A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism in Bacteroides thetaiotaomicron

Abstract

Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron Δ rbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The Δ rbpB mutant had reduced expression of BT1871 , encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes. Importance The human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides, that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.

The RNA-binding protein RbpB is a central regulator of polysaccharide utilization in gutBacteroides

“…Conversely, there is currently little knowledge as to the extent to which post-transcriptional control impacts on Bacteroides metabolic competitiveness. Given that the deletion of the proposed RNA-binder RbpB (BT_1887) in B. thetaiotaomicron led to the differential expression of several PUL genes 15 , we assessed the fitness of a B. thetaiotaomicron ∆rbpB mutant in the mammalian gut in response to distinct diets. As conventionally raised mice are resistant to B. thetaiotaomicron colonization 21 , we treated C57BL/6 mice with an antibiotic cocktail to promote B. thetaiotaomicron engraftment.…”

“…Given that the deletion of the proposed RNA-binder RbpB (BT_1887) in B . thetaiotaomicron led to the differential expression of several PUL genes 15 , we assessed the fitness of a B . thetaiotaomicron Δ rbpB mutant in the mammalian gut in response to distinct diets.…”

“…The present study reports the identification of an in-vivo phenotype, the RNA interactome, a metabolism-associated function, and the underlying molecular mechanism of the first global RNA-binding protein (RBP) in Bacteroides. Specifically, we found that a B. thetaiotaomicron mutant devoid of the RNA recognition motif 1 (RRM-1)-containing protein RbpB 14,15 failed to efficiently colonize the mammalian intestine in a diet-dependent manner.…”

“…Specifically, we found that a B . thetaiotaomicron mutant devoid of the RNA recognition motif 1 (RRM-1)-containing protein RbpB 14,15 failed to efficiently colonize the mammalian intestine in a diet-dependent manner. Cross-linking immunoprecipitation and sequencing (CLIP-seq) of B .…”

Toward Understanding the Alginate Catabolism in Microbulbifer sp. ALW1 by Proteomics Profiling

“…In addition, a significant portion of proteins was related to binding for the intracellular and extracellular proteins. As polysaccharides may serve as the main carbon source for many marine bacteria ( Alderkamp et al, 2007 ; Kabisch et al, 2014 ; Sichert et al, 2020 ), proteins encoded in operons known as polysaccharide utilization loci (PULs) are often enriched to counter the physical barrier of a wide variety of polysaccharides, functioning as carbohydrate-degrading enzymes and binding proteins ( Adams et al, 2021 ). The proteins related to cellular process (GO: 0009987) may participate in material transportation, information transmission, and the maintenance of basic life activities.…”