Ultrastructure and chemical composition of the sheath of Leptothrix discophora SP-6

Abstract

Light microscopy and transmission electron microscopy of thin sections and metal-shadowed specimens showed that the sheath of Leptothrix discophora SP-6 (ATCC 51168) is a tube-like extracellular polymeric structure consisting of a condensed fabric of 6.5-nm-diameter fibrils underlying a more diffuse outer capsular layer. In thin sections, outer membrane bridges seen to contact the inner sheath layer suggested that the sheath fabric was attached to the outer layer of the gram-negative cell wall. The capsular polymers showed an affinity for cationic colloidal iron and polycationic ferritin, indicating that they carry a negative charge. Cell-free sheaths were isolated by treatment with a mixture of lysozyme, EDTA, and N-lauroylsarcosine (Sarkosyl) or sodium dodecyl sulfate (SDS). Both Sarkosyl-and SDS-isolated sheaths were indistinguishable in microscopic appearance. However, the Mn-oxidizing activity of Sarkosyl-isolated sheaths was more stable than that of SDS-isolated sheaths. The Sarkosyl-isolated sheaths also contained more 2-keto-3-deoxyoctanoic acid and more outer membrane protein than SDS-isolated sheaths. The oven-dried mass of detergent-isolated sheaths represented approximately 9% of the total oven-dried biomass of SP-6 cultures; the oven-dried sheaths contained 38% C, 6.9%o N, 6% H, and 2.1% S and approximately 34 to 35% carbohydrate (polysaccharide), 23 to 25% protein, 8% lipid, and 4% inorganic ash. Gas-liquid chromatography showed that the polysaccharide was an approximately 1:1 mixture of uronic acids (glucuronic, galacturonic, and mannuronic acids and at least one other unidentified uronic acid) and an amino sugar (galactosamine). Neutral sugars were not detected.Amino acid analysis showed that sheath proteins were enriched in cysteine (6 mol%). The cysteine residues in the sheath proteins probably provide sulfhydryls for disulfide bonds that play an important role in maintaining the structural integrity of the sheath (D. Emerson and W. C. Ghiorse, J. Bacteriol. 175:7819-7827, 1993 (24,36,39).Leptothrix spp. are similar in physiology and cellular structure to closely related gram-negative aerobic heterotrophic pseudomonads (1, 2, 23, 55). Sheath formation and extracellular metal-oxidizing ability are the two major phenotypic criteria distinguishing them from closely related bacteria. The manganese-and iron-oxidizing activities of L. discophora SS-1 (ATCC 43182) are at least partly determined by metal-oxidizing proteins excreted by the cells in association with extracellular polymers (3, 10, 13). Presumably the excreted metaloxidizing proteins are associated with polymers located in the sheath. However, strain SS-1 irreversibly lost the ability to form a sheath soon after it was isolated (2). Recently we reported the isolation and maintenance of a new strain of L. discophora, SP-6 (ATCC 51168) (19). A sheathless variant, strain SP-6(sl) (ATCC 51169), also was isolated. Taxonomic studies (19,57) showed that strains SP-6 and SP-6(sl) are very similar but not identical to strain SS-1.

Silicon-Rich, Iron Oxide Microtubular Sheath Produced by an Iron-Oxidizing Bacterium, Leptothrix sp. Strain OUMS1, in Culture

“…Leptothrix species deposit copious amounts of iron hydroxide-encrusted sheaths in the environment where they grow [4]. Chemically active saccharic and proteinaceous groups in the secretions of bacteria are thought to play a key role in binding aqueous inorganic ions [4,9,11,12,[34][35][36][37]. Although Hashimoto et al [18] found a variety of chemical bonds such as Fe-O, Fe-O-Si, Fe-O-P and so on in Leptothrix sheaths, it is still unknown how these chemical bonds are formed in the sheaths: are the iron hydroxides and oxides formed first, then other ions bind to them or vice versa?…”

Direct Adherence of Fe(III) Particles onto Sheaths of Leptothrix sp. Strain OUMS1 in Culture

“…Indeed, the sheath material of Leptothrix contains organics such as polysaccharides, proteins, and lipids whose active groups are expected to play critical roles to bind aqueous-phase cations [3,6,18,19]. Chan et al [4] showed a strong correlation between the presence of acidic polysaccharides with carboxyl functional groups to the distribution of iron oxyhydroxides in Leptothrix sheath and Gallionella stalks.…”

Autolysis of Bacterial Cells Leads to Formation of Empty Sheaths by Leptothrix spp.

“…In a separate SEM-EDX observation, these particles were shown to contain Fe, O, P and Si as major elements, suggesting that they could aggregate via autooxidation of aquatic-phase elements and/or unidentified chemical bonds (Appendix Figure A1). When specimens cultured in SGPI broth for 3 days were observed with TEM, some sheaths, characterized by a thin or electron-dense thick wall, enclosed bacterial cells ( Figure 4A, arrows), while other sheaths contained degenerated and deformed cell remnants ( Figure 4A, dotted arrows), as reported for cultured L. discophora SP-6 by Emerson and Ghiorse [7]. As illustrated in a longitudinal section of a sheath ( Figure 4B), a body that maintained the shape of a cell (arrow) occasionally coexisted side-by-side with cell remnants (dotted arrow) within a sheath.…”

“…The formation of a sheath that contains bacterial cells and extracellular metal-oxidizing activities are the two major phenotypic criteria that distinguish Leptothrix species from closely related bacteria [1,7]. Although the presence of a sheath is thought to confer nutritional and ecological advantages to species of Sphaerotilus and Leptothrix and enables the bacterial cells to attach themselves to solid surfaces and protects them against parasites and predators [1,3], sufficient evidence to explain the significance of the sheath in these bacteria is still lacking.…”