title: Mechanisms of hyperproduction of TEM-1 beta-lactamase by clinical isolates of Escherichia coli.
abstract: The mechanisms of hyperproduction (defined as > or = 200 nmoles nitrocefin hydrolysed per minute per mg of protein) of TEM-1 beta-lactamase by 38 isolates of Escherichia coli were investigated. The copy numbers of TEM-encoding plasmids were determined for the hyperproducing isolates and for 39 TEM-1-producing isolates that did not hyperproduce the enzyme. Allele-specific PCR was used to determine if the promoter region of the TEM-1 gene was of the TEM-1 or TEM-2 type. Twenty three of the 38 hyperproducers had the TEM-1-type promoter but 15 had the more efficient TEM-2-type promoter; in contrast, 37 of the 39 isolates with lower activities had the TEM-1-type promoter and only two had the TEM-2-type promoter. Many of the TEM-1-hyperproducing isolates possessed small plasmids (< or = 20 MDal) with high copy numbers, in some cases together with large, low copy number plasmids; the average total copy number of TEM-encoding plasmids was 14; if only isolates with the TEM-1-type promoter were included, average total copy number was 22. Hyperproduction was attributed to high copy number (> or = 10) plasmids in 11 isolates; another seven had plasmids with moderately high copy numbers (4-9). The average total copy number for isolates that produced relatively small amounts of TEM-1 beta-lactamase (< or = 100 nmoles/min/mg protein) was 2.2, and for the 12 isolates with TEM-1 activities of 101-200 nmoles/min/mg protein it was 6.8. We conclude that both high copy number plasmids and a more efficient promoter are common causes of hyperproduction of TEM-1.
authors.affiliation: Department of Microbiology, UMDS, St. Thomas's Campus, London, UK.
authors.forename: P J
authors.initials: PJ
authors.lastname: Wu
authors.forename: K
authors.initials: K
authors.lastname: Shannon
authors.forename: I
authors.initials: I
authors.lastname: Phillips
mesh_terms: D000483:Alleles; D001426:Bacterial Proteins / Q000096:biosynthesis*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1