In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa

Bacterial peptidoglycan is the cell wall component responsible for maintain ing cell integrity against osmotic pressure. Biosynthesis of the cytoplasmi c precursor UDP-N-acetylmuramyl pentapeptide is catalyzed by the Mur enzyme s. Genomic analysis of the three regions encoding Mur proteins was achieved . We have cloned and over-expressed the murA, -B, -D, -E and -F genes of Ps eudomonas aeruginosa in pET expression system by adding a His-Tag to the C- termini of the proteins. Mur proteins were purified to homogeneity by a sin gle chromatographic step on affinity nickel columns. Protein identities wer e verified through N-terminal sequencing. Enzyme activity was proved by the identification of the pathway's final product. (C) 2001 Published by Elsev ier Science BN. on behalf of the Federation of European Microbiological Soc ieties.