In the early 1980s, Lamed and Bayer proposed the term ‘cellulosome’ to describe the discrete multi-enzyme organisation of the cellulose-degrading complex from the anaerobic
bacterium Clostridium thermocellum [169-171]. Cellulosome systems and their organisation
have been extensively studied in other Gram-positive anaerobes belonging to clostridial and ruminococcal species [64]. Initially, it was believed that the cellulosomal complexes exclusively degrade crystalline cellulosic substrates, but it was soon recognised that bacterial cellulosomes contain an array of CAZyme modules (such as GHs, PLs and CEs) with hemicellulase and even pectinase activities [172-174]. Moreover, additional types of enzymes (e.g. peptidases), serpins, and putative structural proteins also appear to be components of cellulosomes.
The cellulosomal organisation of fibre-degrading enzymes was found in anaerobic fungi
from genera Neocallimastix, Piromyces and Orpinomyces, as well as in many representatives of
Clostridiales [64, 175]. For some of these bacteria (Bacteroides cellulosolvens ATCC35603,
Clostridium thermocellum ATCC27405 and DSM1313, C. cellulolyticum H10 ATCC 35319, C. josui, C. clarifavum DSM19732, Clostridium sp. BNL 1100) a recent re-classification to genus
Ruminiclostridium [176] and family Ruminococcaceae [177] has been proposed. Cellulosomal
organisation was also found in other Ruminococcaceae (Acetivibrio cellulolyticus CD2,
Ruminococcus flavefaciens FD-1 and 17) and Clostridiaceae (C. cellulovorans 743B, non-
cellulolytic bacterium C. acetobutylicum ATCC824 and EA2018) [64, 175].
The cellulosome is an intricate extracellular multisubunit complex with an average molecular mass of 2 MDa, involved in the efficient degradation of crystalline cellulose and other associated polysaccharides [175, 178]. The main cellulosomal components are large non- catalytic polypeptides, termed scaffoldins [179], that spatially integrate CBMs and catalytic domains of enzymes and other cellulosomal components into a single functional entity. The scaffoldin subunits are composed of multiple copies of cohesin modules, often in combination with other modules. Scaffoldins may play multiple roles, such as: integration of dockerin module-bearing catalytic subunits into the cellulosomal complex through interaction with its complementary cohesin modules; anchoring of the cellulosome to the cell wall by virtue of its C-terminal dockerin or surface (S)-layer homology (SLH) module, and targeting of the
43 fibrolytic bacterium to the carbohydrate substrate by its CBM. The high-affinity cohesin-
dockerin interaction (Ka ~10
9
– 1012 M-1) is involved in the specific integration of polysaccharide hydrolases into the cellulosome complex, determining the supramolecular architecture of the entire complex and providing its stability [175, 180, 181].
Dockerins are domains 70 amino acid residues in length, present in a single copy at the C-terminus of cellulosomal enzymes and are comprised of two duplicated segments (repeats), each containing a motif about 22 amino acid residues in size, spaced by a linker [182, 183]. The first 12 amino acid residues of each repeat are similar to the eukaryotic EF hand motif and are highly conserved. However, this homology is restricted only to the calcium-binding loop, containing the highly conserved calcium-binding amino acid residues asparagine or aspartate. This is consistent with the calcium-dependence of the cohesin-dockerin interactions [184, 185].
Cohesin modules, approximately 140 amino acid residues in length, are usually tandemly repeated between 4 and 11 times in scaffoldin proteins [186]. Several types of cohesin modules have been distinguished on the basis of their structure and binding specificity. Dockerins are, by definition, designated to be of same type as cohesins they are interacting with. Type I cohesins can be found in the ‘primary’ (enzyme-integrating) scaffoldins of the majority of described cellulosomes [187], while type II cohesins, originally discovered in a group of non- catalytic, cell-surface ‘anchoring’ scaffoldins of C. thermocellum, have been also detected in cellulosomes of A. cellulolyticum and B. cellulosolvens [188]. Type III cohesins have been
described only in Ruminococcus flavefaciens [189]. Regardless of type, comparative studies of
cohesin domains indicate that a common structural feature of the cohesin fold is a nine-stranded β-sandwich with an overall ‘jelly roll’ fold [190]. Both type II and type III cohesins may have several additional structural elements, such as α-helix and two ‘β-flaps’. The crystal structure of
the type III ScaE cohesin from R. flavefaciens showed that the additional α-helix is enveloped
by an extensive N-terminal loop, a feature not seen in any other known cohesins [191-193]. It has been suggested that these structural differences between different types of cohesins have a role in the type-specificity of cohesin-dockerin interactions.
Cohesins and dockerins are well conserved between species [128]. Species-specificity of the cohesin-dockerin interaction, reported for several pairs of clostridial species, depends on conserved amino acid residues at positions 11 and 12 of dockerin repeats [166, 194, 195] and the combination of both segments is important for target recognition [196]. However, several exceptions to this rule are known. For example, Cel9D-Cel44A or Xyn11A dockerin modules of
C. thermocellum can interact with cohesinsfrom C. josui [196, 197]
Cohesins and dockerins were long considered the signature domains for identification of the cellulosome-producing microbes. However, the extensive bioinformatic mining of genomes of Bacteria, Archaea and primitive eukaryotes identified putative non-cellulosomal cohesin and dockerin modules in proteins with unknown or non-fibrolytic predicted functions,
suggesting that the cellulosomal paradigm may be the exception rather than the rule for the use of these modules in the three domains of life [198].
Although it was initially proposed that the main cultured representatives of rumen cellulolytic bacteria could use cellulosome-like complexes for the initial adhesion to, and degradation of substrates [59], these structures appear to be scarce. The cellulosomal
organisation and anchoring of fibre-degrading enzymes via scaffoldins has only been implicated
for several strains of R. flavefaciens [189, 190, 193, 199-201]. In contrast, R. albus [202] and F. succinogenes [15, 203] seem to use a direct cell-surface anchoring of their enzymes.
The cellulosome system of R. flavefaciens strain 17 is very elaborate (Figure 1.3). Its particular feature is a large number of cohesin-dockerin specificities and, in contrast with clostridial systems, not all enzyme-bound dockerins have the same binding specificity, enabling a more ordered arrangement of enzymes in the cellulosome complex [204]. The multiple
scaffoldin-encoding sca gene cluster consists of five genes (scaA-E), encoding proteins bearing
one or more cohesins. The cluster also includes the cttA gene, which encodes a cell wall- anchored substrate-binding protein containing two putative CBMs [205]. The cellulosome is
anchored to the cell wall covalently via a single cohesin (ScaE), and this is mediated through the
sortase mechanism. ScaE binds to the dockerin at the C-terminus of the large scaffoldin, ScaB, and its cohesins accommodate C-terminal dockerins of the smaller scaffoldin ScaA. The ScaA contains three cohesins, which can bind various dockerin-containing enzyme subunits.
Figure 1.3 Schematic overview of the Ruminococcus flavefaciens 17 cellulosome.
The scaffoldin ScaB, and CBM-containing protein CttA, are bound to the bacterial cell wall-
45 with the ScaA dockerins (red), thus increasing the number of components incorporated into the cellulosome. The ScaA cohesins (yellow) bind directly to a group of Cel44A-like enzyme containing dockerin (yellow) or alternatively, they bind to the dockerin (yellow) from ScaC scaffoldin. ScaC has a divergent cohesin type (blue) that recognises and incorporates a different set of dockerin containing enzymes and other components into the cellulosome. Figure taken from [175] with permission.