Graduate Thesis Or Dissertation
 

Characterization of a bacterial agglutinin from legumes

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  • Leaves of Phaseolus vulgaris cv. 'Dark Red Kidney' and cv. 'Red Mexican U. I. 31', Phaseolus lunatus cv. 'Fordhook Bush 242', Phaseolus mungo cv. 'Berken', and Glycine max cv. 'Wilkin' have been shown to contain a factor which consistently agglutinated the saprophyte Pseudomonas putida; another saprophyte Pseudomonas fluorescens was agglutinated variably. The agglutinin was not active against phytopathogenic species of Pseudomonas, Agrobacterium, Erwinia, Corynebacterium, and Xanthomonas, and the saprophytic species Escherichia coli. Agglutinin from Dark Red Kidney and Red Mexican did not distinguish between races of Pseudomonas phaseolicola. The agglutinin may be specific for saprophytic Pseudomonads and responsible for their attachment and immobilization at the plant cell wall. More saprophytic Pseudomonads need to be tested. Isolation and purification of agglutinin involved homogenization of leaves in water, centrifugation, ion exchange chromatography, ethanol precipitation, dialysis and either gel filtration on SepharosR 6B-100 alone or an affinity purification using P. putida followed by gel filtration. Exclusion of agglutinin activity in the void volume indicated that the agglutinin has a molecular weight of a least 4 x 10⁶ daltons. Results of colorimetric assays of active column fractions indicated that the agglutinin is a glycoprotein composed of protein, hexose and uronic acid. From gas chromatographic analyses of alditolacetate derivatives of agglutinin arabinose and galactose were identified as the major neutral sugars present in the carbohydrate component. Sensitivity of the agglutinin to periodate indicated that the carbohydrate component of the agglutinin is essential for its activity. Agglutinin is active against only live bacteria and requires the divalent cation Mg⁺⁺. In the purest agglutinin preparations obtained less than one ug each of protein, hexose, and uronic acid was required to agglutinate 10⁸ cells of P. putida.
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