Journal: Microbiome

Article Title: “ Candidatus Paraporphyromonas polyenzymogenes” encodes multi-modular cellulases linked to the type IX secretion system

doi: 10.1186/s40168-018-0421-8

Figure Lengend Snippet: Ca. P. polyenzymogenes encodes a cellulolytic gene cluster. a Gene organisation of a putative cellulolytic gene cluster encoding four GH5s with varying modular arrangements. For multi-modular ORFs, both C-terminal (c) and N-terminal (n) domains are indicated, whereas “CTD” denotes a carboxy-terminal domain that infers export via T9SS. Hyp indicates domains with no known function. b Products released from crystalline cellulose (Avicel) by Cel5A, Cel5B, Cel5C_N, and Cel5D enzymes. A total of 1% ( w / v ) Avicel was incubated with 1 μM enzyme in 20 mM citrate buffer, pH 5.5 at 40 °C with 1000 rpm horizontal shaking. Products were analyzed by HPAEC-PAD at given time points after stopping the reactions by addition of NaOH to 0.1 M. Error bars represent standard deviations between three replicates (hidden by the markers). c Degradation of Glc(6) by Cel5B and Cel5C_N enzymes, illustrating differences in substrate specificity. Glc(6) (0.1 mg/ml) was incubated with 0.25 μM enzyme in 20 mM citrate buffer pH 5.5. Samples were taken at indicated intervals, and the reaction was stopped by adding NaOH to 0.1 M. Products were analyzed using HPAEC-PAD with cellodextrins as standards. A more complete analysis for all four cellulases shown in Additional file : Figure S7. d Crystal structure of Cel5C_N. Surface view of the structure, seen from above the active-site cleft, and − 45° rotated. The surface of the catalytic residues is shown in red. Figures were created using The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC

Article Snippet: Genes were synthesized by GeneArt (Regensburg, Germany) without predicted signal peptides (SignalP 4.0, [ ]) and T9SS CTDs and cloned into pNIC-CH (Addgene plasmid #26117, a generous gift from Opher Giliadi) using ligation-independent cloning (Additional file : Table S7).

Techniques: Incubation

Journal: Microbiome

Article Title: “ Candidatus Paraporphyromonas polyenzymogenes” encodes multi-modular cellulases linked to the type IX secretion system

doi: 10.1186/s40168-018-0421-8

Figure Lengend Snippet: Selected metabolic features of “ Candidatus Paraporphyromonas polyenzymogenes” (AGa) as inferred from genome and proteome comparisons. Graphical representation of pathways, enzymes, CAZymes, and cellular features are based on functional annotations listed in Additional file : Table S1. Ca. P. polyenzymogenes encoded glucose, mannose, and xylose fermentation capabilities, as well as CAZymes inferred in degradation of cellulose, mannans, and xylans. CAZymes located outside the cell are predicted to be secreted via the T9SS. Metaproteomic analysis (detected genes, enzyme complexes, and transport systems highlighted in green) indicated that Ca. P. polyenzymogenes was metabolically active in rumen samples, with proteins for glycolysis, succinate production, and ammonium metabolism detected, as well as components of the T9SS and T9SS-secreted multimodular CAZymes (details found in Additional file : Table S1, Additional file : Table S4). Features highlighted by yellow boxes indicate detected genes that were identified in only one sample (Additional file : Table S4). Hypothetical domains in multi-modular CAZymes are denoted by “hyp.”, and the red asterisks indicate CAZymes that were encoded in the gene cluster characterized in this study (Fig. ). Broken lines indicate annotations for which representative genes were not identified in the respective reconstructed genomes. Further details on enzyme abbreviations, gene abbreviations, and identification numbers (Integrated Microbial Genomics gene ID) can be found in Additional file : Table S1. The abbreviations for central metabolites are PEP phosphoenolpyruvate, G1P glucose-1-phosphate, G6P glucose-6-phosphate, F6P fructose-6-phosphate, M6P mannose-6-phosphate, G3H d -glyceraldehyde 3-phosphate, E4P d -erythrose 4-phosphate, 5SP d -xylulose 5-phosphate, S7P sedoheptulose 7-phosphate, 5RP d -ribulose 5-phosphate, and RP5 d -ribose 5-phosphate

Article Snippet: Genes were synthesized by GeneArt (Regensburg, Germany) without predicted signal peptides (SignalP 4.0, [ ]) and T9SS CTDs and cloned into pNIC-CH (Addgene plasmid #26117, a generous gift from Opher Giliadi) using ligation-independent cloning (Additional file : Table S7).

Techniques: Functional Assay, Metabolic Labelling