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

Abstract

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.

Studies of the Extracellular Glycocalyx of the Anaerobic Cellulolytic Bacterium Ruminococcus albus 7

“…In this regard, certain "cellulosespecific" genes (e.g., those encoding the major exoglucanase CelS and the scaffoldin protein CipA) in the phylogenetically related Clostridium thermocellum have been shown to be expressed during growth in cellobiose-limited chemostats at low dilution rates (Ͻ0.06 h Ϫ1 ), but not at higher dilution rates (45). Because R. albus 20 has been shown to contain a glycosylated protein, GP25 (since renamed PilA1) (41), that may be involved in binding of this strain to cellulose (33), it is worth considering if the monosaccharide residues observed on acid treatment of the R. albus 7 cellulose fermentation were merely released from a similar glycoprotein. However, because the amount of nonglucan carbohydrate present in the cellulose fermentation residues was substantial (Table 1), and because boiling of the residue in neutral detergent solution resulted in removal of essentially all of the protein but retention of this carbohydrate, it is likely that the nonglucan carbohydrate in R. albus 7 is present in a form distinct from that associated with a GP25 homolog, although its exact form and distribution remain to be elucidated.…”

“…However, even among wild-type R. albus isolates, the relationship between adherence and cellulolytic capability varies substantially by strain, leading Miron et al and Morrison and Miron (27,32) to suggest that multiple mechanisms may be involved in adherence. Structures thought to be involved in adherence by this species include pilin-like proteins associated with fimbriae (32,38,40,41), CBMs of polysaccharide hydrolases (8,27,28,32,52), and an extracellular glycocalyx (6,8,33,37,49). Despite the importance of adherence in anaerobic cellulolysis, the characteristics of these glycocalyces are largely unknown.…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…The two genes are 73% identical, but analysis of mRNA levels revealed that pilA2 was expressed at a level 73-fold greater than that of pilA2. While transcripts can be monocistronic (pilA1 only) or dicistronic, the former is predominant (325). Localization studies showed that while the pilus is composed of PilA1, PilA2 is confined to membrane fractions.…”

“…Ruminococcus albus has two pilA loci, pilA1 and pilA2 (325). The pilA2 gene is located ϳ115 bp downstream of the pilA1 stop codon, with a putative hairpin loop separating them.…”

Muramidases found in the foregut microbiome of the Tammar wallaby can direct cell aggregation and biofilm formation

“…Adhesion mechanisms and biofilm formation on surfaces by commensal gut bacteria are crucial for many ecological functions, including plant biomass degradation, protection against environmental fluctuations and/or pathogens, and for establishing host-microbe interactions (Costerton et al, 1995). Various mechanisms of cell adhesion to plant and epithelial surfaces have now been elucidated, which include surface-associated carbohydrate-binding modules, glycocalyx containing extracellular polymeric substances and organelle-type structures (for example, Pegden et al, 1998;Miron, 2000, Miron et al, 2001;Mosoni and Gaillard-Martinie, 2001;Rakotoarivonina et al, 2005;Weimer et al, 2006). In other microbial environments, the extracellular polymeric substance matrix, which can constitute up to 90% of the biofilm biomass, is a complex mixture of exopolysaccharides, proteins and other macromolecules (Sutherland, 2001).…”