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Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Peptidoglycan synthesis and degradation is a process in bacteria that involves multiple enzymes under strict temporal and spatial regulation. We used a set of small molecules that inhibit the transglycosylation step of peptidoglycan synthesis to discover genes that help to regulate this process. We identified a gene responsible for the susceptibility of Escherichia coli cells to killing by gtycolipid derivatives of vancomycin, thus establishing a genetic basis for activity differences between these compounds and vancomycin.
Vancomycin (Fig. IA) is the drug of last resort for treating resistant Gram-positive bacterial infections, and the emergence of vancomycin resistance presents a serious threat to public health. Vancomycin inhibits the maturation of the peptidoglycan layer surrounding bacterial cells by binding to D-AlaD-Ala, a dipeptide found in peptidoglycan precursors (Fig. 113) (1). Resistance to vancomycin arises when microorganisms acquire genes that lead to the substitution of D-AlaD-Ala by D-Ala-D-Lac (2), which vancomycin does not bind. Remarkably, vancomycin derivatives with a hydrophobic substituent on the carbohydrate moiety are active against vancomycin-resistant strains (3) even though they contain the same peptide binding pocket as vancomycin. The mechanism of action of these derivatives may be fundamentally different from that of vancomycin (4). Unlike vancomycin, they retain activity against both vancomycin-sensitive and vancomycin-resistant strains even when the peptide binding pocket is damaged (5). In vitro, they block a different step of peptidoglycan synthesis than does vancomycin (5). In addition, they kill bacteria very rapidly, whereas vancomycin only stops growth (6).
Because vancomycin and its derivatives affect cells differently (i.e., produce different phenotypes), it might be possible, using a chemical genetics approach, to identify genes involved in the cellular response to these compounds. The synthesis of peptidoglycan from its disaccharide precursor involves numerous enzymes with overlapping functions that are subject to tight temporal and spatial regulation (7). Most of the major enzymes in peptidoglycan synthesis-the transglycosy-- lases and transpeptidases-have been identified, but how these enzymes are regulated remains poorly understood. In developing an experimental approach to probe the cellular response to glycolipid derivatives of vancomycin, we focused on the following facts: Vancomycin blocks the transpeptidation step of peptidoglycan synthesis and kills cells slowly; glycolipid derivatives of vancomycin block...