MOLECULAR ANALYSIS OF HIGH-LEVEL STREPTOMYCIN RESISTANCE IN ERWINIA-AMYLOVORA

Streptomycin-resistant and -sensitive strains of Erwinia amylovora fro m New Zealand and five states in the United States were compared on st reptomycin- and myomycin-amended media. Strains with high resistance t o streptomycin (HR strains) were insensitive to myomycin, an antibioti c resembling streptomycin in its mode of action, whereas strains with medium resistance (MR) or sensitivity (S) to streptomycin were sensiti ve to myomycin. No mutations were found in the 16S rRNA gene of nine H R strains of E. amylovora that could account for streptomycin resistan ce. Nucleotide sequence and allele-specific amplification-polymerase c hain reaction analyses were used to examine S, MR, and HR strains for mutations in the rpsL gene. All 102 HR strains of E. amylovora contain ed a single base-pair mutation in codon 43 of their rpsL gene that res ulted in an amino acid substitution in ribosomal protein S12. Codon 43 , which encodes lysine in S and MR strains, was converted to a codon f or arginine in 96 HR strains, a codon for asparagine in three HR strai ns, and a codon for threonine in three HR strains. In gene complementa tion studies, sensitivity to streptomycin and myomycin was restored to E. amylovora and Escherichia coli HR strains with a plasmid carrying the wild-type E. amylovora rpsL gene. Conversely, resistance to strept omycin and insensitivity to myomycin in E. amylovora and E. coli S str ains was restored only when the plasmid carried a mutant rpsL gene wit h the lysine-to-arginine substitution in codon 43, We conclude that mu tations in a single codon of ribosomal protein S12 gene rpsL have resu lted in high-level streptomycin resistance in E. amylovora. Two geneti c bases of streptomycin resistance have now been identified in E, amyl ovora. These results indicate that the genetic mechanism determines th e level of streptomycin resistance and the expression of myomycin resi stance and that the presence or relative importance of the two mechani sms differs among various geographic regions.