Adherence of the Gram-Positive Bacterium Ruminococcus albus to Cellulose and Identification of a Novel Form of Cellulose-Binding Protein Which Belongs to the Pil Family of Proteins

Abstract

ABSTRACT The adherence of Ruminococcus albus 8 to crystalline cellulose was studied, and an affinity-based assay was also used to identify candidate cellulose-binding protein(s). Bacterial adherence in cellulose-binding assays was significantly increased by the inclusion of either ruminal fluid or micromolar concentrations of both phenylacetic and phenylpropionic acids in the growth medium, and the addition of carboxymethylcellulose (CMC) to assays decreased the adherence of the bacterium to cellulose. A cellulose-binding protein with an estimated molecular mass following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ∼21 kDa, designated CbpC, was present in both cellobiose- and cellulose-grown cultures, and the relative abundance of this protein increased in response to growth on cellulose. Addition of 0.1% (wt/vol) CMC to the binding assays had an inhibitory effect on CbpC binding to cellulose, consistent with the notion that CbpC plays a role in bacterial attachment to cellulose. The nucleotide sequence of the cbpC gene was determined by a combination of reverse genetics and genomic walking procedures. The cbpC gene encodes a protein of 169 amino acids with a calculated molecular mass of 17,655 Da. The amino-terminal third of the CbpC protein possesses the motif characteristic of the Pil family of proteins, which are most commonly involved with the formation of type 4 fimbriae and other surface-associated protein complexes in gram-negative, pathogenic bacteria. The remainder of the predicted CbpC sequence was found to have significant identity with 72- and 75-amino-acid motifs tandemly repeated in the 190-kDa surface antigen protein of Rickettsia spp., as well as one of the major capsid glycoproteins of the Chlorella virus PBCV-1. Northern blot analysis showed that phenylpropionic acid and ruminal fluid increase cbpC mRNA abundance in cellobiose-grown cells. These results suggest that CbpC is a novel cellulose-binding protein that may be involved in adherence of R. albus to substrate and extends understanding of the distribution of the Pil family of proteins in gram-positive bacteria.