Molecular-Based Analysis of Cellulose Binding Proteins Involved with Adherence to Cellulose by Ruminococcus albus

At the beginning of this project, it was clear that R. albus adhered tightly to cellulose and its efficient degradation of this polysaccharide was dependent on micromolar concentrations of phenylacetic acid (PAA) and phenylpropionic acid (PPA). The objectives for our research were: i) to identify how many different kinds of cellulose binding proteins are produced by Ruminococcus albus; ii) to isolate and clone the genes encoding some of these proteins from the same bacterium; iii) to determine where these various proteins were located and; iv) quantify the relative importance of these proteins in affecting the rate and extent to which the bacterium becomes attached to cellulose. BARD support has facilitated a number of breakthroughs relevant to our fundamental understanding of the adhesion process. First, R. albus possesses multiple mechanisms for adhesion to cellulose. The P.I.'s laboratory has discovered a novel cellulose-binding protein (CbpC) that belongs to the Pil-protein family, and in particular, the type 4 fimbrial proteins. We have also obtained genetic and biochemical evidence demonstrating that, in addition to CbpC-mediated adhesion, R. albus also produces a cellulosome-like complex for adhesion. These breakthroughs resulted from the isolation (in Israel and the US) of spontaneously arising mutants of R. albus strains SY3 and 8, which were completely or partially defective in adhesion to cellulose, respectively. While the SY3 mutant strain was incapable of growth with cellulose as the sole carbon source, the strain 8 mutants showed varying abilities to degrade and grow with cellulose. Biochemical and gene cloning experiments have been used in Israel and the US, respectively, to identify what are believed to be key components of a cellulosome. This combination of cellulose adhesion mechanisms has not been identified previously in any bacterium. Second, differential display, reverse transcription polymerase chain reaction (DD RT-PCR) has been developed for use with R. albus. A major limitation to cellulose research has been the intractability of cellulolytic bacteria to genetic manipulation by techniques such as transposon mutagenesis and gene displacement. The P.I.'s successfully developed DD RT- PCR, which expanded the scope of our research beyond the original objectives of the project, and a subset of the transcripts conditionally expressed in response to PAA and PPA have been identified and characterized. Third, proteins immunochemically related to the CbpC protein of R. albus 8 are present in other R. albus strains and F. intestinalis, Western immunoblots have been used to examine additional strains of R. albus, as well as other cellulolytic bacteria of ruminant origin, for production of proteins immunochemically related to the CbpC protein. The results of these experiments showed that R. albus strains SY3, 7 and B199 all possess a protein of ~25 kDa which cross-reacts with polyclonal anti-CbpC antiserum. Several strains of Butyrivibrio fibrisolvens, Ruminococcus flavefaciens strains C- 94 and FD-1, and Fibrobacter succinogenes S85 produced no proteins that cross-react with the same antiserum. Surprisingly though, F. intestinalis strain DR7 does possess a protein(s) of relatively large molecular mass (~200 kDa) that was strongly cross-reactive with the anti- CbpC antiserum. Scientifically, our studies have helped expand the scope of our fundamental understanding of adhesion mechanisms in cellulose-degrading bacteria, and validated the use of RNA-based techniques to examine physiological responses in bacteria that are nor amenable to genetic manipulations. Because efficient fiber hydrolysis by many anaerobic bacteria requires both tight adhesion to substrate and a stable cellulosome, we believe our findings are also the first step in providing the resources needed to achieve our long-term goal of increasing fiber digestibility in animals.