The Effect of Calcium Nutrition on the Production of Diffusible Antigens by Rhizobium trifolii

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Abstract
Rhizobium trifolii grown in a defined medium deficient in calcium yielded additional diffusible antigens as compared with organisms receiving calcium or strontium. The two kinds of organisms were indistinguishable in respect of their somatic agglutinogens. The same additional precipitinogens were released from Ca-adequate bacteria by mechanical disintegration, freezing, drying, lysozyme or chloroform. Their occurrence in the untreated Ca-deficient bacteria is attributed to autolysis of these more fragile bacteria. The precipitinogen ("a" band) that was common to the Ca-adequate and Ca-deficient bacteria was not shared by a second strain of R. trifolii nor by 2-strains of R. meliloti. This "a" antigen was heat stable and appeared to be a component of the cell wall, from which it could be separated in diffusible form by formamide extraction of whole bacteria, trypsin digestion of broken bacteria and by mechanical disintegration of prepared cell walls. Neither of the additional antigens ("b", "c") was related to agglutination; both were found in Ca-deficient or mechanically broken bacteria of the other strain of R. trifolii; "c" only was similarly shared with R. meliloti. The "b" band antigen appeared to be an intracellular component and was heat labile. Like "a", "c" was heat stable and associated with cell wall, but evidently was not ordinarily exposed, although readily released by lysis or mechanical breakage. The extracellular polysaccharide was neither antigenic nor haptenic, but the preparation obtained from Ca-deficient and mechanically broken Ca-adequate organisms was persistently associated with a co-precipitating antigenic component (chiefly "a" band).

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