Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii

Abstract

Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energy from dietary starches that escape digestion by host enzymes via its exceptional activity against particulate "resistant" starches. Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family (GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructose as an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptides were detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted to carry dockerin modules, with one, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediate the formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerins from R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymes Amy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wall-anchoring protein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylases Amy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, as its dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides the first example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an "amylosome."

Importance: Fermentation of dietary nondigestible carbohydrates by the human colonic microbiota supplies much of the energy that supports microbial growth in the intestine. This activity has important consequences for health via modulation of microbiota composition and the physiological and nutritional effects of microbial metabolites, including the supply of energy to the host from short-chain fatty acids. Recent evidence indicates that certain human colonic bacteria play keystone roles in degrading nondigestible substrates, with the dominant but little-studied species Ruminococcus bromii displaying an exceptional ability to degrade dietary resistant starches (i.e., dietary starches that escape digestion by host enzymes in the upper gastrointestinal tract because of protection provided by other polymers, particle structure, retrogradation, or chemical cross-linking). In this report, we reveal the unique organization of the amylolytic enzyme system of R. bromii that involves cohesin-dockerin interactions between component proteins. While dockerins and cohesins are fundamental to the organization of cellulosomal enzyme systems of cellulolytic ruminococci, their contribution to organization of amylases has not previously been recognized and may help to explain the starch-degrading abilities of R. bromii.

Copyright © 2015 Ze et al.

Figures

FIG 1

Major extracellular amylases of R. bromii L2-63. Modular organization is shown for seven predicted gene products that carry GH13 catalytic modules and signal peptides (SP). The cohesin (COH) of Amy4, the CBMs from family 26 or family 48, and the dockerins (red double ellipses) are shown schematically. aa, amino acids.

FIG 2

Phylogenetic tree comparing GH13 enzymes from Ruminococcus bromii and seven other bacterial genomes. R. bromii L2-63 (RB) GH13 sequences (labeled Amy1, Amy2, Amy3, etc.) are compared with those from other human gut species (the starch-utilizing strains Eubacterium rectale A1-86 [ER], Bacteroides thetaiotaomicron VPI-5482 [BT], and Bifidobacterium adolescentis ATCC15703 [BfA] and two other human colonic Ruminococcaceae species, Ruminococcus champanellensis 18P13 [RC] and Faecalibacterium prausnitzii L2-6 [FP]) and also with those from two non-gut species, Bacillus halodurans C-125 (BH) and Paenibacillus terrae HPL-003 (PT), that were known from the results of BLASTp queries of the NCBI database to possess proteins that were the closest matches to R. bromii Amy4. R. bromii sequences that are predicted to possess signal peptides are underlined. Sequences with functions concerned with glycogen or trehalose metabolism (including R. bromii L2-63 Amy 13, Amy14, and Amy15) that are predicted by KEGG GH annotation have been omitted from the tree. Sequences marked with an asterisk (*) encode enzymes that have been experimentally characterized. Bootstrap values, expressed as a percentage of 1,000 replications, are given at the branching nodes. This tree is unrooted and was constructed using the maximum-likelihood method. The scale bar (center of tree) refers to the number of amino acid differences per position.

FIG 3

Schematic representation of cohesin-carrying proteins of Ruminococcus bromii L2-63. The four proteins are designated scaffoldins. Scaffoldin 1 (Sca1) contains a GH13 amylase module and is synonymous with the amylase Amy4. Sca2 carries a predicted C-terminal sortase signal (indicated by an arrow). The X25 domains in Sca3 show some similarity to starch-specific CBMs found in the Bacteroides thetaiotaomicron proteins SusE and SusF (10).

FIG 4

Detection of major R. bromii amylases by zymogram analysis and sequencing. (a) Zymogram showing activity of amylases against RS3 for R. bromii L2-63 cells grown for 24 h on 0.2% fructose or 0.2% RS3. Values on the left correspond to the molecular masses determined by staining the gel with Coomassie blue, prior to staining the gel with iodine to visualize clear zones of amylase activity. sup, supernatant proteins; cell, cell-associated proteins. Bands a to f, visible active bands. (b) Identification of amylolytic enzymes from excised bands by LC-MS/MS. In addition, a homologue of a Cna (collagen adhesion)-type protein was detected in band a, a hypothetical protein (RBR_05030) in band b, RNA polymerase subunit B in band d, and a hypothetical protein (RBR_07100) in band e.

FIG 5

Interactions of recombinant dockerins and cohesins from R. bromii L2-63. Selected cohesin-dockerin interactions were examined by ELISA experiments. (a and b) Amy4 (a) and Amy9 (b) dockerins interact strongly with all four cohesins of R. bromii. (c) Strong interaction of the Amy12 dockerin with Coh3, moderate interaction with Coh4, and negligible interaction with Coh1 and Coh2. CohE from R. flavefaciens FD1 was included in the experiment as a negative control. Error bars indicate the standard deviations from the means of the results determined for triplicate samples from one experiment.

FIG 6

Identification of native R. bromii proteins that interact with overexpressed Amy4-Coh1. Cell-associated or cell-free supernatant proteins of R. bromii L2-63 grown with boiled Novelose RS3 starch as the energy source were incubated with His6-tagged Coh1 at 37°C as described in Materials and Methods. Proteins binding to Coh1 were recovered and separated by SDS-PAGE, followed by proteomic analysis of individual bands. Protein identifiers (ID), molecular-mass values, and conserved modules for identified proteins are shown on the right of the figure. Numbers on the left indicate sizes and positions of molecular mass markers. SP, signal peptide; Amy, amylase; Coh, cohesin; Doc, dockerin; Anc, cell wall surface anchor; CBM, carbohydrate-binding module; Tri-tyrosine, triple-tyrosine motif.

FIG 7

Potential dockerin-mediated interactions of R. bromii amylases with cohesin-carrying proteins and with each other. On the basis of observed interactions with recombinant cohesins and dockerins (Table 4 and Fig. 5), we can predict that the Amy4 and Amy9 enzymes are likely to bind to the cell surface via the Sca2 scaffoldin protein. Further complexes are likely to form between the Amy4, Amy9, and Amy10 proteins and between Amy4, Amy9, Amy10, and Amy12 and the Sca3 and Sca4 proteins. Binding of the enzymes to Sca3 would presumably confer starch-binding features to the resultant complex. Binding of dockerin-bearing amylases Amy9 and Amy10 to the cohesin of Amy4 would result in the formation of multienzyme complexes that can be part of a cell-bound or cell-free system. Amy4 also has the potential to self-aggregate through interactions between its own cohesin and dockerin.