Ruminococcus albus 8 Mutants Defective in Cellulose Degradation Are Deficient in Two Processive Endocellulases, Cel48A and Cel9B, Both of Which Possess a Novel Modular Architecture

Abstract

The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B 2 (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.

Kinetic Studies of Polyhydroxybutyrate Granule Formation in Wautersia eutropha H16 by Transmission Electron Microscopy

“…In any of these scenarios, the mediation elements would not be observed at the late stage of PHB biosynthesis. The concept of scaffolds has been exemplified by the cellulosome, the multicomponent, multienzyme complex from anaerobic cellulolytic bacteria that is used to degrade plant cell wall polysaccharides (2,8,21). The main component of the cellulosome is a multienzyme integrating protein unit called scaffoldin.…”

Rumen microbial (meta)genomics and its application to ruminant production

“…R. albus 7 has 29 dockerin-containing proteins, of which 25 are on the main The smaller number of dockerin proteins presumably indicates that R. albus has a less well-developed cellulosome compared with R. flavefaciens. The draft R. albus 8 genome sequence has been used previously to identify proteins involved in cellulose adhesion and degradation (Devillard et al, 2004). Compared with the wild-type strain, mutant strains of R. albus 8 that are defective in cellulose degradation lack two largemolecular-weight proteins, which were identified as endocellulases Cel48A and Cel9B.…”

Laboratory Evolution and Multi-platform Genome Re-sequencing of the Cellulolytic Actinobacterium Thermobifida fusca

“…Of the identified cellulases, three are endocellulases, Cel9B, Cel6A, and Cel5A (21)(22)(23)(24)(25), two of them are exocellulases, Cel6B and Cel48A (24,25), and one is a novel processive endocellulase, Cel9A (26). Importantly, CelR (Tfu_0938) has previously been identified as a regulator of several cellulase genes, is a member of the lactose repressor family, and binds cellobiose with a mM K d (27)(28)(29).…”