Genes for gentamicin-(3)-N-acetyl-transferases III and IV

Summary

The direction of transcription, exact location of the decoding region and nucleotide sequences of the aacC3 genes cloned from R-plasmids pWP14a, pWP116a, and pWP113a were determined. The respective fragments could code for a protein of 30.5 kd molecular weight. which was in agreement with the size of the polypeptide expressed by these plasmids in minicells of Escherichia coli. The aacC3 genes, including the promoters, were completely identical in pWP14a and pWP116a. In contrast, in the respective homologous segment in pWP113a were 3.3% of the nucleotides different, leading to ten exchanges in the proposed amino acid sequence for the AAC(3)-III enzyme. Comparison of the aacC3 sequence with another functionally related amoniglycoside resistance determinant, aacC4 (Bräu et al. 1984), revealed only a distant relationship. A hypothetical genealogy of the aacC genes is proposed.

In pWP113a no good correlation with the consensus sequence for—35 boxes of E. coli promoters was found However, in pWP14a and pWP116a, also expressing higher gentamicin resistance, a-35 sequence (5′TTGCAA3′) was complemented by an IS140 element inserted at exactly the same position in both cases. The element bears palindromic —35 boxes at both ends, inside its inverted repeats. In pWP116a, IS140 was inverted relative to its orientation in pWP14a and most of it, together with part of a structure related to Tn3, was deleted during a process leading to fusion of the two transposable elements.

Downstream from the aacC3 genes, an open reading frame seperated by a possible intercistronic region of 12 bp and preceded by a translational initiation site exists in both pWP113a and pWP14a.

There is a discussion of the possible preference of the IS140 element for nucleotide sequences similar to—35 boxes of E. coli promoters as target sites for transposition.