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Cellulase system of a free-living mesophilic Clostridium species and interactions with a commensal bacterium
Abstract
The cellulase system of Clostridium strain C7, a free-living, mesophilic bacterium that was isolated from freshwater sediment, was studied. The enzymatic activity responsible for crystalline cellulose degradation (Avicelase activity) by Clostridium strain C7 was present in culture supernatant fluid. The extracellular cellulase system of strain C7 was fractionated by Sephacryl S-300 gel filtration, and the fractions were assayed for Avicelase and carboxymethylcellulase activities. The Avicelase activity coincided with an absorbance (A$\sb{280}$) peak which eluted in the 700,000 M$\sb{\rm r}$ region. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecylsulfate-PAGE analysis of the 700,000 M$\sb{\rm r}$ fractions showed that Avicelase was present as a multiprotein aggregate. An Avicelase-deficient mutant of strain C7 (strain LS), which was not capable of degrading crystalline cellulose, lacked the Avicelase-active 700,000 M$\sb{\rm r}$ peak. The results indicated that an exctracellular 700,000 M$\sb{\rm r}$ multiprotein complex, consisting of at least 15 proteins, is utilized by Clostridium strain C7 for the hydrolysis of crystalline cellulose. Electron microscopic analysis of the 700,000 M$\sb{\rm r}$ protein complex revealed particles apparently composed of subunit structures. In addition to crystalline cellulose, the 700,000 M$\sb{\rm r}$ protein complex hydrolyzed carboxymethylcellulose, cellodextrins, xylan, and xylooligosaccharides. Main products were celobiose from cellulose, and xylobiose from xylan. The Avicelase activity of the 700,000 M$\sb{\rm r}$ complex required Ca$\sp{2+}$ and dithiothreitol. The temperature and pH optima for Avicelase and xylanase activities were 45 and 55$\sp\circ$C, and 5.6 and 6.5, respectively. The results indicated that the multiprotein complex had multiple enzyme activities and that, possibly, the hydrolysis of each substrate involved different active sites. It was thought likely that, in nature, strain C7 derives advantages from releasing its enzyme complex into the external environment. To study this possibility, a noncellulolytic bacterium (strain W1) was isolated from freshwater mud samples, and was grown in stable coculture with strain C7 in a chemically defined medium containing cellulose as the fermentable substrate. The results indicated the occurrence of mutually beneficial interactions in which cellulolytic strain C7 provided fermentable substrates (cellulose-derived sugars) for strain W1 cell growth, and strain W1 produced two vitamins (d-biotin and p-aminobenzoic acid) required for the growth of cellulolytic strain C7.
Subject Area
Microbiology|Ecology
Recommended Citation
Cavedon, Katherine, "Cellulase system of a free-living mesophilic Clostridium species and interactions with a commensal bacterium" (1990). Doctoral Dissertations Available from Proquest. AAI9022669.
https://scholarworks.umass.edu/dissertations/AAI9022669