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

M Stephen Trent,Robert J Cotter,Shanhua Lin,Christian R.H Raetz,Anthony A Ribeiro

doi:10.1074/jbc.m106961200

Abstract

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia 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 membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors. No soluble factors 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-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid 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 its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

Highlights

Attachment of the cationic sugar 4-amino-4-deoxy-Larabinose (L-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium
Substituents that can be attached to lipid A in both Escherichia coli and Salmonella typhimurium include 4-amino-4-deoxy-Larabinose (L-Ara4N)1 [7,8,9,10,11], phosphoethanolamine [8, 9, 11], and palmitate [5, 9, 12] (Fig. 1)
Membranes of S. typhimurium CS022 [47], in which PhoP is constitutively active (PhoPC), converted a measurable portion of the [4Ј-32P]lipid IVA probe to a more hydrophilic product migrating like lipid IIA (Fig. 4, lane 3), a well characterized substance that accumulates in Kdo-deficient mutant of S. typhimurium [9, 46, 48]

Summary

The abbreviations used are

L-Ara4N, 4-amino-4-deoxy-L-arabinose; MES, 2-[N-morpholino]ethanesulfonic acid; Kdo, 3-deoxy-D-manno-octulosonic acid; PCR, polymerase chain reaction; pEtN, phosphoethanolamine; MALDI/TOF mass spectrometry, matrix assisted laser desorption-ionization/time of flight mass spectrometry. The enzyme adds a single L-Ara4N unit to the 1-phosphate moiety of the tetraacylated lipid A precursor, lipid IVA, which lacks Kdo (Fig. 3). Lipid A molecules containing a Kdo disaccharide are modified with two L-Ara4N units, indicating that the addition of L-Ara4N to the 4Ј-position is Kdo-dependent. Transferase activity is greatly elevated when the orf5(pmrK) gene of S. typhimurium [6, 24], renamed arnT (Fig. 2), is expressed behind a T7lac promoter in E. coli BLR(DE3), which is itself shown here to be a polymyxin-resistant strain containing L-Ara4N modified lipid A. The L-Ara4N transferase is not dependent upon added soluble factors. As shown in the following article [32], ArnT utilizes the novel lipid, undecaprenyl phosphate-␣-L-Ara4N, as its donor substrate (Fig. 2)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION