Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

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. Dans le plasmide pUC8ksgA7 [1], la région de codage du gène ksgA est précédée par le promoteur lac (Plac) ainsi que par un petit cadre ouvert de lecture (ORF) de 15 codons composés de nucléotides derivés du gene lacZ, d'un site de clonage multiple et du géne ksgA lui-même. Le cadre de lecture commence par le codon d'initiation ATG de lacZ et se termine á quelques nucléotides au-delá du codon de départ ATG de ksgA. Les cellules transformées avec le pUC8ksgA7 produisent de la méthylase active, le produit du géne ksgA, bien qu'aucun signal Shine-Dalgarno (SD) ne précède le géne ksgA. L'introduction d'un codon d'arrêt TAA en phase dans le petit cadre de lecture ouvert abolit la production de méthylase dans les cellules transformées. Sur le plasmide pUC8ksgA5, contenant la région ksgA en entier [1], on a trouvé, par deux méthodes indépenda