 inoculated soils contain a substance which inhibits a trifolitoxin-sensitive strain of Rhizobium but not a trifolitoxin-resistant strain; and (ii) sterile soils inoculated with a Tn5 mutant of T24 that lacks trifolitoxin production (T24::Tn5 1) did not contain any detectable trifolitoxin activity during the 4 days of the experiment. These data demonstrate that trifolitoxin is produced by T24 in unamended as well as amended soils, although much larger amounts are detected in the amended soil.

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.

Isolation of genes involved in nodulation competitiveness from Rhizobium leguminosarum bv. trifolii T24

Rhizobium leguminosarum bv. trifolii T24 produces a potent anti-rhizobial compound, trifolitoxin, and exclusively nodulates clover roots when in mixed inoculum with trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii [Schwinghamer, E. A. & Belkengren R. P. (1968) Arch. Mikrobiol. 64, 130-145]. In the present study, the isolation of trifolitoxin production and resistance genes is described. A cosmid genomic library of T24 was prepared in pLAFR3. No trifolitoxin expression was observed in the resulting Escherichia coli cosmid clones. One cosmid clone was identified that restored trifolitoxin production and nodulation competitiveness in three nonproducing mutants of T24. The recombinant plasmid from this cosmid clone, pTFX1, also conferred trifolitoxin production and resistance when transferred to symbiotically effective strains of R. leguminosarum bvs. trifolii, phaseoli, and viceae. Cosmid pTFX1 also conferred expression of trifolitoxin production when present in strains of Rhizobium meliloti and Agrobacterium tumefaciens. No trifolitoxin expression was observed in strains of Bradyrhizobium japonicum or Rhizobium sp. (cowpea) with pTFX1. Southern blot analysis with a biotinylated pTFX1 probe did not suggest that these genes were plasmid-borne. Transfer of pTFX1 to T24 or its derivatives resulted in 6- to 10-fold higher level of trifolitoxin production than wild-type T24.

Trifolitoxin Production and Nodulation Are Necessary for the Expression of Superior Nodulation Competitiveness by Rhizobium leguminosarum bv. trifolii Strain T24 on Clover

Rhizobium leguminosarum bv. trifolii T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain Rhizobium strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of R. leguminosarum bv. trifolii. A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive R. leguminosarum bv. trifolii when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of R. meliloti did not nodulate alfalfa when co-inoculated with T24 and a trifolitoxin-resistant strain of R. meliloti. Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of R. leguminosarum bvs. trifolii, viceae, and phaseoli tested. Strains of all 13 biotypes of R. leguminosarum bv. trifolii were inhibited by trifolitoxin. Three strains of R. fredii were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate Trifolium ambiguum, and induced partially effective nodules on Trifolium micranthum. Since T24 produced partially effective nodules on T. micranthum and since a trifolitoxin-minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24.