BIOSYNTHESIS OF SALMONELLA-LIPOPOLYSACCHARIDE - IN-VITRO TRANSFER OF PHOSPHATE TO HEPTOSE MOIETY OF CORE

An enzyme involved in the biosynthesis of the core part of Salmonella lipopolysaccharide, an ATP‐dependant lipopolysaccharide phosphorylating enzyme, has been studied in an in vitro system. The system requires as acceptor the heated 20 000 ×g cell wall fraction from Salmonella minnesota mR5, a mutant which lacks the phosphotransferase (P–), and as enzyme source 1000 00 ×g supernatant of protein extracts from rough P+ mutants of either S. minnesota or S. typhimurium. Phosphate donor is ATP labelled with 32P in the γ‐position. The enzyme system requires a high concentration of Mg++ ions (0.1 M) for optimal functioning. The phosphotransferase is solubilised by repeated washings of cell debris obtained after lysis of the cells by EDTA and lysozyme with dilute Tris buffer. Isolation and characterisation of two fractions consisting of heptose phosphate from the lipopolysaccharide of S. minnesota mRz, a UDP‐synthetase‐less P+ mutant, is described. Products of partial hydrolysis of acceptor after 32P‐incorporation were compared with these fractions and found to be identical. In this way the incorporation product was identified to be core‐lipopolysaccharide (glycolipid) which is phosphorylated at the heptose moiety. Copyright © 1969, Wiley Blackwell. All rights reserved