In red algae, the most abundant principal cell wall polysaccharides are mixed galactan agars, of which agarose is a common component. While bioconversion of agarose is predominantly catalyzed by bacteria that live in the oceans, agarases have been discovered in microorganisms that inhabit diverse terrestrial ecosystems, including human intestines. Here we comprehensively define the structure–function relationship of the agarolytic pathway from the human intestinal bacterium Bacteroides uniformis (Bu) NP1. Using recombinant agarases from Bu NP1 to completely depolymerize agarose, we demonstrate that a non-agarolytic Bu strain can grow on GAL released from agarose. This relationship underscores that rare nutrient utilization by intestinal bacteria is facilitated by the acquisition of highly specific enzymes that unlock inaccessible carbohydrate resources contained within unusual polysaccharides. Intriguingly, the agarolytic pathway is differentially distributed throughout geographically distinct human microbiomes, reflecting a complex historical context for agarose consumption by human beings.

Gut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as “medium grower” and “high grower.” Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and “omics” approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem.

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Genetically engineering intestinal bacteria, such as Bacteroides thetaiotaomicron (B. theta), holds potential for creating new classes of biological devices, such as diagnostics or therapeutic delivery systems. Here, we have developed a series of B. theta strains that produce functional transgenic enzymes in response to dextran and arabinogalactan, two chemically distinct glycans. Expression systems for single glycan induction, and a novel “dual-glycan” expression system, requiring the presence of both dextran and arabinogalactan, have been developed. In addition, we have created two different chromosomal integration systems and one episomal vector system, compatible with engineered recipient strains, to improve the throughput and flexibility of gene cloning, integration, and expression in B. theta. To monitor activity, we have demonstrated the functionality of two different transgenic enzymes: NanoLuc, a luciferase, and BuGH16C, an agarase from the human intestinal bacterium, Bacteroides uniforms NP1. Together this expression platform provides a new collection of glycan-responsive tools to improve the strength and fidelity of transgene expression in B. theta and provides proof-of-concept for engineering more complex multi-glycan expression systems.

34Gut microbiomes have vast catabolic potential and are essential to host health and nutrition. 35 An in-depth understanding of the metabolic pathways in these ecosystems will enable us to 36 design treatments (i.e. prebiotics) that influence microbiome structure and enhance host 37 109 (Hehemann et al., 2010;Hehemann et al., 2012; Pluvinage et al., 2018), facilitating their 110 persistence within highly competitive ecosystems and adaption to spatially and culturally 111 diversified diets. 112 Although major advances have been made in understanding the diversity of metabolic 113 potential in symbiotic bacteria and the mechanisms of prebiotic utilization, establishing stabile 114 engineered microbiomes in complex ecosystems, such as the rumen, will require more detailed 115 knowledge of the competitive and complementary processes that drive metabolic phenotypes 116 at the strain level. To achieve this, "next-generation physiology" (Hatzenpichler, Krukenberg, 117 Spietz, & Jay, 2020) based approaches that identify metabolic potentials of individual bacteria, 118 thereby providing critical insights of cellular functions and assigning cellular phenotypes, must 119 be developed. One such approach is the application of fluorescently labeled polysaccharides 120 (FLA-PS). FLA-PS were initially developed to demonstrate selfish uptake of marine 121 polysaccharides in marine Bacteroidetes (Reintjes, Arnosti, Fuchs, & Amann, 2017), and have 122 also been applied to the gut bacterium BtVPI-5482 to confirm that YM metabolism also occurs 123 through a selfish mechanism (Cuskin, Lowe, et al., 2015a). 124Here, for the first time, we apply FLA-PS as a next-generation physiology approach to 125 directly visualize YM metabolism by single cells in a complex rumen community and 126 subsequently classify populations of cells using FISH. We combine this analysis with a multi-127 tiered study of the evolution and function of YM metabolism in bovine-adapted B. theta strains 128 (Bt Bov ), which adopt one of two dichotomous growth phenotypes, referred to as "High Grower" 129 (HG) or "Medium Grower" (MG), based on the optical density of cultures after 24 hrs. Using 130 genomics, transcriptomics, and CAZyme fingerprinting, multiple MAN-PUL architectures 131 were identified in this study that are consistent with reports for human-associated BtVPI-5482 132 6 (Cuskin, Lowe, et al., 2015a) and key differences in the YM utilization systems between MGs 133and HGs were uncovered. To define the mechanisms that contribute to these growth 134 phenotypes, we present a new quantitative application of FLA-PS, which we believe has far-135 reaching implications for elucidating differences in substrate utilization of individual cells 136 within complex microbial communities. 137 Results 138 Ex vivo visualization of YM-metabolising taxa within the rumen community 139To assess the capability of rumen microbiota to metabolize YM, extracted rumen 140 samples were incubated with FLA-YM and visualized on food particles and in solution ( Fig. 141 1...