A bacterial pioneer produces cellulase complexes that persist through community succession

Cultivation of microbial consortia provides low-complexity communities that can serve as tractable models to understand community dynamics. Time-resolved metagenomics demonstrated that an aerobic cellulolytic consortium cultivated from compost exhibited community dynamics consistent with the definition of an endogenous heterotrophic succession. The genome of the proposed pioneer population, ‘Candidatus Reconcilibacillus cellulovorans’, possessed a gene cluster containing multidomain glycoside hydrolases (GHs). Purification of the soluble cellulase activity from a 300litre cultivation of this consortium revealed that ~70% of the activity arose from the ‘Ca. Reconcilibacillus cellulovorans’ multidomain GHs assembled into cellulase complexes through glycosylation. These remarkably stable complexes have supramolecular structures for enzymatic cellulose hydrolysis that are distinct from cellulosomes. The persistence of these complexes during cultivation indicates that they may be active through multiple cultivations of this consortium and act as public goods that sustain the community. The provision of extracellular GHs as public goods may influence microbial community dynamics in native biomass-deconstructing communities relevant to agriculture, human health and biotechnology.


Supplementary Tables
Supplementary Table 1. Genome information of the bins extracted from the 15 L cultivation.
Supplementary Table 10B. Sequences and properties of individual unique spectra of CelB. Mapping of these spectra to the CelB sequence is found in Supplementary Figure  9B.
Supplementary Table 10C. Sequences and properties of individual unique spectra of CelC. Mapping of these spectra to the CelC sequence is found in Supplementary Figure  9C.
Supplementary Table 10D. Sequences and properties of individual unique spectra of XynA. Mapping of these spectra to the XynA sequence is found in Supplementary Figure  9D.

Supplementary Figures
Supplementary Figure 1    Supplementary Figure 5 Comparison of glycoside hydrolase gene clusters. Molecular organization of glycoside hydrolase gene clusters of 'Ca. Reconcilibacillus cellulovorans', Caldicellulosiruptor bescii, Caldicellulosiruptor obsidiansis, and Caldicellulosiruptor saccharolyticus. CDS coding for glycoside hydrolases (yellow) are complemented with individual active sites (grey), poly-proline stretches (green), and cellulose binding motifs (red). CDS encoding non-GHs are indicated in brown. Genomic data for Caldicellulosiruptor isolates was obtained from IMG (https://img.jgi.doe.gov/). (A) Evidence for glycoside hydrolase glycosylation. Coomassie stain and glycoprotein stain of SDS-PAGE of CelABC complexes obtained by affinity digestion. Lane 1: MW markers (kDa); Lane 2: Coomassie staining of AD fraction (7.5 µg); Lane 3: periodic acid-Schiff base staining of AD fraction (7.5 µg). CelC and CelB are identified in Lane 3. The gel was run at 140 V for 3 h to separate CelA, which is not glycosylated, and CelB. This gel was representative of four gels representing four technical replicates of the glycosylation stain. The image is cropped for clarity and the original gel image is in Supplementary Figure 16.

Supplementary Figure 11
Beta-elimination to identify O-linked glycans. A) Annotated MALDI-TOF MS spectrum of permethylated glycans from the sample obtaining via beta-elimination. All annotated ions are [M+Na] + . The number indicated above the peak in the spectra is the m/z value of the ion that has been detected by the mass spectrometer. Because the linkage (linear or branched) of the galactoses in these glycans is not clear, the annotation is simplified by using a disaccharide with the remaining hexoses listed outside a bracket. B) Annotated ESI MS/MS spectrum of the permethylated glycan at m/z 493.2 from the sample obtaining via beta elimination. Assignments of the possible fragment ions are indicated on the cartoons and on the spectra the horizontal arrows show antennae losses. The number indicated above the peak is the m/z value of the fragment ion (resulting ion) that has been detected by the mass spectrometer. Data were acquired in forms of Proteinase K digestion of an AD preparation (7.5 µg, CelABC complex in box), CelA (2 µg), CelC (2 µg) and a mixture of CelA and CelC (1:1 ratio; 2 µg each). All proteins were analyzed by SDS PAGE in the presence (+) or absence (−) of Proteinase K (PK) (187.5 µg/ml at 50 °C for 1 h). MW markers in lane 1 are in kDa, and lane 2 was left blank. This gel was representative of three gels representing technical replicates of the protease reactions. The image is cropped for clarity and the original gel image is in Supplementary Figure 17