Degree Name

Master of Science (MS)

Semester of Degree Completion


Thesis Director

Russell Carlson


The ever-present specter of the inavailability of food production to increase in direct proportion to population growth haunts practically every nation of the world. As more and more tracts of land are incorporated to accommodate expanding cities, they become less available for food production. Emphasis, therefore, is placed on obtaining a higher yield per acreage planted. Since fertilization is not reliable in the long term due to an eventual depletion of raw materials for fertilizers, the ability of plants like legumes to assimilate free soil nitrogen on their own has become increasingly important [1,11,14,17]. However, the mechanisms involved are still not well-understood, so more research have to be done to make this the cornerstone of increasing plant productivity.

Indeed, the importance of understanding biological nitrogen fixation is accentuated by the number of researchers involved biochemists, plant physiologists, molecular geneticists, and biologists, to name a few. Countries around the world, from Australia to Japan to Sweden to Argentina, have research projects underway involving biological nitrogen fixation and/or plant productivity.

In our lab, the research focus is from both biochemical and immunological point of views. This particular work involves the study of the biochemistry of a cell surface macropolysaccharide – the capsular polysaccharide or CPS. This is obtained from a pure culture of Rhizobium trifolii, a hac+, nod+, fix+ Gram negative bacteria that could be found naturally in the soil. Together with its nod- mutants, comparisons were done on the polysaccharide make-up of the CPS [25]. The CPS does not constitute a part of the cell membrane like the LPS nor is it excreted freely into the media like the EPS. Rather, it adheres on the surface of the bacteria in the form of capsules [7,15].

From the isolated crude CPS, EPS-like materials called acidic CPS (ACPS) and LPS-like materials called neutral CPS (NCPS) were obtained upon treatment with CTAB. GC analysis revealed that the ACPS contains galactose and glucose whereas the NCPS has 2-0-methyl-6-deoxyhexose, fucose, mannose, galactose, 3-N-methyl-3-amino-3,6-dideoxyhexose, and glucose in both parent and mutant strains. KDO, acetyl groups, pyruvic acid, and uronic acid were present in both ACPS and NCPS of all strains using specific colorimetric assays.

Mild acid hydrolysis showed that the polysaccharide fractions of both ACPS and NCPS may be attached to a lipid, perhaps via KDO linkages. SDS-PAGE analyses showed different patterns of molecular aggregation between the ACPS and the NCPS but not within strains. Methanolysis of the samples revealed that the uronic acids in ACPS’s are galacturonic acid while in NCPS’s, they are both galacturonic and glucuronic acids.

These results suggest that the CPS of R. trifolii may have a dual character resembling that of E. coli and N. meningitaides, both Gram negative animal pathogens [24,33]. Whether the CPS in R. trifolii functions in the same manner still remains to be determined, as is its mode of attachment to the bacterial cell surface.

Lastly, the role of the acetyl groups merits careful investigation since these, together with heptose, correlate proportionally with phage-binding abilities [23]. Overall, the study of the CPS is still in a stage of infancy and a lot more research have to be done to determine whatever role it plays in the R. trifolii -clover symbiosis [7,15,35].

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