Rhizobium-legume symbioses involve processes that are fundamental to all bacterial-host interactions. These include (a.) prokaryotic-eukaryotic recognition, (b.) interaction with the host defense mechanism, and (c.) bacterial and host differentiation processes (i.e. formation of nitrogen-fixing bacteroids and root nodules, respectively). As with animal pathogens, the invading rhizobia must adapt to the environment of the host to avoid destruction and persist in the host cell. These adaptations involve changes to cell surface polysaccharides, such as lipopolysaccharides (LPSs) and capsular polysaccharides. Rhizobium-legume symbiosis is an excellent model system to examine the molecular bases by which Gram-negative bacteria infect and survive within their host since both the bacteria and the host cells are viable throughout the infection process, and the LPSs from the invading bacteria can be isolated and characterized as a function of the infection process. The symbiosis system is particularly relevant to a number of intracellular pathogens that form chronic infections such as species of Brucella, Bartonella, Legionella, as well as Francisella. We have observed that unique structural features of Rhizobium LPSs are also present on LPSs from these pathogens. Our project focuses on the adaptation that rhizobia (R. etli and R. leguminosarum; symbionts of bean and pea, respectively) make to their LPSs as they encounter the stressful environment (e.g. changes in pH, osmolarity, oxygen tension, etc.) of the host cell. We hypothesize that unique structural features of rhizobial LPSs are essential for infection and persistence in the legume hosts. Unique structural features include a replacement of phosphate with galacturonic acid residues, oxidation of the lipid-A proximal glucosamine residue to 2-aminogluconate, and the presence of the very long chain fatty acid, 27- hydroxyoctacosaonic acid (27OHC28:0), on the lipid-A. Methylation and acetylation changes to the O-chain polysaccharide and an overall change in the hydrophobicity of the LPS and the whole rhizobial cell occur during symbiosis.
Our Specific Aims are: (1.) characterize the modifications to the O-chain polysaccharide of the LPS during symbiosis, (2.) create specific LPS mutants and characterize their structural and symbiotic phenotypes, and (3.) determine how a 27OHC28:0-lacking mutant, acpXL::kan, is able to activate an alternative mechanism within the host cell. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Host Interactions with Bacterial Pathogens Study Section (HIBP)
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Marino, Pamela
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University of Georgia
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Bourassa, Dianna V; Kannenberg, Elmar L; Sherrier, D Janine et al. (2017) The Lipopolysaccharide Lipid A Long-Chain Fatty Acid Is Important for Rhizobium leguminosarum Growth and Stress Adaptation in Free-Living and Nodule Environments. Mol Plant Microbe Interact 30:161-175
Brown, Dusty B; Muszynski, Artur; Carlson, Russell W (2013) Elucidation of a novel lipid A ?-(1,1)-GalA transferase gene (rgtF) from Mesorhizobium loti: Heterologous expression of rgtF causes Rhizobium etli to synthesize lipid A with ?-(1,1)-GalA. Glycobiology 23:546-58
Brown, Dusty B; Muszynski, Artur; Salas, Omar et al. (2013) Elucidation of the 3-O-deacylase gene, pagL, required for the removal of primary ?-hydroxy fatty acid from the lipid A in the nitrogen-fixing endosymbiont Rhizobium etli CE3. J Biol Chem 288:12004-13
Brown, Dusty B; Forsberg, L Scott; Kannenberg, Elmar L et al. (2012) 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 me J Biol Chem 287:935-49
Muszynski, Artur; Laus, Marc; Kijne, Jan W et al. (2011) Structures of the lipopolysaccharides from Rhizobium leguminosarum RBL5523 and its UDP-glucose dehydrogenase mutant (exo5). Glycobiology 21:55-68
Brown, Dusty B; Huang, Yu-Chu; Kannenberg, Elmar L et al. (2011) 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. J Bacteriol 193:4766-78
Vanderlinde, Elizabeth M; Harrison, Joe J; Muszy?ski, Artur et al. (2010) Identification of a novel ABC transporter required for desiccation tolerance, and biofilm formation in Rhizobium leguminosarum bv. viciae 3841. FEMS Microbiol Ecol 71:327-40
Vanderlinde, Elizabeth M; Muszynski, Artur; Harrison, Joe J et al. (2009) Rhizobium leguminosarum biovar viciae 3841, deficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility. Microbiology 155:3055-69
Forsberg, L Scott; Carlson, Russell W (2008) Structural characterization of the primary O-antigenic polysaccharide of the Rhizobium leguminosarum 3841 lipopolysaccharide and identification of a new 3-acetimidoylamino-3-deoxyhexuronic acid glycosyl component: a unique O-methylated glycan of uniform s J Biol Chem 283:16037-50
D'Haeze, Wim; Leoff, Christine; Freshour, Glenn et al. (2007) Rhizobium etli CE3 bacteroid lipopolysaccharides are structurally similar but not identical to those produced by cultured CE3 bacteria. J Biol Chem 282:17101-13

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