The Ruminococcus albus pilA1–pilA2 locus: Expression and putative role of two adjacent pil genes in pilus formation and bacterial adhesion to cellulose

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