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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Publications at this Location » Publication #182111
Title: DETERMINATION OF THE AMINO ACID RESIDUES REQUIRED FOR THE ACTIVITY OF THE ANTI-RHIZOBIAL PEPTIDE ANTIBIOTIC TRIFOLITOXIN

Author
item SCUPHAM, ALEXANDRA
item TRIPLETT, ERIC - UNIV FLORIDA-GAINESVILLE

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/16/2005
Publication Date: 3/20/2006
Citation: Scupham, A.J., Triplett, E.W. 2006. Determination of the amino acid residues required for the activity of the anti-rhizobial peptide antibiotic trifolitoxin. Journal of Applied Microbiology. 100(3):500-507.

Interpretive Summary: Peptide antibiotics provide powerful defense against pathogens for animals lacking adaptive immune systems, such as amphibians and invertebrates. This class of antibiotic has also been found in bacteria, among them microcin B17 made by Escherichia coli and the lantibiotics. Trifolitoxin (TFX), an 11 amino acid peptide antibiotic produced by the legume symbiont Rhizobium leguminosarum bv. trifolii T24, was discovered to confer a competitive advantage for the colonization of the legume nodule niche by inhibiting the growth of closely related Rhizobium strains. Transfer of the antibiotic-production capability into Rhizobium strains that are highly efficient nitrogen fixers has been shown to increase alfalfa and bean yields and may decrease agricultural dependence on nitrogen fertilizers. In addition, avirulent Agrobacterium vitus strain F2/5 producing trifolitoxin was found to be an effective biological control agent against virulent Agrobacterium strains causing crown gall disease on grape and tobacco crops. Finally, TFX was shown to be effective against many members of the alpha-proteobacteria, including the animal pathogen Brucella, and it may have a future use for treatment of this disease. The trifolitoxin structure and mechanism of action have not yet been resolved, nor has the mechanism of resistance. In this work site-specific mutagenesis was used to identify the active sites of the molecule. It was determined that the central residues arginine, glutamine, glycine and cysteine, which are believed to be post-translationally modified to form thiazole and UV-absorbing rings, are required for biological activity of the molecule and the C-terminal alanine is necessary for optimal activity. In addition, it was determined that changes to the N-terminus of the molecule were not inhibitory, suggesting sufficient quantities of the peptide may be isolated for structural analysis using histidine tagging.

Technical Abstract: Trifolitoxin is a ribosomally synthesized, post-translationally modified peptide antibiotic produced by Rhizobium leguminosarum bv. trifolii T24. The structural gene, tfxA, codes for a 42 residue peptide, which is cleaved and modified to produce an active peptide containing 11 amino acid residues. The amino acid sequence of the unmodified 11-mer peptide is DIGGSRQGCVA. Previous structural analysis of the antibiotic showed that the arginine (R37), glutamine (Q38), glycine (G39), and cysteine (C40) residues are post-translationally modified to form a UV-absorbing chromophore of unknown structure and a thiazoline ring. In this work the unmodified peptide was shown to lack biological activity, indicating that the post-translational modifications are required for biological activity. An expression system for site directed mutagenesis was developed to show that these residues are required for activity. Conservative amino acid substitutions of the R37, Q38, G39, and C40 residues resulted in loss of antibiotic activity. Conservative and non-conservative substitutions of the S36 residue reduced activity, and a conservative substitution of the A42 residue also decreased TFX activity. None of the substitutions tested enhanced activity. Thus all of those amino acid residues that are post-translationally modified to produce active TFX are either essential for activity or necessary for optimal activity.