Elucidation of the 3-O-deacylase gene, pagL, required for the removal of primary ß-hydroxy fatty acid from the lipid A in the nitrogen-fixing endosymbiont Rhizobium etli CE3.

Until now, the gene responsible for the 3-O-deacylation of lipid A among nitrogen-fixing endosymbionts has not been characterized. Several Gram-negative animal pathogens such as Salmonella enterica, Pseudomonas aeruginosa, and Bordetella bronchiseptica contain an outer membrane 3-O-deacylase (PagL) that has been implicated in host immune evasion. The role of 3-O-deacylated lipid A among nitrogen-fixing endosymbionts, plant endophytes, and plant pathogens has not been studied. However, D'Haeze et al. (D'Haeze, W., Leoff, C., Freshour, G., Noel, K. D., and Carlson, R. W. (2007) J. Biol. Chem. 282, 17101-17113) reported that the lipopolysaccharide from Rhizobium etli CE3 bacteroids isolated from host bean root nodules contained exclusively tetraacylated lipid A that lacked a lipid A ß-hydroxymyristyl residue, an observation that is consistent with the possibility of PagL activity being important in symbiosis. A putative pagL gene was identified in the R. etli genome sequence. With this information, we created a pagL(-) mutant strain derived from R. etli CE3. Using mass spectrometry, we demonstrated that the mutant lacks 3-O-deacylated lipid A. The parent and mutant LPS were very similar as determined by gel electrophoresis and glycosyl composition analysis using gas chromatography/mass spectrometry. However, fatty acid analysis showed that the mutant lipid A contained larger amounts of ß-hydroxypentadecanoic acid than that of the parent. Furthermore, the mutant was adversely affected in establishing symbiosis with its host, Phaseolus vulgaris.

Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni.

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/ß/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.

Characterization of galacturonosyl transferase genes rgtA, rgtB, rgtC, rgtD, and rgtE responsible for lipopolysaccharide synthesis in nitrogen-fixing endosymbiont Rhizobium leguminosarum: lipopolysaccharide core and lipid galacturonosyl residues confer membrane stability.

Rhizobium lipopolysaccharide (LPS) contains four terminally linked galacturonic acid (GalA) residues; one attached to the lipid A and three attached to the core oligosaccharide moiety. Attachment of the GalA residues requires the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA), which is synthesized by the GalA transferase RgtE reported here. The galacturonosyl transferases RgtA, -B, and -C utilize Dod-P-GalA to attach GalAs on the LPS core region, and RgtD attaches GalA to the lipid A 4' position. As reported here, the functions of the rgtD and rgtE genes were determined via insertion mutagenesis and structural characterization of the mutant lipid A. The rgtE(-) mutant lacked Dod-P-GalA as determined by mass spectrometry of total lipid extracts and the inability of rgtE(-) mutant membranes to provide the substrate for heterologously expressed RgtA activity. In addition, we created single mutations in each of the rgtA, -B, -C, -D, and -E genes to study the biological function of the GalA residues. The structures of the core oligosaccharide region from each of the rgt mutants were elucidated by glycosyl linkage analysis. Each mutant was assayed for its sensitivity to sodium deoxycholate and to the antimicrobial cationic peptide, polymyxin B (PmxB). The rgt mutants were more sensitive than the parent strain to deoxycholate by varying degrees. However, the rgtA, -B, and -C mutants were more resistant to PmxB, whereas the rgtD and E mutants were less resistant in comparison to the parent strain.

Elucidation of a novel lipid A α-(1,1)-GalA transferase gene (rgtF) from Mesorhizobium loti: Heterologous expression of rgtF causes Rhizobium etli to synthesize lipid A with α-(1,1)-GalA.

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.

An acpXL mutant of Rhizobium leguminosarum bv. phaseoli lacks 27-hydroxyoctacosanoic acid in its lipid A and is developmentally delayed during symbiotic infection of the determinate nodulating host plant Phaseolus vulgaris.

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 membrane-derived 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.

Top-Down Strategies for the Structural Elucidation of Intact Gram-negative Bacterial Endotoxins.

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.