MUTANT SEQUENCES IN THE RPSL GENE OF ESCHERICHIA-COLI B/R - MECHANISTIC IMPLICATIONS FOR SPONTANEOUS AND ULTRAVIOLET-LIGHT MUTAGENESIS

Mutants able to grow in the presence of 1.2 mg/ml streptomycin were isolated from Escherichia coli WP2 after exposure to ultraviolet light (UV) or in the absence of any treatment (spontaneous), and from a umuC derivative after exposure to UV and delayed photoreversal. These mutants, characterized as streptomycin resistant (Sm(r)) or dependent (Sm(d)), carry mutations in the rpsL gene. This gene was amplified using the polymerase chain reaction and sequenced. Mutations induced by UV were largely (76%) of the Sm(r) phenotype, all of which were changes at an A:T base pair at codons 42 or 87. Mutations induced by UV plus delayed photoreversal in the non-UV-mutable umuC122 derivative of WP2 were exclusively of the Sm(d) phenotype and all occurred at G:C base pairs at codons 41, 90 or 91. These results are consistent with current understanding of the mechanism of mutagenesis by UV and delayed photoreversal. A broader spectrum of mutations was seen in the spontaneous series including three-base deletions leading to amino acid loss (2 of codon 93, 1 of codon 87). Of particular note was the number of intragenic second site mutations in the spontaneous series, most if not all of which appeared to be silent with respect to streptomycin phenotype. It is necessary to postulate a high rate of formation of such mutations at some stage during the experiment. One possibility is that spontaneous mutation may often occur in bursts when an error correction mechanism (eg., proofreading, mismatch correction) is temporarily inactive. In all, 12 different mutations conferring the ability to grow in the presence of streptomycin were identified, five Sm(r) and seven Sm(d), including two three-base deletions. The mutations fall into two small regions of the gene, one spanning codons 40-43, the other 87-93. Both Sm(r) and Sm(d) mutations were found in each region. The sequence of the wild-type rpsL gene of E. coli WP2 (a B/r strain) was identical to that of E. coli K12, in contrast to the situation with a number of other genes. The sequence conservation may be a consequence of codon usage bias in this highly expressed gene.