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Sporulation capability and amylosome conservation among diverse human colonic and rumen isolates of the keystone starch-degrader Ruminococcus bromii.

ABSTRACT: Ruminococcus bromii is a dominant member of the human colonic microbiota that plays a 'keystone' role in degrading dietary resistant starch. Recent evidence from one strain has uncovered a unique cell surface 'amylosome' complex that organizes starch-degrading enzymes. New genome analysis presented here reveals further features of this complex and shows remarkable conservation of amylosome components between human colonic strains from three different continents and a R. bromii strain from the rumen of Australian cattle. These R. bromii strains encode a narrow spectrum of carbohydrate active enzymes (CAZymes) that reflect extreme specialization in starch utilization. Starch hydrolysis products are taken up mainly as oligosaccharides, with only one strain able to grow on glucose. The human strains, but not the rumen strain, also possess transporters that allow growth on galactose and fructose. R. bromii strains possess a full complement of sporulation and spore germination genes and we demonstrate the ability to form spores that survive exposure to air. Spore formation is likely to be a critical factor in the ecology of this nutritionally highly specialized bacterium, which was previously regarded as 'non-sporing', helping to explain its widespread occurrence in the gut microbiota through the ability to transmit between hosts.

SUBMITTER: Mukhopadhya I

PROVIDER: S-EPMC5814915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledRuminococcus 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."ImportanceFermentation 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.

Project description:Dietary and host glycans shape the composition of the human gut microbiota with keystone carbohydrate-degrading species playing a critical role in maintaining the structure and function of gut microbial communities. Here, we focused on two major human gut symbionts, the mucin-degrader Ruminococcus gnavus ATCC 29149, and R. bromii L2-63, a keystone species for the degradation of resistant starch (RS) in human colon. Using anaerobic individual and co-cultures of R. bromii and R. gnavus grown on mucin or starch as sole carbon source, we showed that starch degradation by R. bromii supported the growth of R. gnavus whereas R. bromii did not benefit from mucin degradation by R. gnavus. Further we analyzed the growth (quantitative PCR), metabolite production (1H NMR analysis), and bacterial transcriptional response (RNA-Seq) of R. bromii cultured with RS or soluble starch (SS) in the presence or absence of R. gnavus. In co-culture fermentations on starch, 1H NMR analysis showed that R. gnavus benefits from transient glucose and malto-oligosaccharides released by R. bromii upon starch degradation, producing acetate, formate, and lactate as main fermentation end-products. Differential expression analysis (DESeq 2) on starch (SS and RS) showed that the presence of R. bromii induced changes in R. gnavus transcriptional response of genes encoding several maltose transporters and enzymes involved in its metabolism such as maltose phosphorylase, in line with the ability of R. gnavus to utilize R. bromii starch degradation products. In the RS co-culture, R. bromii showed a significant increase in the induction of tryptophan (Trp) biosynthesis genes and a decrease of vitamin B12 (VitB12)-dependent methionine biosynthesis as compared to the mono-culture, suggesting that Trp and VitB12 availability become limited in the presence of R. gnavus. Together this study showed a direct competition between R. bromii and R. gnavus on RS, suggesting that in vivo, the R. gnavus population inhabiting the mucus niche may be modulated by the supply of non-digestible carbohydrates reaching the colon such as RS.

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Sporulation capability and amylosome conservation among diverse human colonic and rumen isolates of the keystone starch-degrader Ruminococcus bromii.

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