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<p>Abstract</p> <p>Background</p> <p>rRNA adenine dimethyltransferases, represented by the <it>Escherichia coli </it>KsgA protein, are highly conserved phylogenetically and are generally not essential for growth. They are responsible for the post-transcriptional transfer of two methyl groups to two universally conserved adenosines located near the 3'end of the small subunit rRNA and participate in ribosome maturation. All sequenced genomes of <it>Chlamydia </it>reveal a <it>ksgA </it>homolog in each species, including <it>C. trachomatis</it>. Yet absence of a S-adenosyl-methionine synthetase in <it>Chlamydia</it>, the conserved enzyme involved in the synthesis of the methyl donor S-adenosyl-L-methionine, raises a doubt concerning the activity of the KsgA homolog in these organisms.</p> <p>Results</p> <p>Lack of the dimethylated adenosines following <it>ksgA </it>inactivation confers resistance to kasugamycin (KSM) in <it>E. coli</it>. Expression of the <it>C. trachomatis </it>L2 KsgA ortholog restored KSM sensitivity to the <it>E. coli ksgA </it>mutant, suggesting that the chlamydial KsgA homolog has specific rRNA dimethylase activity. <it>C. trachomatis </it>growth was sensitive to KSM and we were able to isolate a KSM resistant mutant of <it>C. trachomatis </it>containing a frameshift mutation in <it>ksgA</it>, which led to the formation of a shorter protein with no activity. Growth of the <it>C. trachomatis ksgA </it>mutant was negatively affected in cell culture highlighting the importance of the methylase in the development of these obligate intracellular and as yet genetically intractable pathogens.</p> <p>Conclusion</p> <p>The presence of a functional rRNA dimethylase enzyme belonging to the KsgA family in <it>Chlamydia </it>presents an excellent chemotherapeutic target with real potential. It also confirms the existence of S-adenosyl-methionine - dependent methylation reactions in <it>Chlamydia </it>raising the question of how these organisms acquire this cofactor.</p>