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Antimicrobial Agents and Chemotherapy, January 2009, p. 193-201, Vol. 53, No. 1
0066-4804/09/$08.00+0 doi:10.1128/AAC.00873-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Inactivation of KsgA, a 16S rRNA Methyltransferase, Causes Vigorous Emergence of Mutants with High-Level Kasugamycin Resistance 
Kozo Ochi,1*
Ji-Yun Kim,1,2
Yukinori Tanaka,1,3
Guojun Wang,1
Kenta Masuda,4
Hideaki Nanamiya,4
Susumu Okamoto,1
Shinji Tokuyama,3
Yoshikazu Adachi,2 and
Fujio Kawamura4
National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan,1 Laboratory of Animal Health, School of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan,2 Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka 422-8529, Japan,3 Laboratory of Molecular Genetics and Research Information Center for Extremophiles, College of Science, Rikkyo University, Tokyo 171-8501, Japan4
Received 2 July 2008/ Returned for modification 2 September 2008/ Accepted 31 October 2008
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 (Tspr and Rifr), 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.
* Corresponding author. Mailing address: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Phone: 81-29-838-8125. Fax: 81-29-838-7996. E-mail: kochi{at}affrc.go.jp
Published ahead of print on 10 November 2008.
Antimicrobial Agents and Chemotherapy, January 2009, p. 193-201, Vol. 53, No. 1
0066-4804/09/$08.00+0 doi:10.1128/AAC.00873-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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