Table 1.
Characteristics of the primers used in the analysis of RT-qPCR.
Fig 1.
Annotation of genes clustered in “PULs-like” associated with CAZyme GH2 β-mannosidase CB10-00347, obtained from Chitinophaga sp. CB10.
The annotation was performed using the CGC-Finder program. The GH2 sequences identified in previous studies (unpublished data) were annotated using the dbcan2 tool [30]. The prediction of putative PUL was proceeded by the CGC-Finder [31]. The CAZyme gene clusters (CGCs) are annotated based on related signature genes (i) CAZymes using CAZy database [32]; (ii) Transporter Classification (TCs) were searched against TCDB [33], and Transcription Factors (TFs) searched against CollectTF [34], DBTBS [35] and RegulonDB [36]. The annotation for CGCs was performed using the dbcan2 tool [30]. Legend: CAZyme, “Carbohydrate-Active Enzymes”; null, non-annotated; STP, signal transduction proteins; TC, Transporter classification; TF, Transcription Factor.
Fig 2.
Phenetic dendrogram generated by the MEGA 5 program for β-mannosidases.
The analysis was carried out based on the distance matrix generated from an alignment made by the T-coffee, using the Neighbor Joining clustering optimization method [47]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10,000 replicates) is shown next to the branches [48]. The tree is drawn to scale, with branch lengths in the same units, based on similarity; distances were computed using the Poisson correction method [49] and are in the units of the number of amino acid substitutions per site. The analysis involved 36 amino acid sequences. All positions containing gaps and missing data were eliminated. There was a total of 330 positions in the final dataset. The tree was conducted in MEGA7 [39]. Circle and Triangle: respectively, β-mannosidases CB10-153.446 and CB10-00347.
Fig 3.
BHB agar medium plates supplemented with different substrates (10.0 g L−1), resulting from cultivation after 96 hours, respectively, (A) Glucose, (B) Galactomannan (Gum Locust bean from Ceratonia siliqua), (C) Carboxymethylcellulose (CMC), (D) Aloe vera and (E) Starch. The lower plates depict the B-E treatments stained with Congo Red (1%).
Fig 4.
Chromatograms of the sugar profile of the culture medium with galactomannan grown with CB10 were detected by HPLC-RID, demonstrating the degradation of the polysaccharide generating mannose (Blue), as was evinced by the comparison of the overlapping of the pure solution pattern that demonstrates the location of the peak mannose (black), which is absent in the control sample (culture medium without cultivation, red).
Fig 5.
Bacterial growth curves of Chitinophaga sp. (CB10), in a solid BHB culture medium with sugarcane bagasse by Colony Counting Unit (CFU) and in a liquid BHB culture medium added with glucose by reading Optical Density (DO600).
Fig 6.
Level of expression of the β-mannosidases genes, respectively associated (CB10-00347) and not associated (CB10-153.446) to PUL-like clusters, calculated using the 2-ΔΔCT method for Chitinophaga sp. (CB10) in a liquid BHB culture medium with sugarcane bagasse in relation to endogenous control (SigmaA) and negative enzyme induction (glucose), in the periods of 24; 96 and 144 hours.