Downloaded from www.microbiologyresearch.org by IP: 93.91.26.109 On: Fri, 23 Oct 2015 20:34:20 Microbiology (2002), 148, 1871–1880 Printed in Great Britain Adhesion to cellulose of the Gram-positive bacterium Ruminococcus albus involves type IV pili Harivony Rakotoarivonina, 1 Gre  gory Jubelin, 1 Michel Hebraud, 2 Brigitte Gaillard-Martinie, 1 Evelyne Forano 1 and Pascale Mosoni 1 Author for correspondence : Pascale Mosoni. Tel : 33 4 73 62 40 45. Fax: 33 4 73 62 45 81. e-mail : pmosoniclermont.inra.fr Unite  de Microbiologie 1 and Unite  de Recherches sur la Viande, Equipe Microbiologie 2 , INRA, Centre de Recherches de Clermont- Ferrand-Theix, 63122 Saint- Gene  s-Champanelle, France This study was aimed at characterizing a cell-surface 25 kDa glycoprotein (GP25) that was previously shown to be underproduced by a spontaneous adhesion-defective mutant D5 of Ruminococcus albus 20. An antiserum against wild-type strain 20 was adsorbed with the mutant D5 to enrich it in antibodies ‘ specific ’ to adhesion structures of R. albus 20. The resulting antiserum, called anti-Adh serum, blocked adhesion of R. albus 20 and reacted mainly with GP25 in bacterial and extracellular protein fractions of R. albus 20. The N-terminal sequence of purified GP25 was identical to that of CbpC, a 21 kDa cellulose- binding protein (CBP) of R. albus 8. The nucleotide sequence of the gp25 gene was determined by PCR and genomic walking procedures. The gp25 gene encoded a protein of 165 aa with a calculated molecular mass of 16 940 Da that showed 729 % identity with CbpC and presented homologies with type IV pilins of Gram-negative pathogenic bacteria. Negative-staining electron microscopy revealed fine and flexible pili surrounding R. albus 20 cells while mutant cells were not piliated. In addition, immunoelectron microscopy showed that the anti-Adh serum probing mainly GP25, completely decorated the pili surrounding R. albus 20, thereby showing that GP25 was a major pilus subunit. This study shows for the first time the presence of pili at the surface of R. albus and identifies GP25 as their major protein subunit. Though GP25 was not identified as a CBP, isolated pili were shown to bind cellulose. In conclusion, these pili, which belong to the family of type IV pili, mediate adhesion of R. albus 20 to cellulose. Keywords : adhesion-defective mutant, cell-surface glycoprotein, cloning of type IV pilin, immunoelectron microscopy INTRODUCTION Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes are the three cellulolytic bac- terial species considered today to play a major role, with rumen fungi, in fibre breakdown in the rumen of herbivores (Lee et al., 2000). They have been shown to adhere to cellulose and to possess cellulolytic and xylanolytic enzymes that enable them to efficiently ................................................................................................................................................. Abbreviations : CBP, cellulose-binding protein ; GP25, cell-surface 25 kDa glycoprotein. The EMBL accession number for the sequence reported in this paper is AJ416469. degrade plant cell wall polysaccharides (Forsberg et al., 2000). R. albus was shown to be far more abundant than either R. flavefaciens and F. succinogenes in the bovine rumen whatever diet the animals were fed (Weimer et al., 1999). In vitro studies confirmed that R. albus out- competed the two other species and became predomi- nant in cultures where the three species were grown in the presence of cellulose as a growth substrate, alone or associated with non-cellulolytic species (Chen & Weimer, 2001). These studies tend to show that R. albus, R. flavefaciens and F. succinogenes do not act synergistically, but rather compete for their substrate, and that R. albus displays several ecological advantages that allow it to compete successfully. We previously showed that R. albus surpassed R. flavefaciens and 0002-5356  2002 SGM 1871