Isolation and characterization of mutants with impaired regulation of rpsA, the gene encoding ribosomal protein S1 of Escherichia coli

Abstract

In order to select mutants that would help to characterize the post-transcriptional regulation of rpsA, we constructed a strain in which the growth rate on lactose minimal medium is determined by the amount of an rpsA-lacZ′ α-fragment fusion protein produced, even when this is encoded by a high-copy-number plasmid. In the parental strain, synthesis of the fusion protein is repressed by a wild-type rpsA gene, present on a compatible plasmid. Twenty-eight spontaneous and independent mutants, all of them mapping in the rpsA leader region, were isolated as strains that showed higher growth rates, on lactose medium, due to increased synthesis of the rpsA lacZ′ fusion protein. Among these mutants only three sequence changes were found, mapping 9, 10 and 27 bases upstream of the rpsA start codon. At both the −9 and −10 positions an A to G transition and at −27 a C to G transversion all resulted in a sequence with better complementarity to the 3′ end of 16S rRNA. We also isolated two mutations mapping in the plasmid-encoded rpsA structural gene: an ochre nonsense mutation in codon 15 of the rpsA gene and a frameshift mutation, deleting the T residue at position + 1186. To facilitate the in vitro assay of α-fragment activity we also constructed a strain that overproduces the α-acceptor fragment four-fold relative to a strain that is diploid for this lacZΔM15 allele.