Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of Escherichia coli

Three ribosomal RNA mutations conferring resistance to the antibiotic kasug amycin were isolated using a strain of Escherichia coli in which all of the rRNA is transcribed from a plasmid-encoded rrn operon. The mutations, A794 G, G926A, and A1519C, mapped to universally conserved sites in the 16 S RNA gene. Site-directed mutagenesis studies showed that virtually all mutation s at these three sites conferred kasugamycin resistance and had very slight effects on cell growth. It has been known for many years that the absence of post-transcriptional modification at A1519 and the adjacent A1518 in str ains lacking a functional KsgA methylase produces a kasugamycin resistance phenotype. Mutations at A1519 conferred kasugamycin resistance and had mino r effects on cell growth, whereas mutations at 1518 did not confer resistan ce and increased the doubling time of the cells dramatically. Expression of mutations at A1518/A1519 in a methylase deficient kcsgA(-) strain had dive rgent effects on the phenotype of the rRNA mutants, suggesting that the bas e identity at either position does not affect methylation at the adjacent s ite. Residues A794 and G926 are protected from chemical modification by kas ugamycin and tRNA, and have been implicated in the initiation of protein sy nthesis. Despite the universal conservation and functional importance of th ese residues, the results presented here show that the identity of the base s is not critical for ribosomal function. (C) 1999 Academic Press.