Genome-wide analysis of acetivibrio cellulolyticus provides a blueprint of an elaborate cellulosome system

Abstract

Background: Microbial degradation of plant cell walls and its conversion to sugars and other byproducts is a key step in the carbon cycle on Earth. In order to process heterogeneous plant-derived biomass, specialized anaerobic bacteria use an elaborate multi-enzyme cellulosome complex to synergistically deconstruct cellulosic substrates. The cellulosome was first discovered in the cellulolytic thermophile, Clostridium thermocellum, and much of our knowledge of this intriguing type of protein composite is based on the cellulosome of this environmentally and biotechnologically important bacterium. The recently sequenced genome of the cellulolytic mesophile, Acetivibrio cellulolyticus, allows detailed comparison of the cellulosomes of these two select cellulosome-producing bacteria.

Results: Comprehensive analysis of the A. cellulolyticus draft genome sequence revealed a very sophisticated cellulosome system. Compared to C. thermocellum, the cellulosomal architecture of A. cellulolyticus is much more extensive, whereby the genome encodes for twice the number of cohesin- and dockerin-containing proteins. The A. cellulolyticus genome has thus evolved an inflated number of 143 dockerin-containing genes, coding for multimodular proteins with distinctive catalytic and carbohydrate-binding modules that play critical roles in biomass degradation. Additionally, 41 putative cohesin modules distributed in 16 different scaffoldin proteins were identified in the genome, representing a broader diversity and modularity than those of Clostridium thermocellum. Although many of the A. cellulolyticus scaffoldins appear in unconventional modular combinations, elements of the basic structural scaffoldins are maintained in both species. In addition, both species exhibit similarly elaborate cell-anchoring and cellulosome-related gene- regulatory elements.

Conclusions: This work portrays a particularly intricate, cell-surface cellulosome system in A. cellulolyticus and provides a blueprint for examining the specific roles of the various cellulosomal components in the degradation of complex carbohydrate substrates of the plant cell wall by the bacterium.

Figure 1

Modular architecture of the array of scaffoldins identified in the A. cellulolyticus CD2 genome and their homologs from C. thermocellum ATCC 27405. Putative A. cellulolyticus scaffoldins were identified bioinformatically (see Materials and Methods for their accession numbers). Binding specificities of the indicated (black spots) cohesin and dockerin modules were determined previously [17-19]. The sca gene cluster is framed in a shaded box. All proteins have an N-terminal signal peptide except for ScaI. Acronyms: GH9, family-9 glycoside hydrolase; CBM(n), carbohydrate-binding module (family number); Cu, Copper amine oxidase; FN3, Fibronectin type III domain; Peptidase, S8 subtilisin-like peptidase; PPC, bacterial pre-peptidase C-terminal domain; Rhs, Rhs repeat domain. Accession numbers of the A. cellulolyticus scaffoldins are: [GenBank: ZP_09464033-30 (ScaA-D), ZP_09465494 (ScaE), ZP_09464236 (ScaF), ZP_09464788 (ScaG), ZP_09462752 (ScaH), ZP_09463446 (ScaI), ZP_09462222 (ScaJ), ZP_09464725 (ScaK), ZP_09464968 (ScaL), ZP_09463433 (ScaM), ZP_09463827 (ScaN), ZP_09462124 (ScaO), ZP_09461865 (ScaP)]. Accession numbers of the C. thermocellum scaffoldins are: [GenBank: CAA47840 (CipA), YP_001039467 (OlpB), ABN54275 (Orf2p), YP_001039469 (OlpA), YP_001037164 (Cthe_0736), YP_001037732 (SdbA), YP_001036883 (OlpC) and YP_001037163 (Cthe_0735)]

Figure 2

Relationship of all cohesin modules from A. cellulolyticusand C. thermocellum. Sequence-based dendrogram of cohesin modules from A. cellulolyticus (red) and C. thermocellum (blue). See scheme and key in Figure 1. Only significant bootstrap values are shown

Figure 3

Sequence conservation pattern of dockerin modules. The two internal dockerin repeats of A. cellulolyticus (based on 137 sequences) and C. thermocellum (71 sequences) are represented by sequence logos. Positions of calcium binding residues are shown in cyan, and putative recognition residues are shown in yellow