Multiplicity and organisation of plant cell wall degrading enzymes in Ruminococcus flavefaciens 17
The Gram-positive, strictly anaerobic bacterium, R. flavefaciens, plays an important role in the degradation of plant cell wall polysaccharides in the rumen. There is a paucity of information available, however, regarding the multiplicity and organisation of R. flavefaciens cellulolytic and xylanolytic enzyme systems. A technique involving PCR amplification of DNA with primers designed from conserved sequences, followed by hybridisation of the PCR products to chromosomal DNA, has led to an estimate of xylanase gene multiplicity in R. flavefaciens. The xylanase-specific primers were also useful in the isolation and sequencing of a partial xylanase gene, xynC. Although R. flavefaciens 17 appears to produce a cellulose-binding enzyme-complex, none of the individual enzymes examined was found to bind cellulose in isolation. However, a 210 kDa protein which is present in the complex was shown to bind cellulose after isolation from a renatured SDS-gel. In order to look for genetic evidence for a cellulose-binding mechanism, sequencing of the R. flavefaciens 17 endoglucanase gene, endA, was completed from PCR products. The carboxy-terminus of the predicted endA product consists of a domain which is similar to dockerins found in Clostridium thermocellum polysaccharidases. Homologous domains are also found in the R. flavefaciens xylanases, XynB and XynD. As the C. thermocellum dockerin domains mediate binding to the 210 kDa scaffolding protein in the cellulosome complex, it is likely that the R. flavefaciens domains play a similar role in assembly of a cellulosome-like complex (Lamed and Bayer, 1994). A gene which maps approximately 1.5 kb downstream from endA on the R. flavefaciens 17 chromosome was sequenced and found to be homologous to nifS genes from nitrogen-fixing bacteria (Zheng et al, 1993). The R. flavefaciens NifS product catalyses the production of sulphur from cysteine, and is suspected to partake in the assembly of iron-sulphur clusters. The precise role of NifS is not yet known, but may be related to the degradation of crystalline cellulose.