Inactivation of KsgA, a 16S rRNA Methyltransferase, Causes Vigorous Emergence of Mutants with High-Level Kasugamycin Resistance

Abstract

The methyltransferases RsmG and KsgA methylate the nucleotides G535 (RsmG) and A1518 and A1519 (KsgA) in 16S rRNA, and inactivation of the proteins by introducing mutations results in acquisition of low-level resistance to streptomycin and kasugamycin, respectively. In a Bacillus subtilis strain harboring a single rrn operon (rrnO), we found that spontaneous ksgA mutations conferring a modest level of resistance to kasugamycin occur at a high frequency of 10 ؊6 . More importantly, we also found that once cells acquire the ksgA mutations, they produce high-level kasugamycin resistance at an extraordinarily high frequency (100-fold greater frequency than that observed in the ksgA ؉ strain), a phenomenon previously reported for rsmG mutants. This was not the case for other antibiotic resistance mutations (Tsp r and Rif r ), indicating that the high frequency of emergence of a mutation for high-level kasugamycin resistance in the genetic background of ksgA is not due simply to increased persistence of the ksgA strain. Comparative genome sequencing showed that a mutation in the speD gene encoding S-adenosylmethionine decarboxylase is responsible for the observed high-level kasugamycin resistance. ksgA speD double mutants showed a markedly reduced level of intracellular spermidine, underlying the mechanism of high-level resistance. A growth competition assay indicated that, unlike rsmG mutation, the ksgA mutation is disadvantageous for overall growth fitness. This study clarified the similarities and differences between ksgA mutation and rsmG mutation, both of which share a common characteristic-failure to methylate the bases of 16S rRNA. Coexistence of the ksgA mutation and the rsmG mutation allowed cell viability. We propose that the ksgA mutation, together with the rsmG mutation, may provide a novel clue to uncover a still-unknown mechanism of mutation and ribosomal function.

The Novel Kasugamycin 2′- N -Acetyltransferase Gene aac(2′)-IIa , Carried by the IncP Island, Confers Kasugamycin Resistance to Rice-Pathogenic Bacteria

“…Previous in vitro studies showed that spontaneous KSM-resistant mutants of E. amylovora, E. coli, and Bacillus subtilis harbored mutations in the ksgA methyltransferase gene (29,33). However, our results clarified that a nucleotide substitution of the ksgA gene is not involved in KSM resistance in field isolates of B. glumae.…”

“…Bacterial resistance to KSM by inactivation of the dimethyltransferase gene (ksgA) is the most frequent mechanism of spontaneous mutation in vitro (29,33). We detected nucleotide variations, but not changes in amino acids, in ksgA gene sequences between KSM-susceptible B. glumae strains 210 and 3 (see Fig.…”

Identification and Characterization of a Novel Multidrug Resistance Operon,mdtRP(yusOP), ofBacillus subtilis

“…However, the importance of studying low-level resistance to antibiotics has increased in both medical and industrial microbiology. For example, we recently found that low-level resistance to streptomycin was due to a mutation in the rsmG gene, which encodes a 16S rRNA methyltransferase, and that low-level resistance to kasugamycin was due to a mutation in the speD gene, which encodes S-adenosylmethionine decarboxylase (25,26,29). These findings provided clues to study the mechanism of high-frequency appearance of highlevel streptomycin resistance in Mycobacterium tuberculosis (30) and the overproduction of antibiotics by Streptomyces coelicolor (25).…”

Regulation of expression and catalytic activity of Escherichia coli RsmG methyltransferase

“…Loss of RsmG activity confers low-level streptomycin resistance and inexplicably prompts the appearance of high-level streptomycin resistance mutations in the S12-encoding gene, rpsL (Nishimura et al 2007a,b;Okamoto et al 2007;Gregory et al 2009;Wong et al 2011). Moreover, the effects of rsmG mutations on bacterial fitness and translational accuracy have been reported as being variable, depending on the bacterial species (Nishimura et al 2007b;Okamoto et al 2007;Gregory et al 2009;Ochi et al 2009). We recently found that a null rsmG mutation produces an error-prone phenotype in certain genetic backgrounds, which suggests that modification m 7 G527 is important for ribosomal accuracy and that pleitropy may explain, at least partially, the observed variability (A Benítez-Páez, M Villarroya, and M-E Armengod, in prep.…”