Role of disulfide bonds in maintaining the structural integrity of the sheath of Leptothrix discophora SP-6

Abstract

Isolated sheaths of Leptothrix discophora SP-6 (ATCC 51168) were tested for susceptibility to degradation by a variety of chemical denaturants and lytic enzymes and found to be resistant to many reagents and enzyme treatments. However, disulfide bond-reducing agents such as dithiothreitol (DTT), P-mercaptoethanol, sodium cyanide, and sodium sulfite degraded the sheath, especially at elevated pH (pH 9) and temperature (50°C). DTT and 1-mercaptoethanol caused more rapid degradation of the sheath than cyanide or sulfite. Treatment of the sheath with 1 N NaOH resulted in rapid breakdown, while treatment with 1 N HCI resulted in slow but significant hydrolysis. Transmission electron microscopy showed that the 6.5-nm fibrils previously shown to be an integral structural element of the sheath fabric (D. Emerson and W. C. Ghiorse, J. Bacteriol. 175:7808-7818, 1993) were progressively dissociated into random masses during DTT-induced degradation. Quantitation of disulfide bonds with DTT showed that the sheaths contained approximately 2.2 ,umol of disulfides per mg of sheath protein. Reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) showed that sheaths also contained approximately 0.8 ,umol of free sulfhydryls per mg of protein. A sulfhydryl-specific fluorescent probe (fluorescein 5-maleimide) showed that the free sulfhydryls in sheathed cell filaments were evenly distributed throughout the sheath. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of ['4C]iodoacetamide-labeled sheaths and DTT-dissociated sheath fibril suspensions showed that the majority of "4C-labeled sulfhydryls in the sheaths did not enter the gel. However, low-molecular-mass silver-staining bands (14 to 45 kDa) did appear in the gels after iodoacetic acid or iodoacetamide alkylation of the dissociated fibrils. These bands did not stain with Coomassie blue. Their migration in gels was slightly affected by digestion with pronase. The fibrils contained 20 to 25% protein. These results confirm that the sheath fibrils consist of high-molecular-weight heteropolysaccharide-protein complexes. We hypothesize that proteins in the fibril complexes provide interfibril cross-linking to maintain the structural integrity of the sheath.Several previous studies of eubacterial sheaths have focused on their ultrastructure and gross chemical composition (1,20,31,33,34,42). These tube-like extracellular structures are composed principally of heteropolysaccharides in a fabric of fine fibrils, which appear to be the primary structural elements (11,20,31). Bacterial sheaths are very durable structures. Indeed, fossilized eubacterial sheaths are frequently reported in the geologic record (17). It seems likely that covalent cross-linking could play a major role in maintaining the structure. However, the manner by which the macromolecular components of the sheath fabric are cross-linked has not been elucidated.In an accompanying paper (15), we show that the fine structure and gross chemical composition of the sheath of Leptothrix discophora SP-6 clos...

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

“…They appeared to be integral structural elements of the inner condensed sheath layer. This interpretation was confirmed in later sheath dissociation studies (20). No subunit structure was FIG.…”

“…Here we confirm that the ultrastructure and chemical com-position of intact cellular filaments and cell-free sheaths of L. discophora sheath proteins contain a high relative abundance of cysteine residues which provide sulfhydryls for the disulfide bonding responsible for maintaining the structural integrity of the sheath (20). (A preliminary report of this work was presented previously [18].)…”

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?…”

Structural Analysis of the Fundamental Polymer of the Sheath Constructed bySphaerotilus natans

“…The uniqueness of the two sheaths may reflect the fact that they are not degraded by the commonly available proteases and glycosidases. 9,25) These structures are drawn based on assumptions that every pentasaccharide repeating unit has a peptide side chain and that the amino sugar residues free of the peptide side chain are not derivatized. The residue numbers in parentheses correspond to those indicated in Table 1.…”