Fluorescein Isothiocyanate-Labeled Lectin Analysis of the Surface of the Nitrogen-Fixing Bacterium Azospirillum brasilense by Flow Cytometry

Abstract

The cell surface of Azospirillum brasilense was probed by using fluorescein isothiocyanate (FITC)-labeled lectins, with binding determined by fluorescence-activated flow cytometry. Cells from nitrogen-fixing or ammonium-assimilating cultures reacted similarly to FITC-labeled lectins, with lectin binding in the following order: Griffonia simplicifolia II agglutinin > Griffonia simplicifolia I agglutinin > Triticum vulgaris agglutinin > Glycine max agglutinin > Canavalia ensiformis agglutinin > Limaxflavus agglutinin > Lotus tetragonolobus agglutinin. The fluorescence intensity of cells labeled with FITC-labeled G. simplicifolia I, C. ensiformis, T. vulgaris, and G. max agglutinins was influenced by lectin concentration. Flow cytometry measurements of lectin binding to cells was consistent with measurements of agglutination resulting from lectin-cell interaction. Capsules surrounding nitrogen-fixing and ammonium-assimilating cells were readily demonstrated by light and transmission electron microscopies.

Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities

“…Also, various lectins may agglutinate with different strengths to the same sugar class. Many bacterial species have different sugar residues on the cell surface such as nitrogen-fixing bacterium Azospirillum brasilense, which was described to bind nearly all of the above-described lectins (Yagoda-Shagam et al, 1988). Additionally, the presence of sugar residues on the bacteria's surface may depend on the growth conditions, influencing therefore the agglutination strength and the intensity of the label in the end (Heine et al, 2009).…”

The effect of wheat germ agglutinin on dinitrogen fixation, glutamine synthetase activity and ammonia excretion in Azospirillum brasilense Sp 245

“…In previous works by Yagoda-Shagam et al [5] and Del Gallo et al [6] fluorescein isothiocyanate-labelled WGA was shown to bind to A. brasilense cells and this process was inhibited by N-acetyl-D-glucosamine. The increase in nitrogenase and GS activities and ammonia excretion observed by us probably results from the WGA binding to bacterial cell surface.…”

“…It remains unclear which structures of the cell surface of A. brasilense are responsible for the binding of WGA. Besides lipopolysaccharides and capsule polysaccharides, whose N-acetyl-Dglucosamine residue are presumed [5,16,17] to interact with WGA, the lectin of A. brasilense [18] may be involved in the reception of WGA. It is a cell surface glycoprotein which incorporates N-acetyl-D-glucosamine.…”

“…Plant lectins attract wide attention as leguminous root lectins are implicated in information exchange in the Rhizobium-legume symbiosis [4]. It has been shown that wheat germ agglutinin (WGA), being 286 the most studied lectin among a number of closely related graminae lectins, binds to ceils of Azospirillum brasilense and Azospirillum lipoferum [5,6]• Besides being found in embryos, WGA is also found in the caps and tips of adventitious roots [7]. The root cap cells are easily sloughed off and eroded.…”

Recent advances of flow cytometry in fundamental and applied microbiology

“…Fluorescent lectins (table II), which bind to the carbohydrate moiety of glycoproteins and glycolipids on the cell membrane outer surface, are now popularly used in conjunction with, or in place of monoclonal antibodies, for the same applications as the latter. Lectins and FCM have been used to characterize the sugars present on the outer surface of the nitrogen-fixing bacterium, Azospirillum brasilense, which is possibly involved in the colonization of host tissue during plant-bacteria symbiotic association [210].…”