Antibiotic resistance is a significant threat to infectious disease control. β-lactamases represent some of the most commonly found enzymes that confer antibiotic resistance. β-lactamases are enzymes that confer resistance to β-lactam antibiotics such as ceftazidime. Studying how these enzymes confer resistance to β-lactam antibiotics is a key factor in fighting the battle against resistance. A novel antibiotic resistance gene, cft2, from a commercial apple orchard encodes an unusual β-lactamase that provides resistance to the cephalosporin antibiotic ceftazidime. Cft2 encodes a 607 amino acid residue class A β-lactamase that is nearly 400 residues longer than typical β-lactamases. The goal of this project is to characterize the novel aspects of the cft2 enzyme. This project focuses on one specific feature mainly a putative transmembrane helix found in cft2. HMMTOP software was used to analyze the Cft2 and identified a transmembrane helix in the Cft2 enzyme at amino acid residues 290-307. The Cft2 enzyme was shown to be in the insoluble fraction of cell lysate from western blot analysis, suggesting this enzyme is embedded in the membrane. That membrane localization is predicted to be critical for function and fits with the bioinformatics prediction. Here, we conducted site-directed mutagenesis to remove the suspected transmembrane region, amino acid residues 211-343. Once expressed in Escherichia coli BL21 the mutated gene was assayed on media supplemented with ceftazidime (2.5 mg/mL) against the non-mutated gene. The deletion mutant lacking the transmembrane helix failed to grow on media supplemented with ceftazidime, whereas the wild type grew. Resistance profile coupled with the bioinformatics analysis suggest the transmembrane domain is essential for conferring resistance to ceftazidime.

Characterization of the transmembrane region of a novel ceftazidime resistance gene

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.