Genomes sequenced by the Consortium
Fibrobacter succinogenes S85 has long been known to degrade crystalline cellulose and plant cell walls at a rate exceeding that of most other microorganisms. Prior to genome sequencing, the process of cellulose hydrolysis by Fibrobacter spp. has been examined by recombinant DNA and cloning methods, as well as isolating mutant strains of F. succinogenes and F. intestinalis, which are defective in growth on cellulose. Collectively, these studies have shown that protein glycosylation is an important prerequisite for bacterial adhesion and measurable activity against crystalline cellulose. Moreover, immunoelectron microscopy has documented that a 180 kDa cellulose-binding glycoprotein is present within complexes on the cell surface, at the site of adhesion to cellulose.
These results support the idea that adhesion and cellulase activity probably resides in a complex(es) in Fibrobacter spp., but its formation seems dependent on protein glycosylation. Although the carboxy-terminal domains of the endoglucanases and xylanases might possess a role similar to the Clostridial Type I dockerins, their primary sequence is very different, and the functionality associated with these domains is unresolved. Taken together, these data show that Fibrobacter species have evolved to possess a novel, and catalytically superior cellulase system, dissimilar from the cellulosomal systems of gram-positive bacteria and fungi, and the free-form cellulases of aerobic bacteria and fungi. However, the genetics and supramolecular structure of the complex have not been elucidated.
Ruminococcus spp. are part of the Bacillus/Clostridium subphylum of Gram-positive eubacteria. A unique feature of cellulose degradation by this bacterium is its requirements for phenylacetic and phenylpropionic acids (PAA/PPA), both of which elicit changes in cell wall ultrastructure, cellulase complex assembly, and increase cell adhesion to cellulose. Prior to genome sequencing, a combination of functional proteomics, differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) and mutational analysis produced evidence suggesting that in addition to a cellulosome-like mechanism, the bacterium possesses other mechanisms for adhesion to plant surfaces.
A novel form of cellulose-binding protein has been isolated from multiple strains and shown to belong to the Pil-protein family, being most similar to the type 4 fimbrial proteins of gram-negative pathogenic bacteria. The genome sequence data has allowed the identification of a novel form of carbohydrate-binding module (CBM37) and despite numerous instances of coding sequences containing modules highly similar to Type I dockerin modules, no evidence has yet been produced for the presence of a gene(s) encoding a functional homolog of cohesin modules or a scaffoldin protein - the other critical structural component of the Clostridial-like cellulosomes. This is stark contrast to the closely related species Ruminococcus flavefaciens, which contains a number of genes encoding scaffoldin (cohesin) functions.
Modern US animal feeding practices are still dependant upon large amounts of grain, which reduces ruminal pH values to 6.0 and below. The increased acidity associated with high grain diets has long been known to inhibit the predominant cellulolytic bacteria, and reduce fiber degradation. Prevotella bryantii is one of the few fiber-degrading ruminal bacteria capable of sustained growth at low pH. A gene encoding a beta-1,4-endoglucanase was isolated from P. bryantii but was found to possess relatively poor catalytic activity towards insoluble forms of cellulose.
However, a hybrid "cellulase" was constructed by fusing the Thermomonospora fusca E2 cellulose-binding domain to the P. bryantii gene and shown to be much more active towards insoluble substrates. Despite the potential associated with reintroducing the recombinant gene in Prevotella spp. and evaluating fiber degradation at low ruminal pH, the progress has been slowed by several fundamental deficiencies in our knowledge of Prevotella genetics. An arduous and low-efficiency system for gene transfer is available for use with P. bryantii, but it has not been successfully used with other, numerically more predominant members of the genus. The acquisition of genome sequence data for these bacteria is perceived as offering the most expedient route to overcoming "roadblocks" to the creation and evaluation of recombinant Prevotella spp. that might improve ruminal fiber degradation at low pH.