Intramembranal events in the biosynthesis of peptidoglycan in Gaffkya homari.

In order to monitor the intermediates involved in nascent peptidoglycan (PG) assembly in Gaffkya homari, a pulse/chase assay utilizing UDP-MurNAc-Ala-DGlu-Lys(N epsilon-Dns)-DAla-DAla [Dns (dansyl) = 5-(dimethylamino)naphthalene-1-sulfonyl] was devised. The perturbation introduced by the dansyl group provided a means for separating the synthesis of nascent PG into discrete stages. Together with paramagnetic quenching of the fluorophore by n-doxylstearic acids (n = 5, 7, 12, 16; doxyl = N-oxy-4',4'-dimethyloxazolidine), this assay allows one to observe the synthesis of undecaprenyl diphosphate-MurNAc-[N epsilon-Dns)pentapeptide)-GlcNAc and its utilization for the formation of dansyl-labeled PG by fluorescence emission and by change in specific positional quenching. The utilization of the dansylated lipid disaccharide-pentapeptide occurs without a lag, whereas the formation of the chromatographically immobile dansylated PG occurs with a lag of 4-6 min. Membrane-associated undecaprenyl diphosphate-MurNAc-(N epsilon-Dns)-pentapeptide was quenched primarily by 7-doxylstearate. In contrast, the fluorophore of the undecaprenyl diphosphate-MurNAc-[N epsilon-Dns)pentapeptide)-GlcNAc was quenched primarily by 5-doxyl- and 16-doxylstearates. In the chase phase of the assay, quenching by 16-doxylstearate decreased at a faster rate than that by 5-doxylstearate during the formation of dansyl-labeled PG.