Biosynthesis of the Core Part of the Lipopolysaccharide of Pseudomonas aeruginosa

Abstract

The incorporation of rhamnose and glucose into the core part of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa was studied using enzyme preparations from strain PAC1 R and LPS-defective mutants derived from it. Crude membrane preparations from the LPS-defective mutant PAC556 transferred rhamnose from dTDP-L-[14C]rhamnose to material insoluble in trichloroacetic acid. The preparations contained both transferase enzyme and acceptor, the former being destroyed by heating. Between 60 and 70% of the radioactive rhamnose transferred to the membranes was extractable by aqueous phenol and non-diffusible. The material extracted did not move in any of the chromatography solvents tested and contained rhamnose as the sole radioactive component. Soluble dTDP-L-rhamnose-LPS rhamnosyltransferase was obtained from the parent strain PAC 1 R by ammonium sulphate precipitation of a 105000 g supernatant fraction from broken bacteria. It was most active at pH 8 with 5 mM-MgC1, and required heat-treated membranes of PAC556 as acceptor. This mutant, whose LPS lacks both 0-antigenic side-chains and rhamnose in the core, was shown to lack either the epimerase or the NADP-dependent oxidoreductase used to synthesize dTDPrhamnose. After preincubation with soluble transferase and UDPglucose, heated membranes of mutant strains PAC6 I 1, PAC6 12 and PAC605 could also act as acceptors for rhamnose. These mutants all lacked some or all of the glucose as well as the rhamnose from the core of their LPS and the experiments thus provided confirmation that rhamnose was the terminal hexose of the core in P. aeruginosa PAC1.

Structure of the Core Oligosaccharide from the Lipopolysaccharide of Pseudomonas aeruginosa PAC1R and Its Defective Mutants

“…Indirect evidence for the sequence and role of the core structure came from our work with mutants of P. aeruginosa PAClR which lacked the 0-antigenic side chains and parts of the core itself [13] and from studies of the pyocin and bacteriophage-binding sites in these mutants [2]. We proposed a core structure for PAC1 R lipopolysaccharide consistent with these studies and with some biosynthestic work on the incorporation of rhamnose and glucose into the core [14]. The present work was undertaken to determine chemically the structure of the core part of the lipopolysaccharide of strain PAClR and some defective mutants derived from it.…”

“…The structures of their L2 fractions suggest that each has lost a specific enzyme for core biosynthesis and give some indication of the sequence involved. Direct evidence of biosynthetic sequence has been obtained for PAC611 and PAC605 whose incomplete lipopolysaccharides require the transfer of glucose from UDPglucose before they can act as acceptors for rhamnose from TDPrhamnose [14]. The structure now proposed suggests that the two glucose units of isomaltose are added sequentially by different enzymes, the first being defective in PAC605 and the second in PAC61 1.…”

“…The only one in which a genetic defect has been identified is PAC556 which cannot synthesize TDPrhamnose [14]. This 0 -a n t i g e n A l a n i n e Fig.4.…”

“…The L2 fraction from mutant PAC609, which otherwise resembled the parent strain in producing 0-antigenic side chains and an L1 fraction, lacked one of the glucose units in the L2 core fraction. The L2 fraction from PAC556, which is defective in TDPrhamnose biosynthesis [14], lacked this sugar but retained three glucose molecules. PAC61 1 contained glucose and heptose in equimolar proportions, while the most defective mutant PAC605 contained heptose as its sole neutral sugar.…”

Receptor Sites for R-type Pyocins and Bacteriophage E79 in the Core Part of the Lipopolysaccharide of Pseudomonas aeruginosa PAC1

“…1). The only mutant whose defect has so far been defined is ~~~5 5 6 which cannot synthesize TDPrhamnose and whose LPS has a vacant acceptor site for rhamnose (Asonganyi & Meadow, 1978). The behaviour of this mutant shows the necessity for rhamnose in R1 sensitivity.…”