Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

Abstract

In the plasmid pUC8ksgA7 [1], the codind region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region [1], the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1 - Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 [2] stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 [3] makes β-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl - Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene. No evidence was found for a feedback regulation of its own synthesis by the ksgA gene product.