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

• Published in: Antimicrobial agents and chemotherapy Vol. 42; no. 12; pp. 3173 - 3178
• Main Authors: VAN BOXTEL, R. A. J, VAN DE KLUNDERT, J. A. M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.12.1998
• Subjects: Antibacterial agents; Antibiotics. Antiinfectious agents. Antiparasitic agents; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Biological and medical sciences; Blotting, Northern; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Drug Resistance, Microbial; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Gene Expression Regulation, Bacterial - drug effects; Genes, Bacterial; Gentamicins - pharmacology; Mechanisms of Resistance; Medical sciences; Pharmacology. Drug treatments; Pseudomonas aeruginosa; Pseudomonas aeruginosa - genetics; Reverse Transcriptase Polymerase Chain Reaction; RNA, Bacterial - genetics; RNA, Bacterial - isolation & purification; Terminator Regions, Genetic; Pseudomonadales; Molecular hybridization; Escherichia coli; Gentamicin; Reverse transcription; Gene expression; In vitro; Northern blotting; Polymerase chain reaction; Resistance; Antibiotic; Messenger RNA; Aminoglycoside; Bacteria; Pseudomonadaceae; Pseudomonas aeruginosa; Antibacterial agent; Enterobacteriaceae
• ISSN: 0066-4804; 1098-6596
• DOI: 10.1128/aac.42.12.3173

The Pseudomonas aeruginosa aacC3 gene was expressed in Escherichia coli after cloning of the single gene behind the strong tac promoter. In the original Pseudomonas strain, aacC3 is preceded by cysC; together they form a single transcription unit. The ribosome-binding site and start codon of aacC3 are involved in a putative intercistronic hairpin, the stability of which interfered with the aminoglycoside resistance level. In Northern blots, full-length transcripts comprising both cysC and aacC3 could not be detected either in the original Pseudomonas strain or in E. coli harboring a plasmid with the cloned operon. In contrast, cysC transcripts were abundant. Cloning of the operon between the tac promoter and a transcription termination signal 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 undetected 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.