Environmental Microbiology

Multi-omic Directed Discovery of Cellulosomes, Polysaccharide Utilization Loci, and Lignocellulases from an Enriched Rumen Anaerobic Consortium

Geizecler Tomazetto, Agnes C. Pimentel, Daniel Wibberg, Neil Dixon, Fabio M. Squina
Charles M. Dozois, Editor
DOI: 10.1128/AEM.00199-20

Article Figures & Data

Figures

  • FIG 1
    FIG 1

    Biochemical assays using the enriched rumen anaerobic consortium (ERAC) metaproteome against nine different substrates. Reducing sugars were released from reactions of the ERAC metaproteome against xylan, lichenan, β-glucan, rye arabinoxylan, xyloglucan, rhamnogalacturonan, pectin, mannan, and carboxymethyl cellulose sodium salt (CMC).

  • FIG 2
    FIG 2

    Scanning electron microscopy images of the sugarcane bagasse prior to incubation (A and B) and after 7 days of incubation (C and D) with the enriched rumen anaerobic consortium (ERAC).

  • FIG 3
    FIG 3

    Relative abundance (%) of the phylum (A) and family (B) taxons identified in the cow rumen sample and enriched rumen anaerobic consortium (ERAC). Abundances were determined based on the 16S rRNA gene amplicon sequences. Phyla represented by less than 1% and families represented by less than 3% of the total reads were combined in the groups named “Phyla < 1%” and “Families < 3%,” respectively.

  • FIG 4
    FIG 4

    Distribution of the percent identity of the carbohydrate-active enzyme (CAZyme) sequences predicted in the enriched rumen anaerobic consortium (ERAC) against the four classes of the CAZy database. Only the maximum percent identities for each CAZyme ERAC were considered. GH, glycoside hydrolase; PL, polysaccharide lyases; CE, carbohydrate esterases; AA, auxiliary activities.

  • FIG 5
    FIG 5

    (A) Distribution of the predicted carbohydrate-active enzymes (CAZymes) found in enriched rumen anaerobic consortium genomes (ERACgs) at the class level. (B) Total CAZymes found in the rumen-derived anaerobic microbial consortium (enriched rumen anaerobic consortium [ERAC]) metagenome data. Red, nonbinned metagenome contigs; green, ERACgs.

  • FIG 6
    FIG 6

    Heat map displaying the distribution of the most abundant glycoside hydrolases (GH1) found in the ERACgs from ERAC. GH families were grouped according to their action on components of the plant cell wall.

  • FIG 7
    FIG 7

    Examples of polysaccharide utilization loci (PUL) predicted in Bacteroidia ERACgs reconstructed from the enriched rumen anaerobic consortium metagenome. To facilitate the visualization of gene arrangements, the predicted proteins were colored according to the function of the encoded proteins: SusC, SusD, glycoside hydrolase (GH), polysaccharide lyase (PL), carbohydrate esterase (CE), peptidase, and regulators (AraC, MaR, LacI). Genes that do not encode PUL components or that encode hypothetical proteins are identified as non-PUL genes. All PULs predicted in Bacteroidia ERACgs are presented in Data Set S2 in the supplemental material.

Tables

  • TABLE 1

    The most common CAZyme modules predicted in the total ERAC metagenome and their relative abundance in ERACgs, according to their representation in the CAZy databasea

    TABLE 1

    • ↵a Abbreviations: ERAC, enriched rumen anaerobic consortium; ERACgs, enriched rumen anaerobic consortium genomes; CAZyme and CAZy, carbohydrate-active enzyme; GH, glycoside hydrolase; CBM, carbohydrate-binding module; CE, carbohydrate esterases; PL, polysaccharide lyases; AA, auxiliary activities.

  • TABLE 2

    Genomic features of ERACgs from ERAC metagenome shotgun sequencinga

    TABLE 2

    • ↵a Abbreviations: ERACg, enriched rumen anaerobic consortium genomes; ERAC, enriched rumen anaerobic consortium.

    • ↵b Total number of carbohydrate-active enzymes (CAZymes) predicted.

  • TABLE 3

    Putative cellulosomal proteins and SusC/SusD families identified by LC-MS/MS from ERAC grown on sugarcane bagassea

    TABLE 3

    • ↵a Abbreviations: cohesin_number, cohesin type number; dockerin_number, dockerin type number; Cthe_2159 represents a novel family of cellulose-binding beta-helix proteins from Clostridium thermocellum; LRR_5, leucine-rich repeats; PUL, polysaccharide utilization loci. Cohesin and dockerin domains are represented with the family number according to their representation in the dbCAN database. The protein set secreted by enriched rumen anaerobic consortium (ERAC) is given in Data Set S3 in the supplemental material.

    • ↵b Prediction of signal peptides based on SignalP analysis.

    • ↵c Metaproteome analysis based on spectral counting.

  • TABLE 4

    CAZy families identified by LC-MS/MS from ERAC grown on sugarcane bagassea

    TABLE 4

    • ↵a Abbreviations: ERAC, enriched rumen anaerobic consortium; ERACg, enriched rumen anaerobic consortium genome; EC, Enzyme Commission; cohesin_number, cohesin type number; dockerin_number, dockerin type number; GH, glycoside hydrolase; CBM, carbohydrate-binding module; CE, carbohydrate esterases; PL, polysaccharide lyases; ND, not determined. CAZymes are represented with the family number according their representation in the CAZy database.

    • ↵b Prediction of signal peptides based on SignalP analysis.

    • ↵c Metaproteome analysis based on spectral counting.

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KEYWORDS

anaerobic consortium
lignocellulose degradation
metagenome
metasecretome
polysaccharide utilization loci
rumen

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