An inner membrane enzyme in Salmonella and Escherichia coli that transfers4-amino-4-deoxy-L-arabinose to lipid A - Induction in polymyxin-resistant mutants and role of a novel lipid-linked donor

Attachment of the cationic sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N) to l ipid A is required for the maintenance of polymyxin resistance in Escherich ia coli and Salmonella typhimurium. The enzymes that synthesize L-Ara4N and transfer it to lipid A have not been identified. We now report an inner me mbrane enzyme, expressed in polymyxin-resistant mutants, that adds one or t wo L-Ara4N moieties to lipid A or its immediate precursors. No soluble fact ors are required. A gene located near minute 51 on the S. typhimurium and E . coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the L-Ar a4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid resid ues in S. typhimurium with 12 possible membrane-spanning regions. ArnT disp lays distant similarity to yeast protein mannosyltransferases. ArnT adds tw o L-Ara4N units to lipid A precursors containing a Kdo disaccharide. Howeve r, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single L-Ara4N residue to the 1-phosphate moiety of lipid IVA, a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are pres ent in genomes of other bacteria thought to synthesize L-Ara4N-modified lip id A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132-43144) , ArnT utilizes the novel lipid undecaprenyl phosphate-alpha -L-Ara4N as it s sugar donor, suggesting that L-Ara4N transfer to lipid A occurs on the pe riplasmic side of the inner membrane.