Resistance to
spectinomycin emerged after widely used for treatment of
gonorrhea. Previous studies revealed that Lysobacter enzymogenes strain C3 (LeC3) exhibited elevated level of intrinsic resistance to
spectinomycin. In this study, we screened a Tn5 transposon mutant library of LeC3 to elucidate the underlying molecular mechanisms of
spectinomycin resistance. Insertion sites in 15 out of 19 mutants recovered with decreased
spectinomycin resistance were located on two
ribosomal RNA operons at different loci, indicating the pivotal role of ribosomal RNAs in conferring
spectinomycin resistance in L. enzymogenes. The other mutants harbored mutations in the tuf, rpoD, mltB, and purB genes. Among them, the tuf and rpoD genes, respectively, encode a translation
elongation factor Tu and an
RNA polymerase primary
sigma factor. They both contribute to protein biosynthesis, where ribosomal RNAs play essential roles. The mltB gene, whose product is involved in cell-wall recycling, was not only associated with resistance against
spectinomycin, but also conferred resistance to osmotic stress and
ampicillin. In addition, mutation of the purB gene, for which its product is involved in the biosynthesis of
inosine and
adenosine monophosphates, led to decreased
spectinomycin resistance. Addition of exogenous
adenine at lower concentration in medium restored the growth deficiency in the purB mutant and increased bacterial resistance to
spectinomycin. These results suggest that while cell-wall recycling and
purine biosynthesis might contribute to
spectinomycin resistance, target rRNAs play critical role in
spectinomycin resistance in L. enzymogenes.