Isolation, Cultural Maintenance, and Taxonomy of a Sheath-Forming Strain of Leptothrix discophora and Characterization of Manganese-Oxidizing Activity Associated with the Sheath

Leptothrix discophora SP-6 was isolated from the outflow reservoir of an artificial iron seep. Its sheathforming phenotype was maintained by slow growth in a mineral salts-vitamin-pyruvate medium under minimal aeration at 20 to 25°C. A sheathless variant, SP-6(sl), was isolated from smooth colonies that appeared on spread plates after rapid growth of SP-6 in well-aerated cultures. SP-6 and SP-6(sl) are closely related but not identical to the previously studied sheathless strain SS-1 (ATCC 43182). Increasing Mn²⁺ concentrations in the growth medium of SP-6 increased the phase density of the sheath, indicating increased Mn oxide deposition in the sheath. Electron microscopy of cultures grown without added Mn²⁺ revealed that the sheath consisted of a well-defined inner layer, 30 to 100 nm thick, and a diffuse outer capsular layer of variable thickness. Mn oxides were identified in the sheath by their characteristic ultrastructure, electron density, and X-ray-dispersive energy spectra. In heavily encrusted sheaths, the Mn oxides were evenly distributed in both layers of the sheath. Sheathed cells retained more Mn-oxidizing activity than did sheathless cells after washing with distilled, deionized water; the sheath retained some of its activity after an EDTA-lysozyme-detergent treatment which removed the cells. An ultrafiltration-dialysis procedure significantly increased the recovery of activity from spent media of SP-6 over that reported previously for SS-1 (L.F. Adams and W.C. Ghiorse, J. Bacteriol. 169:1279-1285, 1987). A 108-kDa Mn-oxidizing protein was identified in concentrated spent media of SP-6 and SP-6(sl), and the activity of the concentrates showed stability in detergents comparable to that of SS-1 and patterns of heat inactivation and chemical inhibition similar to those of SS-1.