Isolation, Expression, and Characterization of a β–lactamase produced by Meiothermus ruber

Abstract

β‐lactamase enzymes are the most significant contributor to antibiotic resistance in bacteria. The pharmaceutical industry's response to antibiotic resistance has been to develop new antibiotics or β–lactamase inhibitors; these successes in the clinical setting require an understanding of the structure and function of the β–lactamases. A β–lactamase gene was identified in Meiothermus ruber, a thermophilic gram‐negative bacterium, with a bright red carotenoid pigment. M. ruber genomic DNA was utilized to clone the β–lactamase gene into the plasmid pKT1. The pKT1 plasmid contains a β–lactamase selection gene, which could potentially obfuscate kinetic assay data. Therefore, the β–lactamase gene from M. ruber was then amplified by PCR, inserted into pET‐26b, a protein‐expression vector containing a Kanamycin resistance selection gene, and transformed into E. coli. Restriction digestion and sequencing were performed on the successful transformants to verify the presence and correct orientation of the M.ruber β–lactamase gene. Activity and substrate preference of the gene product was determined by growing the bacteria in the presence of various β–lactam antibiotics. Preliminary tests suggest the M.ruber β–lactamase gene product can hydrolyze cephalosporin β–lactam antibiotics but not carbapenems. This result is supported by sequence comparison studies that suggest the gene product is a class C (AmpC‐type) β–lactamase. Further experiments are required to understand the structure and function of the β–lactamase from M.ruber.Support or Funding InformationThis work was funded by Augustana College Augustana College Summer Undergraduate Research Funds and the Department of Energy Joint Genome Institute's GEBA project.