Early on, it was recognized that cellulosomes were intimately associated with the bacterial cell surface of C. thermocellum (88, 108, 120). As mentioned above, the molecular mechanism for this phenomenon was later determined. The type-II cohesin-dockerin complex fixes the primary scaffoldin and its complement of enzymes to one of the anchoring proteins, which also contains an S-layer homology (SLH) module that mediates attachment to the peptidoglycan of the cell surface (Figure 13.1) (36, 40-43, 121). Other species of bacteria, e. g., Acetivibrio cellulolyticus and Bacteroides cellulosolvens, possess anchoring protein(s) that exhibit similar type-II cohesins and SLH modules for attachment of the enzyme-bearing primary scaffoldin to the cell surface (88, 108, 120, 122-124). However, this is not the only strategy. As described above, ScaE of R. flavefaciens contains a sortase signal motif through which the scaffoldin is covalently attached to the peptidoglycan (Figure 13.2) (68). Other bacteria, notably C. cellulolyticum and C. cellulovorans, are also known to bear cellulosomes on their cell surface (125, 126), but there is as yet no evidence for involvement of an SLH module or sortase-mediated covalent attachment. The known scaffoldin of both of the latter species includes two and four copies of a hydrophilic domain (37, 46), which, in the case of C. cellulovorans, has been implicated in a cell-attachment function (127), but the true function of this particular domain awaits further experimental verification.

The attachment of the cellulosome to the cell surface, coupled with the CBM of the pri­mary scaffoldin, inferred that the cellulosome is intimately involved in cell adhesion to the insoluble substrate. Indeed, the initial demonstration of the cellulosome in C. thermocel — lum was assisted by the isolation of an adherence defective mutant that was also impaired in its arrangement of cellulosomes on the cell surface (108). Cellulosomes are packaged into polycellulosome-containing protuberances (Figure 13.4), which undergo a dramatic conformational change upon interaction with the cellulose surface (27-29). The protuber­ances thus protract to form “contact corridors” laden with fibrous material that connect between cellulose-bound cellulosomes and the cell surface thus promoting cell uptake of the cellulose degradation products. The cellulosomal enzymes are subject to potent product inhibition (89,111), and cell uptake and assimilation of the cellulose-degradation products (i. e., cellobiose and higher order soluble cellodextrins) relieves the inhibitory effect, thereby allowing facile hydrolysis of the insoluble substrate to proceed unhindered. Moreover, addi­tional saccharolytic bacteria, which share the same ecosystem with the polymer-degrading strains (e. g., C. thermocellum), further purge the immediate environment of the inhibitory sugars, thus propagating substrate degradation even more (19, 128).

The cellulosome-producing cellulolytic bacterium A. cellulolyticus is characterized by an especially elaborate surface morphology; when grown on cellulosic substrates, the

Resting

Protuberances

Protracted

Protuberance

Cellulosomes

.’Ceifutose.

Figure 13.4 Proposed interaction of cellulosome-producing bacteria with cellulosic substrates. (Panel A) Schematic representation of the interaction of cell-surface cellulosome-laden protuberance-like organelles with the cellulose surface. 1) Prior to contact with the cellulosic substrate, the protuberances are compacted in a resting state;2) upon cell-substrate contact, the protuberances protract whereby the cellulosomes interact directly with the cellulose surface;3) eventually, the cell detaches from the substrate, leaving cellulosome clusters on the cellulose surface. (Panel B) Scanning electron microscopy (SEM) of Acetivibrio cellulolyticus bound to cellulose, showing the presence of large characteristic protuberance-like structures on the cell surface. The cellulose-bound cells appear to be connected to the substrate via structural extensions of the cell-surface protuberances.

cellulosome-bearing protuberances inundate the surface of the substrate, a process which appears to assist the cells in overcoming the inherent recalcitrance of the cellulose (Figure 13.4B). During stationary phase growth on cellulose, the cells detach from the substrate, leaving a legacy of attached cellulosomes that continue their hydrolytic action in the absence of the parent cell (18, 28, 129, 130).