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.”

The Sus operon: a model system for starch uptake by the human gut Bacteroidetes

“…The design of the Sus outer membrane protein complex, whereby glycan capture and carbohydrate degradation is spread over multiple polypeptides, is vaguely reminiscent of the cellulosomal architecture [27]. Cellulosomes are multiprotein structures comprised of enzymes and some distinct CBMs that assemble into a complex for efficient cellulose deconstruction [77, 78] and a similar system for starch hydrolysis has recently been identified in the Firmicute Ruminococcus bromii [16]. The cellulosome is held together by a system of complementary protein–protein interaction domains called cohesins and dockerins, motifs that are not found in the Sus.…”

Formate cross‐feeding and cooperative metabolic interactions revealed by transcriptomics in co‐cultures of acetogenic and amylolytic human colonic bacteria

“…Cysteine (0.1 g/100 ml) is added to the medium following boiling and dispensed into Hungate tubes while the tubes are flushed with CO 2 . After autoclaving, filter sterilized solutions of heat labile vitamins were added to give final concentrations of thiamine and riboflavin of 0.05 μgÁml −1 (each), pantothenate and nicotinamide of 1 μgÁml −1 , pantethine of 50 μgÁml −1 and tetrahydrofolic acid of 0.1 μgÁml −1 as described previously (Ze et al, 2015). The final pH of the medium was adjusted to 6.8 AE 0.2.…”

Resistant Starch Regulates Gut Microbiota: Structure, Biochemistry and Cell Signalling

“…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 [32]. Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family.…”

“…Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family. Six GH13 amylases that carry signal peptides were detected by proteomic analysis in R. bromii cultures [32]. …”