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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), beta-mercaptoethanol, sodium cyanide, and sodium sulfite degraded the sheath, especially at elevated pH (pH 9) and temperature (50 degrees C). DTT and beta-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 HCl 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 mumol of disulfides per mg of sheath protein. Reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) showed that sheaths also contained approximately 0.8 mumol 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 [14C]iodoacetamide-labeled sheaths and DTT-dissociated sheath fibril suspensions showed that the majority of 14C-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.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Emerson D,Ghiorse WC

doi

10.1128/jb.175.24.7819-7827.1993

subject

Has Abstract

pub_date

1993-12-01 00:00:00

pages

7819-27

issue

eissn

0021-9193

issn

1098-5530

journal_volume

175

pub_type

杂志文章

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