Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses.

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

SRD5A3 is required for converting polyprenol to dolichol and is mutated in a congenital glycosylation disorder.

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.

Structure and Function of ArnD. A Deformylase Essential for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and Polymyxin Resistance.

Covalent modification of lipid A with 4-deoxy-4-amino-l-arabinose (Ara4N) mediates resistance to cationic antimicrobial peptides and polymyxin antibiotics in Gram-negative bacteria. The proteins required for Ara4N biosynthesis are encoded in the pmrE and arnBCADTEF loci, with ArnT ultimately transferring the amino sugar from undecaprenyl-phospho-4-deoxy-4-amino-l-arabinose (C55P-Ara4N) to lipid A. However, Ara4N is N-formylated prior to its transfer to undecaprenyl-phosphate by ArnC, requiring a deformylase activity downstream in the pathway to generate the final C55P-Ara4N donor. Here, we show that deletion of the arnD gene in an Escherichia coli mutant that constitutively expresses the arnBCADTEF operon leads to accumulation of the formylated ArnC product undecaprenyl-phospho-4-deoxy-4-formamido-l-arabinose (C55P-Ara4FN), suggesting that ArnD is the downstream deformylase. Purification of Salmonella typhimurium ArnD (stArnD) shows that it is membrane-associated. We present the crystal structure of stArnD revealing a NodB homology domain structure characteristic of the metal-dependent carbohydrate esterase family 4 (CE4). However, ArnD displays several distinct features: a 44 amino acid insertion, a C-terminal extension in the NodB fold, and sequence divergence in the five motifs that define the CE4 family, suggesting that ArnD represents a new family of carbohydrate esterases. The insertion is responsible for membrane association as its deletion results in a soluble ArnD variant. The active site retains a metal coordination H-H-D triad, and in the presence of Co2+ or Mn2+, purified stArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. Mutations D9N and H233Y completely inactivate stArnD implicating these two residues in a metal-assisted acid-base catalytic mechanism.

Chasing acyl carrier protein through a catalytic cycle of lipid A production.

Acyl carrier protein represents one of the most highly conserved proteins across all domains of life and is nature's way of transporting hydrocarbon chains in vivo. Notably, type II acyl carrier proteins serve as a crucial interaction hub in primary cellular metabolism by communicating transiently between partner enzymes of the numerous biosynthetic pathways. However, the highly transient nature of such interactions and the inherent conformational mobility of acyl carrier protein have stymied previous attempts to visualize structurally acyl carrier protein tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but also of therapeutic value. For example, acyl carrier protein is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides in Gram-negative microorganisms, which is required for their growth and survival, and is an activator of the mammalian host's immune system, thus emerging as an important therapeutic target. During lipid A synthesis (Raetz pathway), acyl carrier protein shuttles acyl intermediates linked to its prosthetic 4'-phosphopantetheine group among four acyltransferases, including LpxD. Here we report the crystal structures of three forms of Escherichia coli acyl carrier protein engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures show the intricate interactions at the interface that optimally position acyl carrier protein for acyl delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled acyl carrier proteins provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4'-phosphopantetheine groups within the LpxD catalytic chamber shows an unprecedented role of acyl carrier protein in product release.

Crystal structure of LpxK, the 4'-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface.

In Gram-negative bacteria, the hydrophobic anchor of the outer membrane lipopolysaccharide is lipid A, a saccharolipid that plays key roles in both viability and pathogenicity of these organisms. The tetraacyldisaccharide 4'-kinase (LpxK) of the diverse P-loop-containing nucleoside triphosphate hydrolase superfamily catalyzes the sixth step in the biosynthetic pathway of lipid A, and is the only known P-loop kinase to act upon a lipid substrate at the membrane. Here, we report the crystal structures of apo- and ADP/Mg(2+)-bound forms of Aquifex aeolicus LpxK to a resolution of 1.9 Å and 2.2 Å, respectively. LpxK consists of two α/ß/α sandwich domains connected by a two-stranded ß-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide-1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate and Mg(2+) cation using a 25° hinge motion about its base. Activity was severely reduced in alanine point mutants of conserved residues D138 and D139, which are not directly involved in ADP or Mg(2+) binding in our structures, indicating possible roles in phosphoryl acceptor positioning or catalysis. Combined structural and kinetic studies have led to an increased understanding of the enzymatic mechanism of LpxK and provided the framework for structure-based antimicrobial design.

Discovery of a cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates.

Depending on growth phase and culture conditions, cardiolipin (CL) makes up 5-15% of the phospholipids in Escherichia coli with the remainder being primarily phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). In E. coli, the cls and ybhO genes (renamed clsA and clsB, respectively) each encode a CL synthase (Cls) that catalyzes the condensation of two PG molecules to form CL and glycerol. However, a ΔclsAB mutant still makes CL in the stationary phase, indicating the existence of additional Cls. We identified a third Cls encoded by ymdC (renamed clsC). ClsC has sequence homology with ClsA and ClsB, which all belong to the phospholipase D superfamily. The ΔclsABC mutant lacks detectible CL regardless of growth phase or growth conditions. CL can be restored to near wild-type levels in stationary phase in the triple mutant by expressing either clsA or clsB. Expression of clsC alone resulted in a low level of CL in the stationary phase, which increased to near wild-type levels by coexpression of its neighboring gene, ymdB. CL synthesis by all Cls is increased with increasing medium osmolarity during logarithmic growth and in stationary phase. However, only ClsA contributes detectible levels of CL at low osmolarity during logarithmic growth. Mutation of the putative catalytic motif of ClsC prevents CL formation. Unlike eukaryotic Cls (that use PG and CDP-diacylglycerol as substrates) or ClsA, the combined YmdB-ClsC used PE as the phosphatidyl donor to PG to form CL, which demonstrates a third and unique mode for CL synthesis.

LPS remodeling is an evolved survival strategy for bacteria.

Maintenance of membrane function is essential and regulated at the genomic, transcriptional, and translational levels. Bacterial pathogens have a variety of mechanisms to adapt their membrane in response to transmission between environment, vector, and human host. Using a well-characterized model of lipid A diversification (Francisella), we demonstrate temperature-regulated membrane remodeling directed by multiple alleles of the lipid A-modifying N-acyltransferase enzyme, LpxD. Structural analysis of the lipid A at environmental and host temperatures revealed that the LpxD1 enzyme added a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme added a 3-OH C16 acyl group at 18 °C (environment). Mutational analysis of either of the individual Francisella lpxD genes altered outer membrane (OM) permeability, antimicrobial peptide, and antibiotic susceptibility, whereas only the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. Additionally, growth-temperature analysis revealed transcriptional control of the lpxD genes and posttranslational control of the LpxD1 and LpxD2 enzymatic activities. These results suggest a direct mechanism for LPS/lipid A-level modifications resulting in alterations of membrane fluidity, as well as integrity and may represent a general paradigm for bacterial membrane adaptation and virulence-state adaptation.

In vitro assembly of the outer core of the lipopolysaccharide from Escherichia coli K-12 and Salmonella typhimurium.

There are five distinct core structures in the lipopolysaccharides of Escherichia coli and at least two in Salmonella isolates, which vary principally in the outer core oligosaccharide. Six outer core glycosyltransferases, E. coli K-12 WaaG, WaaB, and WaaO and Salmonella typhimurium WaaI, WaaJ, and WaaK, were cloned, overexpressed, and purified. A novel substrate for WaaG was isolated from ΔwaaG E. coli overexpressing the lipid A phosphatase lpxE and the lipid A late acyltransferase lpxM. The action of lpxE and lpxM in the ΔwaaG background yielded heptose2-1-dephospho Kdo2-lipid A, a 1-dephosphorylated hexa-acylated lipid A with the inner core sugars that is easily isolated by organic extraction. Using this structurally defined acceptor and commercially available sugar nucleotides, each outer core glycosyltransferases was assayed in vitro. We show that WaaG and WaaB add a glucose and galactose sequentially to heptose2-1-dephospho Kdo2-lipid A. E. coli K-12 WaaO and S. typhimurium WaaI add a galactose to the WaaG/WaaB product but can also add a galactose to the WaaG product directly without the branched core sugar added by WaaB. Both WaaI and WaaO require divalent metal ions for optimal activity; however, WaaO, unlike WaaI, can add several glucose residues to its lipid acceptor. Using the product of WaaG, WaaB, and WaaI, we show that S. typhimurium WaaJ and WaaK transfer a glucose and N-acetylglucosamine, respectively, to yield the full outer core. This is the first demonstration of the in vitro assembly of the outer core of the lipopolysaccharide using defined lipid A-oligosaccharide acceptors and sugar donors.

The origin of 8-amino-3,8-dideoxy-D-manno-octulosonic acid (Kdo8N) in the lipopolysaccharide of Shewanella oneidensis.

Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-d-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-d-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus Shewanella. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in Shewanella oneidensis. Expression of these genes recombinantly in Escherichia coli resulted in lipid A containing Kdo8N, and in vitro assays confirmed their proposed enzymatic function. Both the in vivo and in vitro data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)(+). Creation of an S. oneidensis in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity.

Mutants resistant to LpxC inhibitors by rebalancing cellular homeostasis.

LpxC, the deacetylase that catalyzes the second and committed step of lipid A biosynthesis in Escherichia coli, is an essential enzyme in virtually all gram-negative bacteria and is one of the most promising antibiotic targets for treatment of multidrug-resistant gram-negative infections. Despite the rapid development of LpxC-targeting antibiotics, the potential mechanisms of bacterial resistance to LpxC inhibitors remain poorly understood. Here, we report the isolation and biochemical characterization of spontaneously arising E. coli mutants that are over 200-fold more resistant to LpxC inhibitors than the wild-type strain. These mutants have two chromosomal point mutations that account for resistance additively and independently; one is in fabZ, a dehydratase in fatty acid biosynthesis; the other is in thrS, the Thr-tRNA ligase. For both enzymes, the isolated mutations result in reduced enzymatic activities in vitro. Unexpectedly, we observed a decreased level of LpxC in bacterial cells harboring fabZ mutations in the absence of LpxC inhibitors, suggesting that the biosyntheses of fatty acids and lipid A are tightly regulated to maintain a balance between phospholipids and lipid A. Additionally, we show that the mutation in thrS slows protein production and cellular growth, indicating that reduced protein biosynthesis can confer a suppressive effect on inhibition of membrane biosynthesis. Altogether, our studies reveal a previously unrecognized mechanism of antibiotic resistance by rebalancing cellular homeostasis.

25-Hydroxycholesterol activates the integrated stress response to reprogram transcription and translation in macrophages.

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a beta-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.

Complex transcriptional and post-transcriptional regulation of an enzyme for lipopolysaccharide modification.

The PhoQ/PhoP two-component system activates many genes for lipopolysaccharide (LPS) modification when cells are grown at low Mg(2+) concentrations. An additional target of PhoQ and PhoP is MgrR, an Hfq-dependent small RNA that negatively regulates expression of eptB, also encoding a protein that carries out LPS modification. Examination of LPS confirmed that MgrR effectively silences EptB; the phosphoethanolamine modification associated with EptB is found in ΔmgrR::kan but not mgrR(+) cells. Sigma E has been reported to positively regulate eptB, although the eptB promoter does not have the expected Sigma E recognition motifs. The effects of Sigma E and deletion of mgrR on levels of eptB mRNA were independent, and the same 5' end was found in both cases. In vitro transcription and the behaviour of transcriptional and translational fusions demonstrate that Sigma E acts directly at the level of transcription initiation for eptB, from the same start point as Sigma 70. The results suggest that when Sigma E is active, synthesis of eptB transcript outstrips MgrR-dependent degradation; presumably the modification of LPS is important under these conditions. Adding to the complexity of eptB regulation is a second sRNA, ArcZ, which also directly and negatively regulates eptB.

Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis.

The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d-glycero-d-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe(2+)/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVAin vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis.

Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide.

Several gram-negative pathogens, including Yersinia pestis, Burkholderia cepacia, and Acinetobacter haemolyticus, synthesize an isosteric analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), known as D-glycero-D-talo-oct-2-ulosonic acid (Ko), in which the axial hydrogen atom at the Kdo 3-position is replaced with OH. Here we report a unique Kdo 3-hydroxylase (KdoO) from Burkholderia ambifaria and Yersinia pestis, encoded by the bamb_0774 (BakdoO) and the y1812 (YpkdoO) genes, respectively. When expressed in heptosyl transferase-deficient Escherichia coli, these genes result in conversion of the outer Kdo unit of Kdo(2)-lipid A to Ko in an O(2)-dependent manner. KdoO contains the putative iron-binding motif, HXDX(n>40)H. Reconstitution of KdoO activity in vitro with Kdo(2)-lipid A as the substrate required addition of Fe(2+), α-ketoglutarate, and ascorbic acid, confirming that KdoO is a Fe(2+)/α-ketoglutarate/O(2)-dependent dioxygenase. Conversion of Kdo to Ko in Kdo(2)-lipid A conferred reduced susceptibility to mild acid hydrolysis. Although two enzymes that catalyze Fe(2+)/α-ketoglutarate/O(2)-dependent hydroxylation of deoxyuridine in fungal extracts have been reported previously, kdoO is the first example of a gene encoding a deoxy-sugar hydroxylase. Homologues of KdoO are found exclusively in gram-negative bacteria, including the human pathogens Burkholderia mallei, Yersinia pestis, Klebsiella pneumoniae, Legionella longbeachae, and Coxiella burnetii, as well as the plant pathogen Ralstonia solanacearum.

Pathway for lipid A biosynthesis in Arabidopsis thaliana resembling that of Escherichia coli.

The lipid A moiety of Escherichia coli lipopolysaccharide is a hexa-acylated disaccharide of glucosamine that makes up the outer monolayer of the outer membrane. Arabidopsis thaliana contains nuclear genes encoding orthologs of key enzymes of bacterial lipid A biosynthesis, including LpxA, LpxC, LpxD, LpxB, LpxK and KdtA. Although structurally related lipid A molecules are found in most other gram-negative bacteria, lipid A and its precursors have not been directly detected in plants previously. However, homozygous insertional knockout mutations or RNAi knock-down constructs of Arabidopsis lpx and kdtA mutants revealed accumulation (or disappearance) of the expected monosaccharide or disaccharide lipid A precursors by mass spectrometry of total lipids extracted from 10-day old seedlings of these mutants. In addition, fluorescence microscopy of lpx-gfp fusions in transgenic Arabidopsis plants suggests that the Lpx and KdtA proteins are expressed and targeted to mitochondria. Although the structure of the lipid A end product generated by plants is still unknown, our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli.

Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are the main lipid components of bacterial outer membranes and are essential for cell viability in most Gram-negative bacteria. Here we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, block the synthesis of LOS in the obligate intracellular bacterial pathogen Chlamydia trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of antichlamydial agents.

Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis.

The sixth step in the lipid A biosynthetic pathway involves phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) intermediate by the cytosol-facing inner membrane kinase LpxK, a member of the P-loop-containing nucleoside triphosphate (NTP) hydrolase superfamily. We report the kinetic characterization of LpxK from Aquifex aeolicus and the crystal structures of LpxK in complex with ATP in a precatalytic binding state, the ATP analogue AMP-PCP in the closed catalytically competent conformation, and a chloride anion revealing an inhibitory conformation of the nucleotide-binding P-loop. We demonstrate that LpxK activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg(2+)-DSMP complex. Using steady-state kinetics, we have identified crucial active site residues, leading to the proposal that the interaction of D99 with H261 acts to increase the pKa of the imidazole moiety, which in turn serves as the catalytic base to deprotonate the 4'-hydroxyl of the DSMP substrate. The fact that an analogous mechanism has not yet been observed for other P-loop kinases highlights LpxK as a distinct member of the P-loop kinase family, a notion that is also reflected through its localization at the membrane, lipid substrate, and overall structure.

The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a ß-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.

Density gradient enrichment of Escherichia coli lpxL mutants.

We previously described enrichment of conditional Escherichia coli msbA mutants defective in lipopolysaccharide export using Ludox density gradients (Doerrler WT (2007) Appl Environ Microbiol 73; 7992-7996). Here, we use this approach to isolate and characterize temperature-sensitive lpxL mutants. LpxL is a late acyltransferase of the pathway of lipid A biosynthesis (The Raetz Pathway). Sequencing the lpxL gene from the mutants revealed the presence of both missense and nonsense mutations. The missense mutations include several in close proximity to the enzyme's active site or conserved residues (E137K, H132Y, G168D). These data demonstrate that Ludox gradients can be used to efficiently isolate conditional E. coli mutants with defects in lipopolysaccharide biosynthesis and provide insight into the enzymatic mechanism of LpxL.