Ruminococcus albus produces fimbria-like structures that are involved with the bacterium's adhesion to cellulose. The subunit protein has been identified in strain 8 (CbpC) and strain 20 (GP25) and both are type IV fimbrial (Pil) proteins. The presence of a pil locus that is organized similarly in both strains is reported here together with the results of an initial examination of a second Pil protein. Downstream of the cbpC/gp25 gene (hereafter referred to as pilA1) is a second pilin gene (pilA2). Northern blot analysis of pilA1 and pilA2 transcripts showed that the pilA1 transcript is much more abundant in R. albus 8, and real-time PCR was used to measure pilA1 and pilA2 transcript abundance in R. albus 20 and its adhesion-defective mutant D5. Similar to the findings with R. albus 8, the relative expression of pilA1 in the wild-type strain was 73-fold higher than that of pilA2 following growth with cellobiose, and there were only slight differences between the wild-type and mutant strain in pilA1 and pilA2 transcript abundances, indicating that neither pilA1 nor pilA2 transcription is adversely affected in the mutant strain. Western immunoblots showed that the PilA2 protein is localized primarily to the membrane fraction, and the anti-PilA2 antiserum does not inhibit bacterial adhesion to cellulose. These results suggest that the PilA2 protein plays a role in the synthesis and assembly of type IV fimbriae-like structures by R. albus, but its role is restricted to cell-associated functions, rather than as part of the externalized fimbrial structure.
INTRODUCTIONThe initial step in the degradation of cellulosic biomass by many anaerobic micro-organisms requires the adhesion of the organism to this substrate. Adhesion facilitates maximal enzyme-substrate interaction and gives the adherent cells an advantage for substrate uptake, and can be a ratelimiting step in polysaccharide hydrolysis. In the gastrointestinal tracts of herbivores and ruminants, Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes are widely believed to coordinate degradation of the cellulosic component of plant biomass. Our understanding of the adhesion mechanism(s) employed by these three bacteria has been limited by the lack of genetic tools available to dissect the process(es), but recent studies have identified a variety of structures including novel cellulosebinding modules present in cellulosomes (Ding et al., 2001;Rincon et al., 2001Rincon et al., , 2003, cell-surface-associated cellulases (Devillard et al., 2004;Mitsumori & Minato, 2000;Malburg et al., 1997), cell-surface exopolysaccharides (Mosoni & Gaillard-Martinie, 2001) and other uncharacterized cellulosebinding proteins (CBPs) (Gong et al., 1996;Miron & Forsberg, 1999). These different adhesion mechanisms are described in recent reviews (Morrison & Miron, 2000;Miron et al., 2001).Recent work in our laboratory has established that type IV Abbreviation: CBP, cellulose-binding protein.