Resistance to Two Vinylglycine Antibiotic Analogs Is Conferred by Inactivation of Two Separate Amino Acid Transporters in

is the causal agent of fire blight of apple and pear trees. Several bacteria have been shown to produce antibiotics that antagonize , including pantocins, herbicolins, dapdiamides, and the vinylglycines, 4-formylaminooxyvinylglycine (FVG) and 4-aminoethoxyvinylglycine (AVG). BRT175 was previously shown to exhibit antibiotic activity against via the production of natural product 1 (PNP-1), later shown to be FVG; however, exposure of to FVG results in spontaneously resistant mutants. To identify the mechanism of resistance, we used genome variant analysis on spontaneous FVG-resistant mutants of and identified null mutations in the l-asparagine permease gene Heterologous expression of in normally resistant was sufficient to impart FVG susceptibility, suggesting that FVG is imported through this permease. Because FVG and AVG are structurally similar, we hypothesized that resistance to AVG would also be conferred through inactivation of ; however, mutants were not resistant to AVG. We found that spontaneously resistant Ea321 mutants also arise in the presence of AVG, with whole-genome variant analysis revealing that resistance was due to inactivation of the arginine ABC transporter permease subunit gene Heterologous expression of the predicted -like transporter encoded within the BRT175 FVG biosynthetic cluster, which is likely responsible for antibiotic export, was sufficient to confer resistance to both FVG and AVG. This work highlights the important roles of amino acid transporters in antibiotic import into bacteria and the potential utility of antimicrobial amino acid analogs as antibiotics. The related antibiotics formylaminooxyvinylglycine (FVG) and aminoethoxyvinylglycine (AVG) have been shown to have activity against the fire blight pathogen ; however, can develop spontaneous resistance to these antibiotics. By comparing the genomes of mutants to those of the wild type, we found that inactivation of the l-asparagine transporter conferred resistance to FVG, while inactivation of the l-arginine transporter conferred resistance to AVG. We also show that the transporter encoded by the FVG biosynthetic cluster can confer resistance to both FVG and AVG. Our work indicates the important role that amino acid transporters play in the import of antibiotics and highlights the possible utility in designer antibiotics that enter the bacterial cell through amino acid transporters.