Resistance to the antimicrobial peptide polymyxin requires myristoylation of Escherichia coli and Salmonella typhimurium lipid A

Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrA(C) strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3'-acyloxyacyl-linked myristate (C(14)) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group.     

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

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.     

High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium

When Escherichia coli are grown on LB broth containing 25 mm NH(4)VO(3), complex modifications to the lipid A anchor of lipopolysaccharide are induced. Six modified lipid As (EV1-EV6) have been purified. Many of these variants possess 4-amino-4-deoxy-l-arabinose (l-Ara4N) and/or phosphoethanolamine (pEtN) substituents. Here we use NMR spectroscopy to investigate the attachment sites of the l-Ara4N and pEtN moieties on underivatized, intact EV3 and EV6 and on precursors II(A) and III(A) from kdsA mutants of Salmonella. CDCl(3)/CD(3)OD/D(2)O (2:3:1, v/v) is shown to be a superior solvent for homo- and heteronuclear one- and two-dimensional NMR experiments. The latter were not feasible previously because available solvents caused sample decomposition. Selective inverse decoupling difference spectroscopy is used to determine the attachment sites of substituents on EV3, EV6, II(A), and III(A). l-Ara4N is attached via a phosphodiester linkage to the 4'-phosphates of EV3 and EV6 and has the beta anomeric configuration. pEtN is attached by a pyrophosphate linkage to the 1-phosphate of EV6. The l-Ara4N and pEtN substituents of lipids II(A) and III(A) are attached in the opposite manner, with l-Ara4N on the 1-phosphate of II(A) and pEtN on the 4'-phosphate of III(A). Determination of the proper attachment sites of these substituents is necessary for elucidating the enzymology of lipid A biosynthesis and for characterizing polymyxin-resistant mutants, in which l-Ara4N and pEtN substituents are greatly increased.     

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.     

Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-L-arabinose

In Escherichia coli and Salmonella typhimurium, addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate group(s) of lipid A is required for resistance to polymyxin and cationic antimicrobial peptides. We have proposed previously (Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. H. (2002) J. Biol. Chem. 277, 2886-2896) a pathway for l-Ara4N biosynthesis that begins with the ArnA-catalyzed C-4" oxidation and C-6" decarboxylation of UDP-glucuronic acid, followed by the C-4" transamination of the product to generate the novel sugar nucleotide UDP-l-Ara4N. We now show that ArnB (PmrH) encodes the relevant aminotransferase. ArnB was overexpressed using a T7lac promoter-driven construct and shown to catalyze the reversible transfer of the amino group from glutamate to the acceptor, uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate), the intermediate that is synthesized by ArnA from UDP-glucuronic acid. A 1.7-mg sample of the putative UDP-l-Ara4N product generated in vitro was purified by ion exchange chromatography, and its structure was confirmed by 1H and 13C NMR spectroscopy. ArnB, which is a cytoplasmic protein, was purified to homogeneity from an overproducing strain of E. coli and shown to contain a pyridoxal phosphate cofactor, as judged by ultraviolet/visible spectrophotometry. The pyridoxal phosphate was converted to the pyridoxamine form in the presence of excess glutamate. A simple quantitative radiochemical assay was developed for ArnB, which can be used to assay the enzyme either in the forward or the reverse direction. The enzyme is highly selective for glutamate as the amine donor, but the equilibrium constant in the direction of UDP-l-Ara4N formation is unfavorable (approximately 0.1). ArnB is a member of a very large family of aminotransferases, but closely related ArnB orthologs are present only in those bacteria capable of synthesizing lipid A species modified with the l-Ara4N moiety.     

Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli. Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose.

Addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate groups of lipid A is implicated in bacterial resistance to polymyxin and cationic antimicrobial peptides of the innate immune system. The sequences of the products of the Salmonella typhimurium pmrE and pmrF loci, both of which are required for polymyxin resistance, recently led us to propose a pathway for l-Ara4N biosynthesis from UDP-glucuronic acid (Zhou, Z., Lin, S., Cotter, R. J., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 18503-18514). We now report that extracts of a polymyxin-resistant mutant of Escherichia coli catalyze the C-4" oxidation and C-6" decarboxylation of [alpha-(32)P]UDP-glucuronic acid, followed by transamination to generate [alpha-(32)P]UDP-l-Ara4N, when NAD and glutamate are added as co-substrates. In addition, the [alpha-(32)P]UDP-l-Ara4N is formylated when N-10-formyltetrahydrofolate is included. These activities are consistent with the proposed functions of two of the gene products (PmrI and PmrH) of the pmrF operon. PmrI (renamed ArnA) was overexpressed using a T7 construct, and shown by itself to catalyze the unprecedented oxidative decarboxylation of UDP-glucuronic acid to form uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate). A 6-mg sample of the latter was purified, and its structure was validated by NMR studies as the hydrate of the 4" ketone. ArnA resembles UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase in oxidizing the C-4" position of its substrate, but differs in that it releases the NADH product.     

Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.     

Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine

Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-d-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl(2) in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mm CaCl(2), is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl(2) is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mm CaCl(2) contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-(32)P]lipid IV(A) as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mm MgCl(2), BaCl(2), or ZnCl(2). Hydrolysis of the in vitro reaction product, pEtN-Kdo(2)-[4'-(32)P]lipid IV(A), at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mm CaCl(2), which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mm CaCl(2) suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS.     

Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence.

We isolated spontaneous mutations (pmrA) in the smooth strain Salmonella typhimurium LT2 that show increased resistance to the cationic antibacterial proteins of human neutrophils and to the drug polymyxin B. The mutation in one strain, JKS5, maps to 93 min on the S. typhimurium chromosome, near the proP gene and the melAB operon. The mutation, designated pmrA505, confers a 1,000-fold increase in resistance to polymyxin B and a 2- to 4-fold increase in resistance to neutrophil proteins. We cloned both the pmrA505 and pmrA+ alleles and found that the pmrA+ gene is partially dominant over pmrA505. DNA sequence analysis of the pmrA505 clone revealed three open reading frames (ORFs). The deduced amino acid sequences indicated that ORF1 encodes a 548-amino-acid (aa) protein with a putative membrane-spanning domain and no significant homology to any known protein. ORF2 and ORF3, which encode 222- and 356-aa proteins, respectively, show strong homology with the OmpR-EnvZ family of two-component regulatory systems. ORF2 showed homology with a number of response regulators, including OmpR and PhoP, while ORF3 showed homology to histidine kinase-sensor proteins EnvZ and PhoR. Genetic analysis of the cloned genes suggested that ORF2 contained the pmrA505 mutation. Comparison of the pmrA505 and pmrA+ ORF2 DNA sequences revealed a single G-A transition, which would result in a His-to-Arg substitution at position 81 in the ORF2 mutant protein. We therefore designate ORF2 PmrA and ORF3 PmrB. The function of ORF1 is unknown.     

A phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant

Addition of a phosphoethanolamine (pEtN) moiety to the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue of lipopolysaccharide (LPS) in WBB06, a heptose-deficient Escherichia coli mutant, occurs when cells are grown in 5-50 mM CaCl2 (Kanipes, M. I., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 1156-1163). A Ca2+-induced, membrane-bound enzyme was responsible for the transfer of the pEtN unit to the Kdo domain. We now report the identification of the gene encoding the pEtN transferase. E. coli yhjW was cloned and overexpressed, because it is homologous to a putative pEtN transferase implicated in the modification of the beta-chain heptose residue of Neisseria meningitidis lipo-oligosaccharide (Mackinnon, F. G., Cox, A. D., Plested, J. S., Tang, C. M., Makepeace, K., Coull, P. A., Wright, J. C., Chalmers, R., Hood, D. W., Richards, J. C., and Moxon, E. R. (2002) Mol. Microbiol. 43, 931-943). In vitro assays with Kdo2-4'-[32P]lipid A as the acceptor showed that YhjW (renamed EptB) utilizes phosphatidylethanolamine in the presence of Ca2+ to transfer the pEtN group. Stoichiometric amounts of diacylglycerol were generated during the EptB-catalyzed transfer of pEtN to Kdo2-lipid A. EptB is an inner membrane protein of 574 amino acid residues with five predicted trans-membrane segments within its N-terminal region. An in-frame replacement of eptB with a kanamycin resistance cassette rendered E. coli WBB06 (but not wild-type W3110) hypersensitive to CaCl2 at 5 mM or higher. Ca2+ hypersensitivity was suppressed by excess Mg2+ in the medium or by restoring the LPS core of WBB06. The latter was achieved by reintroducing the waaC and waaF genes, which encode LPS heptosyl transferases I and II, respectively. Our data demonstrate that pEtN modification of the outer Kdo protected cells containing heptose-deficient LPS from damage by high concentrations of Ca2+. Based on its sequence similarity to EptA(PmrC), we propose that the active site of EptB faces the periplasmic surface of the inner membrane.     

Analysis of lipopolysaccharide biosynthesis in Salmonella typhimurium and Escherichia coli by using agents which specifically block incorporation of 3-deoxy-D-manno-octulosonate.

Antibacterial agents which specifically inhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity were used to block the incorporation of 3-deoxy-D-manno-octulosonate (KDO) into lipopolysaccharide. Lipopolysaccharide synthesis ceased, molecules similar in structure to lipid A accumulated, and bacterial growth ceased following addition of such agents to cultures of Salmonella typhimurium and Escherichia coli. Although four major species of lipid A accumulated in S. typhimurium, their kinetics of accumulation were different. The least polar of the major species was IVA [O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-a lph a- D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'], a molecule previously isolated from bacteria containing a kdsA mutation (C. R. H. Raetz, S. Purcell, M. V. Meyer, N. Qureshi, and K. Takayama, J. Biol. Chem. 260:16080-16088, 1985). Species IVA accumulated first and to the greatest extent following addition of the inhibitor, with other more polar derivatives appearing only after IVA attained half its maximal level. In contrast, only two major species of precursor accumulated in E. coli following addition of the inhibitor. One of these species was identical to IVA from S. typhimurium on the basis of chemical composition, fast atom bombardment mass spectroscopy, and comigration on Silica Gel H, and it also accumulated prior to a more polar species of related structure. We conclude that the addition of KDO to precursor species IVA is the major pathway of lipid A-KDO formation in both S. typhimurium LT2 and E. coli and that accumulation of the more polar species lacking KDO only occurs in response to accumulation of species IVA following inhibition of the normal pathway.     

Synthesis of and evaluation of lipid A modification by 4-substituted 4-deoxy arabinose analogs as potential inhibitors of bacterial polymyxin resistance

Three sets of novel 4-deoxy-l-arabinose analogs were synthesized and evaluated as potential inhibitors of the bacterial resistance mechanism in which lipid A, on the outer membrane, is modified with 4-amino-4-deoxy-l-arabinose (l-Ara4N). One compound diminished the transfer of l-Ara4N onto lipid A. These results suggest that small molecules might be designed that would effect the same reversal of bacterial resistance observed in genetic knockouts.     

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-type Escherichia 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 degrees 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.     

Identification of cptA, a PmrA-regulated locus required for phosphoethanolamine modification of the Salmonella enterica serovar typhimurium lipopolysaccharide core

In response to the in vivo environment, the Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) is modified. These modifications are controlled in part by the two-component regulatory system PmrA-PmrB, with the addition of 4-aminoarabinose (Ara4N) to the lipid A and phosphoethanolamine (pEtN) to the lipid A and core. Here we demonstrate that the PmrA-regulated STM4118 (cptA) gene is necessary for the addition of pEtN to the LPS core. pmrC, a PmrA-regulated gene necessary for the addition of pEtN to lipid A, did not affect core pEtN addition. Although imparting a similar surface charge modification as Ara4N, which greatly affects polymyxin B resistance and murine virulence, neither pmrC nor cptA plays a dramatic role in antimicrobial peptide resistance in vitro or virulence in the mouse model. Therefore, factors other than surface charge/electrostatic interaction contribute to resistance to antimicrobial peptides such as polymyxin B.     

The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica

The PmrA/PmrB regulatory system of Salmonella enterica controls the modification of lipid A with aminoarabinose and phosphoethanolamine. The aminoarabinose modification is required for resistance to the antibiotic polymyxin B, as mutations of the PmrA-activated pbg operon or ugd gene result in strains that lack aminoarabinose in their lipid A molecules and are more susceptible to polymyxin B. Additional PmrA-regulated genes appear to participate in polymyxin B resistance, as pbgP and ugd mutants are not as sensitive to polymyxin B as a pmrA mutant. Moreover, the role that the phosphoethanolamine modification of lipid A plays in the resistance to polymyxin B has remained unknown. Here we address both of these questions by establishing that the PmrA-activated pmrC gene encodes an inner membrane protein that is required for the incorporation of phosphoethanolamine into lipid A and for polymyxin B resistance. The PmrC protein consists of an N-terminal region with five transmembrane domains followed by a large periplasmic region harboring the putative enzymatic domain. A pbgP pmrC double mutant resembled a pmrA mutant both in its lipid A profile and in its susceptibility to polymyxin B, indicating that the PmrA-dependent modification of lipid A with aminoarabinose and phosphoethanolamine is responsible for PmrA-regulated polymyxin B resistance.     

A pmrA constitutive mutant sensitizes Escherichia coli to deoxycholic acid

An Escherichia coli mutant was isolated and shown to be polymyxin B resistant. Mapping and sequence analysis revealed a missense mutation at codon 53 within the pmrA (basR) gene that results in a G-to-V substitution. Fusions of promoters from the pmrC, yibD, and pmrH genes with the lacZ reporter showed that they were constitutively expressed in pmrA53 cells. In pmrA+ strains, these promoters were induced by iron and zinc, while a DeltapmrA mutation blocked induction. The PmrA regulon regulates genes whose products remodel the composition and charge of lipid A and hence the barrier properties of the outer membrane. Along these lines, the pmrA53 mutant was also found to be hypersensitive to the anionic bile detergent deoxycholic acid.     

Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lipid A mutants.

Polymyxin-resistant pmrA mutants of Salmonella typhimurium differed from their parents in that they were resistant to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-lysozyme, tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-deoxycholate, and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-bacitracin. Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate released about 50% less lipopolysaccharide from the pmrA strains than from the parental strains when the bacteria were grown in L-broth containing 2 mM Ca2+. Protamine, polylysine, octapeptin, benzalkonium chloride, cold NaCl, cold MgCl2, or cold tris(hydroxymethyl)aminomethane hydrochloride (pH 7.2) caused no leakage or markedly less leakage of periplasmic beta-lactamase from a pmrA mutant than from its parent strain. pmrA mutants were more resistant than their parent strains to protamine and polylysine but not to octapeptin or benzalkonium chloride, as measured by the ability of these agents to kill the bacteria or to sensitize them to deoxycholate-induced lysis. The pmrA strains did not differ from their parent strains in sensitivity to several antibiotics, in porin function (as measured by cephaloridine diffusion across the outer membrane), or in outer membrane-associated phospholipase A activity.