Molecular Characterization of a Plasmid-Borne (pTE31) Erythromycin Resistance Determinant (erm-31) from Lactobacillus reuteri AE72

Abstract

This study demonstrates that Lactobacillus reuteri AE72 is resistant to erythromycin (Em) with a minimum inhibitory concentration (MIC) of 516 μg/mL. Through a series of analyses including curing, electroporation, hybridization, and cloning experimentation, a 4.4 kb indigenous plasmid (pTE31) of L. reuteri AE72 was successfully identified as Em resistant (Em') A 1.2 kb Hindlll-Pst I DNA fragment containing the Em' determinant (erm-31) from pTE31 was also successfully cloned into E. coli TG1. Determining the nucleotide sequence of erm-31 revealed an open reading frame (ORF) for a putative 250-amino acid rRNA methylase gene (erm). This structural erm gene, 750 bp in length, was highly related (ca. 93% nucleotide and ca. 90% amino acid identity) to the 735 bp erm gene of the Tn917 of Streptococcus faecalis, A recombinant plasmid pUE3112 containing the erm-31 determinant from pTE31 was subjected to maxicell analysis to determine the activity of the putative erm -31 gene. The observed molecular mass of the synthesized protein, based on electrophoretic mobility, correlated with the 27.5 kDa protein predicted from the DNA sequence. To our knowledge, this work characterizes ermAM type of Em' determinant from L. reuteri to be to the nucleotide sequence level for the first time.