Project description:Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to adhere to and degrade RS. Sas20 is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based upon amino acid sequence. Here, we perform a structure-function analysis of Sas20 which features two discrete starch-binding domains separated by a flexible linker. Sas20 domain 1 has an N-terminal β-sandwich followed by a cluster of α-helices and captures the non-reducing end of maltooligosaccharides between these structural features. The crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical 1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose within starch granules. Affinity PAGE and isothermal titration calorimetry demonstrate both domains bind maltoheptaose and soluble starch with relatively high affinity (Kd 20 M) but exhibit limited or no binding to cyclodextrins. Small angle x-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency.
Project description:Strain Ruminococcus bromii TSDC17.2-1.1 (species Ruminococcus bromii) was isolated from the fecal microbiota of a USA female at time point zero (bacterial isolates were sequenced from this donor on day 0 and 49). The species name was assigned by genome clustering.
Project description:Strain Ruminococcus bromii TSDC17.2-1.3 (species Ruminococcus bromii) was isolated from the fecal microbiota of a USA female at time point zero (bacterial isolates were sequenced from this donor on day 0 and 49). The species name was assigned by genome clustering.
Project description:Strain Ruminococcus bromii TSDC10.2-1.1 (species Ruminococcus bromii) was isolated from the fecal microbiota of a USA female at time point zero (bacterial isolates were sequenced from this donor on day 0 and 42). The species name was assigned by genome clustering.
Project description:Strain Ruminococcus bromii TSDC17.2-1.2 (species Ruminococcus bromii) was isolated from the fecal microbiota of a USA female at time point zero (bacterial isolates were sequenced from this donor on day 0 and 49). The species name was assigned by genome clustering.
Project description:Strain Ruminococcus bromii TSDC10.1-1.1 (species Ruminococcus bromii) was isolated from the fecal microbiota of a USA female at time point zero (bacterial isolates were sequenced from this donor on day 0 and 42). The species name was assigned by genome clustering.
Project description:The goal was to assess the impact of carbon source and cross-feeding on transcription profile of both bacteria. R. gnavus was grown in monoculture with glucose (Glc). R. bromii was grown in monoculture with soluble (SS) or resistant (RS) starch. R. bromii and R. gnavus were co-cultured with SS or RS. Total RNA was extracted from a culture sample taken at mid- to late exponential phase of growth. rRNA was depleted and mRNA sequenced. 3 biological replicates were prepared for each condition.
