Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Scupham, A. J. Articles by Triplett, E. W. Search for Related Content PubMed PubMed Citation Articles by Scupham, A. J. Articles by Triplett, E. W. Agricola Articles by Scupham, A. J. Articles by Triplett, E. W.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2002, p. 4334-4340, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4334-4340.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Role of tfxE, but Not tfxG, in Trifolitoxin Resistance

Alexandra J. Scupham,^1, Yuemei Dong,² and Eric W. Triplett^2*

Departments of Bacteriology,¹ Agronomy, University of Wisconsin—Madison, Madison, Wisconsin 53706²

Received 4 March 2002/ Accepted 17 June 2002

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Eight genes, tfxABCDEFG and tfuA, confer production of trifolitoxin (TFX), a ribosomally synthesized, posttranslationally modified peptide antibiotic, in TFX-sensitive -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.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Trifolitoxin (TFX) is a ribosomally synthesized, posttranslationally modified peptide antibiotic that inhibits a specific group of organisms within the -proteobacteria (4, 22). DNA sequence analysis and direct peptide sequencing have shown that the sequence of the peptide prior to posttranslational modification but after cleavage of the leader is DIGGSRQGCVA (3, 4, 13). The addition of the seven genes tfxABCDEFG to strains of -proteobacteria is sufficient to make active TFX and supply resistance for the producing strain (22).

Prior to this work, several lines of indirect evidence suggested roles for TfxE and TfxG in TFX resistance and immunity. Two rounds of transposon mutagenesis of the tfx region showed no inserts in either tfxE or tfxG, suggesting that mutagenesis of either gene led to a lethal phenotype (4). In addition, a tfx region construct lacking part of tfxF and all of tfxG was incapable either of conferring TFX production in a Rhizobium strain or of providing resistance to externally applied TFX (4). A nonpolar insertion in tfxF caused a loss of TFX production but no loss of resistance to externally applied TFX (4). In addition, TfxG possesses significant identity with a protein of unknown function adjacent to a transposon in Rhizobium leguminosarum strain E163N (23). As transposons often contain antibiotic resistance genes, the homology of these two proteins was intriguing. All of these lines of evidence suggested, but did not directly demonstrate, that tfxE and tfxG were involved in immunity to internally produced TFX and resistance to externally applied TFX (4).

TfxG contains a eukaryotic-type serine/threonine kinase motif. Given the proposed role of TfxG in TFX immunity and resistance, a model of TFX resistance and immunity was proposed whereby the serine residue of TFX is phosphorylated intracellularly by TfxG and exported in its phosphorylated form. Upon export, the phosphorylated TFX could be dephosphorylated by the extracellular phosphatases known to be produced by Rhizobium (1). Phosphorylation is a common mechanism for antibiotic inactivation (7, 14, 15, 20).

To test this model, amino acid substitutions in the serine/threonine kinase motif of TfxG were created to determine the role of this motif in TFX resistance. Deletions in tfxE and tfxG were also constructed to test the role of these genes in resistance to TFX. These results showed that TfxE is involved in TFX resistance while TfxG was found to play no role in TFX resistance. Given that TfxG plays no role in resistance, its role in TFX production was assessed.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains, plasmids, and culturing techniques.
All bacterial strains and plasmids used in this work are described in Table 1. Rhizobium strains were grown on Bergersen's minimal media (BSM) at 28°C (2). Escherichia coli strains were grown on Luria-Bertani medium at 37°C (18). Antibiotics were routinely used at the following concentrations: nalidixic acid, 50 µg/ml; streptomycin, 50 µg/ml; kanamycin, 50 µg/ml; tetracycline, 10 µg/ml; ampicillin, 50 µg/ml; and gentamicin, 10 µg/ml.

View this table: [in this window] [in a new window]

TABLE 1. Strains and plasmids used in this work

TFX bioassay.
TFX production and resistance were tested by the plate assay method of Triplett and Barta (21) as modified by Breil et al. (4). For the assays of the tfxG site-directed mutants, wells were bored into the centers of agar-containing petri plates and 50 µl of liquid containing TFX in concentrations of 8.4, 0.8, 0.08, or 0.008 nmol was aliquoted into the wells. The plates were allowed to sit overnight and were subsequently sprayed with one of four strains: ANU794, ANU794(pTFXRWT), ANU794(pRGD126A), or ANU794(pRGD144A).

RNase protection assays.
The Direct Protect Lysate RPA (catalog no. 1420) and MAXIscript (catalog no. 1326) kits from Ambion were used to determine whether the tfxE and tfxG genes are transcribed and whether they are expressed as a single transcript. DNA probes were generated by PCR to genes tfxE, tfxF, and tfxG with plasmid pTFX24 as the template. The same template was used to generate probes to regions between the predicted tfxD, tfxE, tfxF, and tfxG genes. These probes spanned the 3' ends of upstream genes and 5' ends of downstream genes. The primers used for probe synthesis are listed in Table 2. ³²P-labeled RNA probes were generated from the PCR products by using the Ambion MAXIscript kit.

View this table: [in this window] [in a new window]

TABLE 2. Primers used in this work for RNase protection, mutagenesis, and sequencing

TFX-producing strain KIM5s(pTFX6) was harvested from BSM plates and washed twice in phosphate-buffered saline. The cells were then resuspended at a concentration of 0.1 g/ml in lysis-denaturation solution from the MAXIScript kit. The cells were broken by shaking in a bead beater for 10 s at a speed of 4. The ³²P-labeled probes were then hybridized to the cell supernatants and digested with RNase according to the manufacturer's instructions. The reactions were run on 6% acrylamide gels and visualized on X-ray film.

TfxE deletion mutagenesis.
A 171-bp deletion was introduced into the tfxE gene by using a number of steps (Fig. 1). First, all of tfxBCD and part of tfxE were deleted from plasmid pTFX24 by digestion of the plasmid with restriction enzyme AflII and self-ligation of the major fragment. This self-ligation created a new AflII site between tfxA and tfxE and yielded plasmid pTXE21. Plasmid pTXE21 was mutagenized to introduce a second AflII restriction site 171 bp downstream of the existing AflII site. This mutation was generated using the Stratagene QuikChange method. Forward primer EAFL3 and reverse primer EAFL4 (Table 2) were used to introduce the new AflII site, creating plasmid pTXEA2.

View larger version (24K): [in this window] [in a new window]

FIG. 1. Cloning of plasmid pRE171 to create a deletion in the tfxE gene.

TFX-encoding plasmid pTFX24 (4) was digested with NotI and self-ligated, producing pTX1, which lacks all of tfxA and part of tfxB. A fragment containing all of tfxCD and parts of tfxB and tfxE was isolated by digestion of pTX1 with AflII and NotI. Plasmid pTXEA2 was also digested with AflII and NotI. Ligation of the two NotI/AflII fragments created pTXE171, which has a deletion of the tfxAB' genes and contains a 171-bp deletion in tfxE. Sequence analysis with primer TfxE5 (Table 2) was used to confirm that the correct deletion was produced. The TFX genes of pTXE171 were transferred to broad-host-range plasmid pRK415 by digestion with KpnI and ligation into the unique KpnI site of pRK415 to make pRE171.

TfxG site-directed mutagenesis.
Plasmid pTXG containing genes tfxF'G was generated from pTFX24 by digesting the original plasmid with XbaI and self-ligating to remove genes tfxABCDEF' (Fig. 2). Specific point mutations in pTXG were generated with the QuickChange technique (Stratagene). Primers used for the mutageneses were D126AF, D126AR, D144AF, and D144AR (Table 2). PCR conditions included an initial denaturation at 95°C for 5 min followed by 16 cycles of denaturation at 95°C for 1 min and annealing at 60°C for 1 min and elongation at 68°C for 11 min. Primer TfxG3 (Table 2) was used for sequencing to confirm the mutagenesis.

View larger version (28K): [in this window] [in a new window]

FIG. 2. Cloning of the tfxG mutant alleles into the tfx operon.

Cloning of tfxG alleles into non-TFX-producing constructs.
For these experiments, resistance determinants were cloned such that they would be expressed but TFX would not be produced. Previous work (3, 4) showed that the genetic fragment tfxB'CDEFG conferred TFX resistance without antibiotic production. The mutated tfxG alleles D126A and D144A were rejoined with the other TFX genes by doubly digesting the mutated pTXG plasmids with XbaI and SacI (Fig. 2). The fragments were ligated into the XbaI/SacI major fragment of plasmid pTX1 to create pTFXGD126A and pTFXGD144A. These plasmids contained genes tfxB'CDEFG_*, where _* indicates a mutated tfxG. The tfx genes were then excised from pTFXGD126A, pTFXGD144A, and pTX1 with KpnI and ligated into the broad-host-range plasmid pRK415 to form plasmids pRGD126A, pRGD144A, and pTFXRWT (Fig. 2).

TfxG deletion mutagenesis.
The construct pTFXG of the TFX operon was made by deleting tfxG from pTFX24. Digestion of pTFX24 with restriction enzyme Eco47III followed by self-ligation excised the entire tfxG gene from the plasmid without disturbing the other genes. The remaining TFX genes were then excised from pTFXG by digestion with BssHII and cloned into the MluI site of pRKEM for analysis of the TFX production and resistance phenotypes, creating pRTFXG. The complete wild-type tfx operon was moved into plasmid pRKEM by the same method, creating pRTFXWT.

Modification of broad-host-range vector pRK415.
An efficient method for cloning the TFX genes from the pBluescriptII KS+ vector into the broad-host-range vector pRK415 was needed. To this end, the unique EcoRI restriction site was changed to an MluI site via oligo tags. The 10-bp oligo AATTACGCGT was synthesized and phosphorylated with Promega T4 kinase. The tag was then ligated into the pRK415 EcoRI site, creating pRKEM.

In trans complementation of tfxG with mutant alleles.
Plasmids pTFXRWT, pRGD126A, pRGD144A, pRTFXWT, pRTFXD144A, and pRTFXG were moved into TFX-sensitive strain R. leguminosarum ANU794 via triparental mating with E. coli DH5(pRK2013) as the helper strain. The TFX-sensitive strain augmented in this way was then tested for resistance to multiple concentrations of purified TFX by using plate assays. Dilutions of purified TFX were dispensed into wells bored into the centers of agar plates. The plates were allowed to dry and then sprayed with the test strains.

Antibiotic isolation and analysis.
Wild-type TFX and the antibiotic TFX-G produced by ANU794(pRTFXG) were isolated as follows. One hundred petri dishes containing BSM-N agar were inoculated with either KIM5s(pTFX6) or ANU794(pRTFXG). Each of these dishes has a 10-cm diameter. After 4 days of growth, the medium was scraped from the plates and placed into 2 liters of distilled water and soaked overnight at 4°C. The solution was then separated from the cells by filtration and centrifugation. The supernatant was then applied to a preparative C₁₈ reverse-phase column and washed with 2 column volumes of water. TFX activity was eluted with 20% methanol. The 20% methanol wash was concentrated on a rotary evaporator at 50°C to a volume of 10 ml. The sample was treated with 240 µl of BPA-1000 (Supelco, Inc., Bellefonte, Pa.) to decolorize the sample. The BPA-treated sample was allowed to stand for 3 min and centrifuged at 15,000 x g for 3 min. The pellet was discarded. The supernatant was concentrated by rotary evaporation or lyophilization to a volume of 1 ml. The sample was then injected onto a C₁₈ reverse-phase high-pressure liquid chromatography (HPLC) column equilibrated with 5 mM sodium phosphate buffer, pH 5.0. The elution profile was monitored at 302 nm, the maximum absorbance of the UV-absorbing chromophore in TFX. Four TFX active fractions were eluted with a methanol gradient from 15 to 35% in phosphate buffer. The second and third active peaks contained 90% of the total TFX activity. These two compounds are referred to as TFX1 and TFX2, respectively. The sodium phosphate was removed from the sample by first diluting the sample with H₂O to a methanol concentration below 10% followed by absorption on a C₁₈ sep-pack column. TFX was then eluted from the column with 50% methanol. Following methanol removal by evaporation, the sample was resuspended in H₂O and stored at -80°C prior to further analysis. Molecular weights were obtained from matrix-assisted laser desorption ionization (mass spectrometry).

Sequence analysis.
The tfxE and tfxG nucleotide sequences were analyzed against those in GenBank with BLAST by using the blastn nonredundant search. TfxE and TfxG proteins were analyzed against those in GenBank by using the blastp and tblastn nonredundant searches.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Test of wild-type TFX activity—relationship between antibiotic concentration and assay zone size.
Wild-type ANU794 is a TFX-sensitive strain, and in response to the antibiotic, the strain showed zones of growth inhibition of differing radii. A zone of inhibition with a radius of 3.7 cm was measured from the edges of the wells to the edges of bacterial growth with the addition of 8.4 nmol of TFX. The zones decreased in size as the concentration of applied TFX decreased, such that 0.84 nmol of TFX gave a radius of 3.2 cm, 0.08 nmol of TFX gave a radius of 2.3 cm, and 0.008 nmol of TFX gave a radius of 1.5 cm.

Role of TfxE in TFX resistance and immunity by tfxE deletion.
Strain ANU794 containing a 171-bp in-frame deletion of tfxE on plasmid pRE171 was tested for TFX resistance. ANU794(pRE171) showed a 1.6-cm-radius zone of growth inhibition when exposed to the TFX-producing strain Tn5-4 and no zone of inhibition when exposed to the non-TFX-producing strain Tn54A112. Included with these assays were several controls. Zones of inhibition of 2.7 cm in radius were seen in the growth patterns of the TFX-sensitive strains ANU794 and ANU794(pRK415) when tested against the TFX-producing strain Tn5-4. No zones of growth inhibition were apparent when ANU794 and ANU794(pRK415) were tested against the non-TFX-producing strain Tn54A112. Strain ANU794 containing the wild-type determinants of TFX resistance on plasmid pTFXRWT showed no zone of inhibition against either the TFX-producing or non-TFX-producing strains. This indicates the TFX resistance determinants in trans confer TFX resistance to the antibiotic-sensitive strain. Collectively, these results indicate that tfxE is required for resistance to externally applied TFX.

The tfxE nucleotide sequence is unlike any other in GenBank, so the mechanism by which TFX-producing strains are resistant to the antibiotic appears to be novel. Of the seven genes in the tfx cluster, tfxG is the only one with a homolog in the fully sequenced Sinorhizobium meliloti 1021 genome. None of them have homologs in the fully sequenced genome of Mesorhizobium loti strain MAFF303099. As with tfxE, none of the resistance proteins from either the lantibiotic or microcin antibiotic groups have high homologies to sequences in GenBank.

Plate assays showed that the strain with the tfxE deletion was sensitive to TFX. Interestingly, this strain was not as sensitive as the strain containing only the vector. The reason for the decreased sensitivity is not known. Although nearly a third of the protein was removed, it is possible that the truncated TfxE retained some activity. It is also possible that TfxE works in concert with another TFX protein to confer resistance and that this protein alone is able to provide some low level of resistance. A mechanism of this sort is seen among the lantibiotic group of posttranslationally modified antibiotics. Resistance genes have been identified for lantibiotics such as nisin, mutacin II, and pep5, but these genes confer only partial resistance to the antibiotics (6, 16, 17). Complete resistance results only in the presence of genes coding for ABC transport complexes. TfxD is a membrane-bound protein and may serve to export TFX or a derivative of TFX in the presence or absence of an active TfxE.

Role of TfxG in TFX resistance—effect of serine/threonine kinase motif.
Previous evidence suggested that tfxG was involved in TFX resistance (4). In addition, the protein sequence of TfxG indicated it could be a eukaryotic-type serine/threonine kinase (12). TfxG contains aspartic acids D126 and D144 in a motif that appears to place the protein in the eukaryotic serine/threonine kinase family.

The predicted amino acid sequence of TfxG contains the motif IGHCDTGPWNIVC, with an additional aspartic acid 10 residues farther downstream. The initial 13 residues are an 85% match with the diagnostic eukaryotic serine/threonine kinase motif (Table 3). The presence of this motif in TfxG suggested that the protein could have kinase activity, and thus confer immunity through phosphorylation. In this work, a genetic approach was taken to determine whether the serine/threonine kinase motif of tfxG was important in TFX resistance. The TFX peptide contains a serine residue that could serve as a site of phosphorylation and possible inactivation of the peptide.

View this table: [in this window] [in a new window]

TABLE 3. Serine/threonine kinase motif compared to TfxG sequence

Two residues in the serine/threonine kinase motif of TfxG, D126 and D144, are highly conserved in such kinases and are necessary for full kinase activity. The two residues D126 and D144 of TfxG were changed to alanine by site-directed mutagenesis, and the mutant alleles were tested for the ability to confer resistance to exogenously applied TFX. Plasmid pTFXRWT contains the genes tfxB'CDEFG such that all elements necessary for TFX resistance are present on the plasmid. TFX-sensitive strain ANU794 containing this plasmid was resistant to all concentrations of TFX. Finally, plasmids pRGD126A and pRGD144A contain the same set of tfx genes as does pTFXRWT, except that they have the D126A and D144A alleles, respectively, in place of wild-type tfxG. ANU794 harboring these plasmids was as resistant as the wild type to the various levels of TFX present in the plates. These results show that the two mutations D126A and D144A had no effect on the ability of the tfxG gene to confer resistance to exogenously applied TFX.

Role of TfxG in resistance—deletion of TfxG.
Although the serine/threonine kinase motif is not needed for TFX resistance, other portions of the TfxG protein may confer TFX resistance. Constructs lacking the entire tfxG sequence were constructed to test this possibility.

Both the ANU794 TFX-sensitive strain and the ANU794(pRK415) strain exhibited zones of growth inhibition when tested against TFX-producing strain Tn5-4. These zones had radii of 2.9 ± 0.6 and 3.0 ± 0.6 cm. Plasmid pRTFXWT possesses all of the wild-type tfx genes. ANU794(pRTFXWT) was not sensitive to TFX production by Tn5-4, indicating that the plasmid conferred TFX resistance to strain ANU794. ANU794(pTFX131), where pTFX131 lacks tfxG, should not be able to make TFX because half of the tfxF gene is also missing. This allows the role of tfxG to be assessed in the absence of endogenously produced TFX. Tested against the TFX-producing strain Tn5-4, strain ANU794(pTFX131) showed no zone of inhibition. This result indicates that plasmid pTFX131 confers TFX resistance to the strain. Thus, eliminating tfxG did not affect resistance to exogenously applied TFX.

During the course of this work, the complete genome sequence of S. meliloti 1021 was published (10). The annotation of the megaplasmid pSymB (9) from that genome predicts a protein that is 50% identical and 66% homologous to 89% of the coding region of TfxG of R. leguminosarum. Previous results showed that S. meliloti 1021 is resistant to TFX (22). To determine whether tfxG in strain 1021 could be responsible for the TFX resistance phenotype in that strain, 200-bp deletion mutants in tfxG were prepared as described by Scupham and Triplett (19). The deletion mutagenesis of tfxG in strain 1021 had no effect on the TFX resistance phenotype of that strain.

Operon structure of tfxDEFG—RNase protection assays.
The observation that TfxG plays no role in either TFX immunity or resistance led to experiments to determine whether tfxG was cotranscribed with other genes in the tfx region. Figure 3 shows RNase-protected mRNA fragments from genes tfxE, tfxF, and tfxG and the intergenic regions of tfxDE, tfxEF, and tfxFG (Fig. 3, lanes 1). These fragments consist of ³²P-labeled RNA probes annealed to mRNA that was isolated from the TFX-producing Rhizobium strain KIM5s(pTFX6). The probe was also run alone (Fig. 3A, B, C, E, and F, lanes 2, and D, lane 3). The mRNA fragments representing the intergenic spaces and genes tfxE, tfxF, and tfxG all appeared similar in size to the probes run alone in neighboring lanes (Fig. 3). If the genes had been on different transcripts, the mRNAs of the intergenic spaces would have yielded two fragments, the sum of which would be less than or equal to the size of the PCR products. These results show that the genes tfxDEFG are transcribed as a single operon. The RNase fragments were slightly smaller than the probes because the 5' ends of the primers used to generate the probes were not homologous to the tfx transcripts. Controls with RNase-digested probes were designed to show that the RNase was active (Fig. 3A, B, C, E, and F, lanes 3, and D, lane 2).

View larger version (46K): [in this window] [in a new window]

FIG. 3. RNase protection assays of tfxEFG and their intergenic spaces. (A) Intergenic space between tfxD and tfxE. Lane 1, the tfxDE RNase protection fragment is 319 bp; lane 2, the tfxDE probe is 356 bp; lane 3, the tfxDE probe digested with RNase. (B) tfxE. Lane 1, the tfxE RNase protection fragment is 212 bp; lane 2, the tfxE probe is 248 bp; lane 3, the tfxE probe digested with RNase. (C) Intergenic space between tfxE and tfxF. Lane 1, the tfxEF RNase protection fragment is 118 bp; lane 2, the tfxEF probe is 155 bp; lane 3, the tfxEF probe digested with RNase. (D) tfxF. Lane 1, the tfxF RNase protection fragment is 36 bp; lane 2, the tfxF probe digested with RNase; lane 3, the tfxF probe is 29 bp. (E) Intergenic space between tfxF and tfxG. Lane 1, the tfxFG RNase protection fragment is 261 bp; lane 2, the tfxFG probe is 402 bp; lane 3, the tfxFG probe digested with RNase. (F) tfxG. Lane 1, the tfxG RNase protection fragment is 311 bp; lane 2, the tfxG probe is 349 bp; lane 3, the tfxG probe digested with RNase.

TfxG is not required for TFX production.
As tfxG is on the same operon as other tfx genes and as TfxG has no role in TFX resistance or immunity, a role of TfxG in TFX production was assessed.

A construct containing tfxABCDEF but lacking tfxG was tested for its ability to produce TFX. Strain ANU794(pRTFXG) and the appropriate controls were spotted onto agar plates and allowed to grow for 2 days. At that time, slightly turbid suspensions of TFX-sensitive strain 128C1 and TFX-resistant strain Tn5-4 were sprayed onto the plates. Control strains ANU794 and ANU794(pRK415) containing just the cloning vector did not produce zones of growth inhibition, indicating that they do not produce any compounds toxic to Rhizobium strains Tn5-4 or 128C1. Positive-control ANU794(pRTFXWT) containing all of the tfx genes caused zones of inhibition that were 2.4 ± 0.6 cm in radius around sensitive strain 128C1 but not around resistant strain Tn5-4. Zones of growth inhibition of 2.6 ± 0.6 cm were observed around strain ANU794(pTFXG), demonstrating that tfxG lacks an essential role in TFX production.

TfxG is required for production of TFX isomers—structural comparison of TFX and TFX-G.
The antibiotics with TFX activity from ANU794(pRTFXWT) and ANU794(pRTFXG) were purified as described above. Wild-type TFX eluted from the C₁₈ reverse-phase column in two distinct peaks; the molecular weights of these peaks, as determined by matrix-assisted laser desorption ionization (mass spectrometry), were 1,039. These HPLC peaks were first detected by measuring absorbance at 302 nm followed by an assay for TFX activity. TFX-G eluted as a single peak during HPLC separation. Wild-type TFX production results in four peaks of TFX activity, with two of those peaks, referred to as TFX1 and TFX2, making up about 90% of the total TFX activity. TFX-G has a molecular weight of 1,039, which is identical to that of TFX1 and TFX2. Thus, TfxG is involved in the formation of the TFX1 and TFX2 isomers. The tfxG mutant does produce an active TFX peptide.

 
We thank the College of Agricultural and Life Sciences of the University of Wisconsin—Madison and the Consortium for Plant Biotechnology Research for support of this work.

 
* Corresponding author. Mailing address: Department of Agronomy, University of Wisconsin—Madison, 1575 Linden Dr., Madison, WI 53706. Phone: (608) 262-9824. Fax: (608) 262-5217. E-mail: triplett{at}wisc.edu.

Present address: Department of Plant Pathology, University of California, Riverside, CA 92521.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1
1. Abdalla, M. H. 1994. Phosphatases and the utilization of organic phosphorus by Rhizobium leguminosarum biovar viceae. Lett. Appl. Microbiol. 18:294-296.
2
2. Bergersen, F. J. 1961. The growth of Rhizobium in synthetic media. Austral. J. Biol. Sci. 14:349-360.
3
3. Breil, B. T., J. Borneman, and E. W. Triplett. 1996. A newly discovered gene, tfuA, involved in the production of the ribosomally synthesized peptide antibiotic trifolitoxin. J. Bacteriol. 178:4150-4156.[Abstract/Free Full Text]
4
4. Breil, B. T., P. W. Ludden, and E. W. Triplett. 1993. DNA sequence and mutational analysis of genes involved in the production and resistance of the antibiotic trifolitoxin. J. Bacteriol. 175:3693-3702.[Abstract/Free Full Text]
5
5. Chen, H., A. E. Richardson, E. Gartner, M. A. Djordjevic, R. J. Roughly, and B. G. Rolfe. 1991. Construction of an acid-tolerant Rhizobium leguminosarum bv. trifolii strain with enhanced capacity for nitrogen fixation. Appl. Environ. Microbiol. 57:2005-2011.[Abstract/Free Full Text]
6
6. Chen, P., F. Qi, J. Novak, and P. W. Caufield. 1999. The specific genes for lantibiotic mutacin II biosynthesis in Streptococcus mutans T8 are clustered and can be transferred en bloc. Appl. Environ. Microbiol. 65:1356-1360.[Abstract/Free Full Text]
7
7. Davies, J. 1994. Inactivation of antibiotics and the dissemination of resistance genes. Science 264:375-382.[Abstract/Free Full Text]
8
8. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76:1648-1652.[Abstract/Free Full Text]
9
9. Finan, T. M., S. Weidner, K. Wong, J. Buhrmester, P. Chain, F. J. Vorholter, I. Hernandez-Lucas, A. Becker, A. Cowie, J. Gouzy, B. Golding, and A. Pühler. 2001. The complete sequence of the 1,683-kb pSymB megaplasmid from the N₂-fixing endosymbiont Sinorhizobium meliloti. Proc. Natl. Acad. Sci. USA 98:9889-9894.[Abstract/Free Full Text]
10
10. Galibert, F., T. M. Finan, S. R. Long, A. Pühler, P. Abola, F. Ampe, F. Barloy-Hubler, M. J. M. J. Barnett, A. Becker, P. Boistard, G. Bothe, M. Boutry, L. Bowser, J. Buhrmester, E. Cadieu, D. Capela, P. Chain, A. Cowie, R. W. Davis, S. Dreano, N. A. Federspiel, R. F. Fisher, S. Gloux, T. Godrie, A. Goffeau, B. Golding, J. Gouzy, M. Gurjal, I. Hernandez-Lucas, A. Hong, L. Huizar, R. W. Hyman, T. Jones, D. Kahn, M. L. Kahn, S. Kalman, D. H. Keating, E. Kiss, C. Komp, V. Lalaure, D. Masuy, C. Palm, M. C. Peck, T. M. Pohl, D. Portetelle, B. Purnelle, U. Ramsperger, R. R. Surzycki, P. Thebault, M. Vandenbol, F. J. Vorholter, S. Weidner, D. H. Wells, K. Wong, K. C. Yeh, and J. Batut. 2001. The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293:668-672.[Abstract/Free Full Text]
11
11. Keen, N. T., S. Tamaki, D. Kobayashi, and D. Trollinger. 1988. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191-197.[CrossRef][Medline]
12
12. Kennelly, P. J., and M. Potts. 1996. Fancy meeting you here! a fresh look at "prokaryotic" protein phosphorylation. J. Bacteriol. 178:4759-4764.[Abstract/Free Full Text]
13
13. Lethbridge, B. J. 1989. The structure of trifolitoxin. A bacteriocin from Rhizobium leguminosarum biovar trifolii strain T24. Ph.D. thesis. University of Adelaide, Adelaide, Australia.
14
14. McKay, G. A., and G. D. Wright. 1996. Catalytic mechanism of enterococcal kanamycin kinase (APH(3')-IIIa): viscosity, thio, and solvent isotope effects support a Theorell-Chance mechanism. Biochemistry 35:8680-8685.[CrossRef][Medline]
15
15. Mingeot-Leclercq, M., Y. Glupczynski, and P. M. Tulkens. 1999. Aminoglycosides: activity and resistance. Antimicrob. Agents Chemother. 43:727-737.[Free Full Text]
16
16. Pag, U., C. Heidrich, G. Bierbaum, and H. Sahl. 1999. Molecular analysis of the lantibiotic pep5 immunity phenotype. Appl. Environ. Microbiol. 65:591-598.[Abstract/Free Full Text]
17
17. Ra, R., M. M. Beerthuyzen, W. M. de Vos, P. E. J. Saris, and O. P. Kuipers. 1999. Effects of gene disruptions in the nisin gene cluster of Lactococcus lactis on nisin production and producer immunity. Microbiology 145:227-1233.
18
18. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
19
19. Scupham, A. J., and E. W. Triplett. 1997. Isolation and characterization of the UDP-glucose 4'-epimerase-encoding gene, galE, from Brucella abortus 2308. Gene 202:53-59.[CrossRef][Medline]
20
20. Thompson, P. R., D. W. Hughes, N. P. Cianciotto, and G. D. Right. 1998. Spectinomycin kinase from Legionella pneumophila. J. Biol. Chem. 273:14788-14795.[Abstract/Free Full Text]
21
21. Triplett, E. W., and T. M. Barta. 1987. Trifolitoxin production and nodulation are necessary for the expression of superior nodulation competitiveness by Rhizobium leguminosarum bv. trifolii strain T24 on clover. Plant Physiol. 85:335-342.[Abstract/Free Full Text]
22
22. Triplett, E. W., B. T. Breil, and G. A. Splitter. 1994. Expression of tfx and sensitivity to the rhizobial peptide antibiotic trifolitoxin in a taxonomically distinct group of -proteobacteria including the animal pathogen Brucella abortus. Appl. Environ. Microbiol. 60:4163-4166.[Abstract/Free Full Text]
23
23. Ulrich, A., and A. Puehler. 1995. The new class II transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains. Mol. Gen. Genet. 242:505-516.[CrossRef]

---

Applied and Environmental Microbiology, September 2002, p. 4334-4340, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4334-4340.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Scupham, A. J. Articles by Triplett, E. W. Search for Related Content PubMed PubMed Citation Articles by Scupham, A. J. Articles by Triplett, E. W. Agricola Articles by Scupham, A. J. Articles by Triplett, E. W.