Shanhua Lin

An Inner Membrane Enzyme in Salmonellaand Escherichia coli That Transfers 4-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 lipid A is required for the maintenance of polymyxin resistance inEscherichia 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 twol-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on theS. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, oryfbI) 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 IVA, a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesizel-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-α-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L- arabinose, phosphoethanolamine and palmitate

Two-thirds of the lipid A in wild-typeEscherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4′. The remaining lipid A contains a monophosphate substituent at position 4′ and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998)J. Biol. Chem. 273, 12466–12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 °C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-l-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.

Oxygen Requirement for the Biosynthesis of theS-2-Hydroxymyristate Moiety in Salmonella typhimurium Lipid A FUNCTION OF LpxO, A NEW Fe2+/α-KETOGLUTARATE-DEPENDENT DIOXYGENASE HOMOLOGUE

Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium andPseudomonas aeruginosa, may contain secondaryS-2-hydroxyacyl chains. S. typhimurium has recently been shown to synthesize itsS-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O2. Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of 18O2confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/asparaginyl β-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhimuriumaspartyl/asparaginyl β-hydroxylase homologue (designatedlpxO) was cloned into pBluescriptSK and expressed inEscherichia coli K-12, which does not containlpxO. Analysis of the resulting construct revealed thatlpxO expression is sufficient to induce O2-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO very likely is a novel Fe2+/α-ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends. We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase.

A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy-D-manno-octulosonic acid (Kdo) kinase. Possible involvement of kdo phosphorylation in bacterial virulence.

The lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue derivatized with either a phosphate or an ethanolamine pyrophosphate moiety at the 4-OH position. In previous studies, we identified a kinase unique to H. influenzae extracts that phosphorylates Kdo-lipid IVA, a key precursor of lipopolysaccharide in this organism. We have now identified the gene encoding the Kdo kinase by using an expression cloning approach. A cosmid library containing random DNA fragments from H. influenzae strain Rd was constructed in Escherichia coli. Extracts of 472 colonies containing individual hybrid cosmids were assayed for Kdo kinase activity. A single hybrid cosmid directing expression of the kinase was found. The kinase gene was identified by activity assays, sub-cloning, and DNA sequencing. When the putative kinase gene was expressed inE. coli behind a T7 promoter, massive overproduction of kinase activity was achieved (∼8000-fold higher than in H. influenzae membranes). The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IVA as the substrate, were the same as observed withH. influenzae membranes. Unexpectedly, the kinase gene was identical to a previously characterized open reading frame (orfZ), which had been shown to be important for establishing bacteremia in an infant rat model (Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Mol. Microbiol.22, 951–965). However, based solely on the genome sequence of H. influenzae Rd, no biochemical function had been assigned to the product of orfZ, which we now designate kdkA(“Kdo kinase A”). Although Kdo phosphorylation may be critical for bacterial virulence of H. influenzae, it does not appear to be required for growth.

An Outer Membrane Enzyme That Generates the 2-Amino-2-deoxy-gluconate Moiety of Rhizobium leguminosarum Lipid A

The structures of Rhizobium leguminosarum and Rhizobium etli lipid A are distinct from those found in other Gram-negative bacteria. Whereas the more typical Escherichia coli lipid A is a hexa-acylated disaccharide of glucosamine that is phosphorylated at positions 1 and 4′, R. etli and R. leguminosarum lipid A consists of a mixture of structurally related species (designated A–E) that lack phosphate. A conserved distal unit, comprised of a diacylated glucosamine moiety with galacturonic acid residue at position 4′ and a secondary 27-hydroxyoctacosanoyl (27-OH-C28) as part of a 2′ acyloxyacyl moiety, is present in all five components. The proximal end is heterogeneous, differing in the number and lengths of acyl chains and in the identity of the sugar itself. A proximal glucosamine unit is present in B and C, but an unusual 2-amino-2-deoxy-gluconate moiety is found in D-1 and E. We now demonstrate that membranes ofR. leguminosarum and R. etli can convert B to D-1 in a reaction that requires added detergent and is inhibited by EDTA. Membranes of Sinorhizobium meliloti and E. coli lack this activity. Mass spectrometry demonstrates that B is oxidized in vitro to a substance that is 16 atomic mass units larger, consistent with the formation of D-1. The oxidation of the lipid A proximal unit is also demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the positive and negative modes using the model substrate, 1-dephospho-lipid IVA. With this material, an additional intermediate (or by product) is detected that is tentatively identified as a lactone derivative of 1-dephospho-lipid IVA. The enzyme, presumed to be an oxidase, is located exclusively in the outer membrane of R. leguminosarum as judged by sucrose gradient analysis. To our knowledge, an oxidase associated with the outer membranes of Gram-negative bacteria has not been reported previously.