Polysaccharide capsule of Escherichia coli: microscope study of its size, structure, and sites of synthesis

This report describes the structure, size, and shape of the uncollapsed polysaccharide capsule of Escherichia coli strain Bi 161/42 [O9:K29(A):H-], its ultrastructural preservation as well as the filamentous components of the isolated capsular material. In a temperature-sensitive mutant, sites were localized at which capsular polysaccharide is "exported" to the cell surface. The highly hydrated capsule of the wild-type cells was visible in the uncollapsed state after freeze-etching, whereas dehydration in greater than or equal to 50% acetone or alcohol caused the capsule to collapse into thick bundles. This was prevented by pretreatment of the cell with capsule-specific immunoglobulin G; the capsule appeared as a homogeneous layer of 250- to 300-nm thickness. The structural preservation depended on the concentration of the anti-capsular immunoglobulin G. Temperature-sensitive mutants, unable to produce capsular antigen at elevated temperatures, showed, 10 to 15 min after shift down to permissive temperature, polysaccharide strands with K29 specificity appearing at the cell surface at roughly 20 sites per cell; concomitantly, capsule-directed antibody started to agglutinate the bacteria. The sites at which the new antigen emerged were found in random distribution over the entire surface of the organism. Spreading of purified polysaccharide was achieved on air-water interfaces; after subsequent shadow casting with heavy metal, filamentous elements were observed with a smallest class of filaments measuring 250 nm in length and 3 to 6 nm in width. At one end these fibers revealed a knoblike structure of about 10-nm diameter. The slimelike polysaccharides from mutants produced filamentous bundles of greater than 100-microns length, with antigenic and phage-receptor properties indistinguishable from those of the wild-type K29 capsule antigen.