Members of the human gut microbiome enzymatically process many bioactive molecules in the gastrointestinal tract. Most gut bacterial modifications characterized so far are hydrolytic or reductive in nature. Here we report that abundant human gut bacteria from the phylum Bacteroidetes perform conjugative modifications by selectively sulfonating steroidal metabolites. While sulfonation is a ubiquitous biochemical modification, this activity has not yet been characterized in gut microbes. Using genetic and biochemical approaches, we identify a widespread biosynthetic gene cluster that encodes both a sulfotransferase (BtSULT, BT0416) and enzymes that synthesize the sulfonate donor adenosine 3′-phosphate-5′-phosphosulfate (PAPS), including an APS kinase (CysC, BT0413) and an ATP sulfurylase (CysD and CysN, BT0414–BT0415). BtSULT selectively sulfonates steroidal metabolites with a flat A/B ring fusion, including cholesterol. Germ-free mice monocolonized with Bacteroides thetaiotaomicron ΔBT0416 exhibited reduced gastrointestinal levels of cholesterol sulfate (Ch-S) compared with wild-type B. thetaiotaomicron-colonized mice. The presence of BtSULT and BtSULT homologues in bacteria inhibited leucocyte migration in vitro and in vivo, and abundances of cluster genes were significantly reduced in patients with inflammatory bowel disease. Together, these data provide a mechanism by which gut bacteria sulfonate steroidal metabolites and suggest that these compounds can modulate immune cell trafficking in the host. Characterization of a biosynthetic pathway for the sulfonation of steroidal metabolites, such as cholesterol, by gut bacteria may have implications for immune cell trafficking and inflammatory bowel disease. 

A biosynthetic pathway for the selective sulfonation of steroidal metabolites by human gut bacteria

Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/mmi.14793

Comparative genomics provides structural and functional insights into Bacteroides RNA biology

The bacterial strain of Microbulbifer sp. ALW1 has demonstrated visible ability of degrading the cell wall of Laminaria japonica, and biochemical characterization has been performed on some individual enzymes to elucidate its genetic basis. However, it still remains elusive how strain ALW1 successfully breaks down the major cell wall component alginate polysaccharide and colonizes on its marine host. In this study, a mass spectrometry-based quantitative analysis of the extracellular and intracellular proteomes was introduced to elucidate the alginate degradation pathway in ALW1 strain. Mass spectrometry and biochemical assays indicated that strain ALW1 could effectively degrade alginate polysaccharide into disaccharides and trisaccharides within 12 h. Proteome analysis identified 156 and 1,047 proteins exclusively localized in extracellular and intracellular compartments, respectively, with 1,086 protein identities of dual localization. Functional annotation of the identified proteins suggested the involvement of diverse catalytic enzymes and non-catalytic molecules for the cleavage and metabolism of alginate polysaccharide. A simplified pathway was constructed to demonstrate the extracellular digestion, active transport, and intracellular conversion of alginate polysaccharide and its fragmented oligosaccharides, casting a picture of genetic loci controlling alginate catabolism by ALW1 strain. This study aims to provide a guide for utilization and genetic manipulation of the bacterial strain ALW1 for efficient alginate oligosaccharides production by fermentation.

https://www.frontiersin.org/articles/10.3389/fbioe.2022.829428/pdf

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

The last two decades have seen numerous studies connecting physiological behaviors in Bacteroides-including polysaccharide degradation and capsule production-with elements of global regulation, but a complete model is still elusive. A new study by Adams et al. in this issue of the Journal of Bacteriology reveals another layer of regulation by describing a novel family of RNA-binding proteins in Bacteroides thetaiotaomicron that modify expression of genes involved in carbohydrate utilization and capsule expression, among others (A. N. D. Adams, M. S. Azam, Z. A. Costliow, X. Ma, et al., J Bacteriol 203:e00217-21, 2021, https://doi.org/10.1128/JB.00217-21).

Small RNAs Go Global in Human Gut Bacteroides.

ABSTRACT The mammalian gut microbiota is a critical human health determinant with therapeutic potential for remediation of many diseases. The host diet is a key factor governing the gut microbiota composition by altering nutrient availability and supporting the expansion of distinct microbial populations. Diets rich in simple sugars modify the abundance of microbial subsets, enriching for microbiotas that elicit pathogenic outcomes. We previously demonstrated that diets rich in fructose and glucose can reduce the fitness and abundance of a human gut symbiont, Bacteroides thetaiotaomicron, by silencing the production of a critical intestinal colonization protein, called Roc, via its mRNA leader through an unknown mechanism. We have now determined that dietary sugars silence Roc by reducing the activity of BT4338, a master regulator of carbohydrate utilization. Here, we demonstrate that BT4338 is required for Roc synthesis, and that BT4338 activity is silenced by glucose or fructose. We show that the consequences of glucose and fructose on orthologous transcription factors are conserved across human intestinal Bacteroides species. This work identifies a molecular pathway by which a common dietary additive alters microbial gene expression in the gut that could be harnessed to modulate targeted microbial populations for future therapeutic interventions.

https://www.tandfonline.com/doi/pdf/10.1080/19490976.2023.2221484?needAccess=true&role=button

Dietary sugars silence the master regulator of carbohydrate utilization in human gut Bacteroides species

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 ΔrbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The ΔrbpB 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.

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

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 ΔrbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The ΔrbpB 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.

/pdf/a-novel-family-of-rna-binding-proteins-regulate-2dtut9gxuc.pdf

Amanda N.D. Adams

Papers

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

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