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Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules
Abstract:
Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.
Insights
Rhizobium meliloti exo mutants unable to produce succinoglycan cannot infect alfalfa root nodules. Characterizing polysaccharide defects revealed specific mutations affecting cyclic beta-D-glucan and lipopolysaccharide (LPS) are key to nodule entry.
Area of Science:
- Microbiology
- Plant-microbe interactions
- Bacterial cell surface polysaccharides
Background:
- Rhizobium meliloti succinoglycan is crucial for alfalfa root nodule symbiosis.
- Defects in succinoglycan production lead to impaired nodule entry.
- Understanding polysaccharide roles requires detailed analysis of exo mutants.
Purpose of the Study:
- To characterize the polysaccharide profiles of Rhizobium meliloti exo mutants.
- To correlate specific polysaccharide defects with nodule entry failures.
- To elucidate the roles of cyclic beta-D-glucan and lipopolysaccharide in symbiosis.
Main Methods:
- Analysis of periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan in exo mutants.
- Lipopolysaccharide (LPS) profiling of representative exo mutants.
- Assay of high- and low-molecular-weight succinoglycans in various mutants.
Main Results:
- exoC mutant lacked cyclic beta-D-glucan and had abnormal LPS lacking O side chains.
- exoB mutant exhibited unique LPS species distinct from wild-type and exoC.
- exoA, exoD, exoF, and exoH mutants had normal glucan and LPS, ruling them out as causes of entry defects.
- Most exo mutants lacked both high- and low-molecular-weight succinoglycans.
- exoD succinoglycan form depended on growth medium; exoH lacked succinyl modification.
Conclusions:
- Specific defects in cyclic beta-D-glucan and LPS are linked to nodule entry failure in certain exo mutants.
- The exoC and exoB mutations significantly alter polysaccharide composition.
- While succinoglycan is important, other polysaccharide factors (glucan, LPS) are critical for successful nodule colonization.