Expression of the Pseudomonas aeruginosa gentamicin resistance gene aacC3 in Escherichia coli

The Pseudomonas aeruginosa aacC3 gene was expressed in Escherichia coli aft er cloning of the single gene behind the strong tac promoter. In the origin al Pseudomonas strain, aacC3 is preceded by cysC; together they form a sing le transcription unit. The ribosome-binding site and start codon of aacC3 a re involved in a putative intercistronic hairpin, the stability of which in terfered with the aminoglycoside resistance level. In Northern blots, full- length transcripts comprising both cysC and aacC3 could not be detected eit her in the original Pseudomonas strain or in E. call harboring a plasmid wi th the cloned operon. In contrast, cysC transcripts mere abundant. Cloning of the operon between the tac promoter and a transcription termination sign al resulted in higher mRNA levels and phenotypic expression in E. coli. The absence of a transcription termination signal in the wild-type cysC-aacC3 sequence is associated with transcripts of heterogeneous size that were und etected in Northern blots. Our results shed more light on the expression of this gentamicin resistance determinant, although the discrepancies between its expression in E, coli and Pseudomonas are not fully solved.