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Cytoplasmic-Membrane Anchoring of a Class A ß-Lactamase and Its Capacity in Manifesting Antibiotic Resistance

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556

Received 4 January 2007/ Returned for modification 5 March 2007/ Accepted 4 May 2007

Bacterial ß-lactamases are the major causes of resistance to ß-lactam antibiotics. Three classes of these enzymes are believed to have evolved from ancestral penicillin-binding proteins (PBPs), enzymes responsible for bacterial cell wall biosynthesis. Both ß-lactamases and PBPs are able to efficiently form acyl-enzyme species with ß-lactam antibiotics. In contrast to ß-lactamases, PBPs are unable to efficiently turn over antibiotics and therefore are susceptible to inhibition by ß-lactam compounds. Although both PBPs and gram-negative ß-lactamases operate in the periplasm, PBPs are anchored to the cytoplasmic membrane, but ß-lactamases are not. It is believed that ß-lactamases shed the membrane anchor in the course of evolution. The significance of this event remains unclear. In an attempt to demonstrate any potential influence of the membrane anchor on the overall biological consequences of ß-lactamases, we fused the TEM-1 ß-lactamase to the C-terminal membrane-anchor of penicillin-binding protein 5 (PBP5) of Escherichia coli. The enzyme was shown to express well in E. coli and was anchored to the cytoplasmic membrane. Expression of the anchored enzyme did not result in any changes in antibiotic resistance pattern of bacteria or growth rates. However, in the process of longer coincubation, the organism that harbored the plasmid for the anchored TEM-1 ß-lactamase lost out to the organism transformed by the plasmid for the nonanchored enzyme over a period of 8 days of continuous growth. The effect would appear to be selection of a variant that eliminates the problematic protein through elimination of the plasmid that encodes it and not structural or catalytic effects at the protein level. It is conceivable that an evolutionary outcome could be the shedding of the sequence for the membrane anchor or alternatively evolution of these enzymes from nonanchored progenitors.

Published ahead of print on 14 May 2007.