The foodborne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a soluble sulfatase-like catalytic domain in the periplasm. The atomic structure of the catalytic domain of EptC (cEptC) was crystallized and solved to a resolution of 2.40 Å. cEptC adopts the α/β/α fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr266 residue was observed that was hypothesized to mimic a covalent pEtN-enzyme intermediate. The requirement for Thr266 as well as the nearby residues Asn308, Ser309, His358 and His440 was ascertained via in vivo activity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors.

Re-modelling of lipopolysaccharides, which are the primary constituent of the outer cell membrane of Gram-negative bacteria, modulates pathogenesis and resistance to microbials. Reported herein is the characterization of intact Gram-negative bacterial lipooligosaccharides (LOS) via a new strategy utilizing online liquid chromatography (LC) coupled with ultraviolet photodissociation (UVPD) mass spectrometry. Compared to collision-based MS/MS methods, UVPD and UVPD/HCD promoted a greater array of cleavages within both the glycan and lipid moieties, including C-C, C-N, C-O cleavages in the acyl chains as well as glycosidic and cross-ring cleavages, thus providing the most far-reaching structural characterization of LOS. This LC-MS/MS strategy affords a robust analytical method to structurally characterize complex mixtures of bacterial endotoxins that maintains the integrity of the core oligosaccharide and lipid A domains of LOS, providing direct feedback about the cell envelope architectures and LOS modification strategies involved in resistance host innate immune defense.

An unusual α-(1,1)-galacturonic acid (GalA) lipid A modification has been reported in the lipopolysaccharide of a number of interesting Gram-negative bacteria, including the nitrogen-fixing bacteria Azospirillum lipoferum, Mesorhizobium huakuii and M. loti, the stalk-forming bacterium Caulobacter crescentus and the hyperthermophilic bacterium Aquifex aeolicus. However, the α-(1,1)-GalA transferase (GalAT) gene, which we have named RgtF, was not identified. Species of the Rhizobium genera produce lipid A with α-(1,4')-GalA but not α-(1,1)-GalA. The Rhizobium GalAT, RgtD, is the lipid A α-(1-4')-GalAT which utilizes the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA) for GalA transfer. An additional Rhizobium GalAT, RgtE, is required for the biosynthesis of Dod-P-GalA. We predicted candidate rgtF genes in bacterial species known to produce lipid A with α-(1,1)-GalA. In order to determine the predicted rgtF gene function, we cloned the M. loti rgtF gene into an expression plasmid and introduced that plasmid into Rhizobium etli strains that do not contain the rgtF gene nor produce lipid A α-(1,1)-GalA. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis combined with NMR studies revealed that the lipid As from these rgtF-complemented strains were modified with an additional α-(1,1)-GalA attached to the proximal glucosamine.

Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC 28:0 ) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC 28:0 in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC 28:0 from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC 28:0 to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC 28:0 , which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC 28:0 -containing lipid A in the Rlv22 acpXL bacteroids.Rhizobium leguminosarum cells have an envelope similar to that of other gram-negative bacteria. Lipopolysaccharide (LPS) is the primary component of the bacterial outer leaflet and is comprised of three structural regions: the O-chain polysaccharide, the core oligosaccharide, and lipid A. The lipid A region is anchored in the bacterial outer membrane, and the carbohydrate portion projects from the outer surface into the surrounding milieu and is the primary immunogenic determinant. Correlating LPS structure with function has been difficult as LPS is a very complex molecule and LPS preparations consist of structurally heterogeneous mixtures of molecules. There are pronounced variations in LPS structure from strain to strain, and even within a strain there are different sizes and compositions of LPS (23). Interestingly, there are marked differences between LPSs from free-living cultures and LPSs from nitrogen-fixing bacteroids in terms of size, composition, and antigenic properties (10,32,33). It has been shown that the rhizobial LPS undergoes structural modifications during the formation of bacteroids and that there are composition differences between the bacterial and bacteroid LPSs (10, 32).Variation in LPS structure due to environmental changes has been studied in cultured rhizobia by altering the growth conditions, such as lowering the oxygen level, lowering the pH, altering the carbon source, or adding plant-derived compounds (2,14,21,23,27). Such studies have shown that cues from the environment play an import...

Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membranederived subcellular compartments. Notably, an unusual very-long-chain fatty acid (VLCFA) is found in the lipid A of R. leguminosarum as well as in the lipid A of the medically relevant pathogens Brucella abortus, Brucella melitensis, Bartonella henselae, and Legionella pneumophila, which are also able to persist within intracellular host-derived membranes. These bacterial symbionts and pathogens each contain a homologous gene region necessary for the synthesis and transfer of the VLCFA to the lipid A. Within this region lies a gene that encodes the specialized acyl carrier protein AcpXL, on which the VLCFA is built. This study describes the biochemical and infection phenotypes of an acpXL mutant which lacks the VLCFA. The mutation was created in R. leguminosarum bv. phaseoli strain 8002, which forms symbiosis with Phaseolus vulgaris, a determinate nodulating legume. Structural analysis using gas chromatography and mass spectrometry revealed that the mutant lipid A lacked the VLCFA. Compared to the parent strain, the mutant was more sensitive to the detergents deoxycholate and dodecyl sulfate and the antimicrobial peptide polymyxin B, suggesting a compromise to membrane stability. In addition, the mutant was more sensitive to higher salt concentrations. Passage through the plant restored salt tolerance. Electron microscopic examination showed that the mutant was developmentally delayed during symbiotic infection of the host plant Phaseolus vulgaris and produced abnormal symbiosome structures.Rhizobium leguminosarum is a Gram-negative alphaproteobacterium capable of forming nitrogen-fixing symbioses with plants found in the Leguminosae family. The symbiotic process involves the formation of root nodules and simultaneous invasion of the legume root by compatible symbiotic bacteria. The invading bacteria are eventually endocytosed by specialized root nodule tissue, where they develop into nitrogen-fixing bacteroids. Bacteroids persist within plant-derived membranes (peribacteroid membrane), which together form organelle-like structures (symbiosomes) for which the primary function is to provide the plant with nitrogen through the conversion of atmospheric nitrogen to ammonia via the bacteroid-derived nitrogenase enzyme. For a recent review of the rhizobial infection process, refer to the study by Oldroyd and Downie (26).Depending on the legume host plant, two nodule types, indeterminate and determinate, are known, and key fundamental differences exist between the two nodule types. Indeterminate systems are characterized as having an active meristem during the entire life cycle of the nodule. Conversely, meristematic cells from determinate root nodules stop dividing well before nodule senescence. Indeterminate nodules have an oblong shape and harbor terminally differentiated bacteroids (inhibited in cell divisio...