Pan-Cellulosomics of Mesophilic Clostridia: Variations on a Theme

Abstract

The bacterial cellulosome is an extracellular, multi-enzyme machinery, which efficiently depolymerizes plant biomass by degrading plant cell wall polysaccharides. Several cellulolytic bacteria have evolved various elaborate modular architectures of active cellulosomes. We present here a genome-wide analysis of a dozen mesophilic clostridia species, including both well-studied and yet-undescribed cellulosome-producing bacteria. We first report here, the presence of cellulosomal elements, thus expanding our knowledge regarding the prevalence of the cellulosomal paradigm in nature. We explored the genomic organization of key cellulosome components by comparing the cellulosomal gene clusters in each bacterial species, and the conserved sequence features of the specific cellulosomal modules (cohesins and dockerins), on the background of their phylogenetic relationship. Additionally, we performed comparative analyses of the species-specific repertoire of carbohydrate-degrading enzymes for each of the clostridial species, and classified each cellulosomal enzyme into a specific CAZy family, thus indicating their putative enzymatic activity (e.g., cellulases, hemicellulases, and pectinases). Our work provides, for this large group of bacteria, a broad overview of the blueprints of their multi-component cellulosomal complexes. The high similarity of their scaffoldin clusters and dockerin-based recognition residues suggests a common ancestor, and/or extensive horizontal gene transfer, and potential cross-species recognition. In addition, the sporadic spatial organization of the numerous dockerin-containing genes in several of the genomes, suggests the importance of the cellulosome paradigm in the given bacterial species. The information gained in this work may be utilized directly or developed further by genetically engineering and optimizing designer cellulosome systems for enhanced biotechnological biomass deconstruction and biofuel production.

Keywords: cellulosomes; cohesin; dockerin; glycoside hydrolases; scaffoldin.

Figure 1

Similar and modular organization of the cellulosomal gene clusters (sca) of mesophiles. Schematic representation of the gene cluster harboring the major scaffoldin, and followed by genes coding for dockerin-containing cellulolytic enzyme, which are organized in a similar sequence along the gene cluster of the marked species. The major scaffoldin gene is represented by cip; numbers denote the family of glycoside hydrolases; X stands for the orfX gene; asterisks (*) mark draft genomes that have more than two contigs; slashes (//) indicate that the ORF may not be complete, because it was located at the end of contig.

Figure 2

Modular and domain architectures of the primary scaffoldins of mesophilic cellulosome-producing bacteria. Schematic representation of the functional protein modules comprising the primary scaffoldin protein of cellulosome-producing mesophiles. Slashes (//) denote the end of a contig. Asterisks (*) mark draft genomes that have more than two contigs. GenBank accession numbers for the scaffoldins are as follows: C. papyrosolvens, 325985039; C. cellulolyticum, AAC28899.2; Clostridium sp. BNL1100, 373945107; C. josui, 640241850; C. cellulovorans, 302578508; C. acetobutylicum, 336290364; C. acetobutylicum, 15894197; C. acetobutylicum, 325508325; C. saccharoperbutylacetonicum, 451784659; C. termitidis, 474480363; C. sufflavum, Ga0056032 and C. bornimense, 584458187. C. cellobioparum was omitted, because the gene encoding its scaffoldin was fragmented in the draft genome sequence.

Figure 3

Sequence conservation of the major σ^A-dependent promoters upstream of the respective cip gene cluster. (A) The σ^A (RpoD)-dependent promoter and cognate transcription start site (S1) have been experimentally identified as a major region of the C. cellulolyticum H10 cipC gene [52] and its orthologs [54]. The two T nucleotides of S1 are underlined, as well as sequences predicted to be −35, −16 and −10 elements of the cipC promoter; (B) aligned sequences are related to the recently identified RpoD-dependent promoter of the C. thermocellum cipA gene [54]. TSS2 is a transcriptional start site position, while −35 and −10 elements are elements of the cipA promoter. In both panels (A and B), 5′ UTR (untranslated regions) are shown partially, and numbers between the last nucleotide of each sequence and the predicted initial codon for methionine (Met) are provided. The two WebLogos were generated, with the sequences shown in each alignment, and they suggest putative promoter consensuses in the two groups of cellulolytic species. The promoter has two patterns of conservation, one in the related mesophiles, and a second in thermophiles and other complex cellulosomes (denoted ^† in designated species as follows). Cce, C. cellulolyticum; Cpa1, C. papyrosolvens DSM 2782; Cpa2, C. papyrosolvens C7; Csp, Clostridium sp. strain BNL1100; Cjo, C. josui; Ccb, C. cellobioparum; Cte, C. termitidis; Cth ^†, C. thermocellum DSM 1313; Cst ^†, C. straminisolvens JCM 21531; Ccl, C. clariflavum DSM 19,732; Ace ^†, Acetivibrio cellulolyticus CD2; Ccv, C. cellulovorans; Cac, C. acetobutylicum; Pce ^†, Pseudobacteroides (Bacteroides) cellulosolvens ATCC 35603 (DSM 2933). Asterisks (*) indicate sequenced positions with identical nucleotides.

Figure 4

Phylogenetic relation of cohesin modules from the major scaffoldins of mesophilic cellulolytic clostridia. Protein sequences of major scaffoldin cohesins were aligned and analyzed by PhyML. Bootstrap values are denoted, and branches below 80% bootstrapping were collapsed. Two major branches of the dendogram (red and blue) separate between C. acetobutylicum, C. cellulovorans, C. bornimense, and C. saccharoperbutylacetonicum from the other mesophiles.

Figure 5

Conserved sequence features of dockerin modules in cellulolytic species. Aligned sequences of the dockerin module within each species were visualized by WebLogo. Similar profiles of dockerins were observed among the species, in particularly, the conservation of putative cohesin–dockerin binding positions at the Ca-binding loop (in yellow). Number of aligned sequences species is marked in brackets. Dockerin segments (b–d at top) are labelled according to Pagès et al. [39]. C. bornimense was omitted, because it contains only five sequences.

Figure 6

Arrangement of cohesins and dockerins along the bacterial chromosomes of cellulosome-producing mesophiles. Cohesins (blue triangles) and dockerin modules (red triangles) were searched by BLAST and located on the bacterial chromosome. Known clusters of dockerins (the xyl-doc cluster) and the sca gene cluster are marked in blue and black rectangles, respectively, whereas most other dockerin-containing genes were distributed along the chromosome.

Figure 7

Frequency of CAZY modules identified in mesophiles. (A) Number of Carbohydrate-Active enZYmes (CAZyme modules) is denoted for each genome of the mesophilic clostridia. Precise numbers are available in Table 1. (B) A detailed count of CAZYmes and their assignment to the different family types. Glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), carbohydrate-binding modules (CBM).