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      Streamlined Genetic Manipulation of Diverse Bacteroides and Parabacteroides Isolates from the Human Gut Microbiota

      research-article
      a , a ,
      (Solicited external reviewer), (Solicited external reviewer)
      mBio
      American Society for Microbiology
      Bacteroides, microbiota, mutant construction, vectors

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          Abstract

          We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.

          ABSTRACT

          Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis. Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools.

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          Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont.

          The distal human gut is a microbial bioreactor that digests complex carbohydrates. The strategies evolved by gut microbes to sense and process diverse glycans have important implications for the assembly and operation of this ecosystem. The human gut-derived bacterium Bacteroides thetaiotaomicron forages on both host and dietary glycans. Its ability to target these substrates resides in 88 polysaccharide utilization loci (PULs), encompassing 18% of its genome. Whole genome transcriptional profiling and genetic tests were used to define the mechanisms underlying host glycan foraging in vivo and in vitro. PULs that target all major classes of host glycans were identified. However, mucin O-glycans are the principal host substrate foraged in vivo. Simultaneous deletion of five genes encoding ECF-sigma transcription factors, which activate mucin O-glycan utilization, produces defects in bacterial persistence in the gut and in mother-to-offspring transmission. Thus, PUL-mediated glycan catabolism is an important component in gut colonization and may impact microbiota ecology.
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            The evolution of cooperation within the gut microbiota

            Cooperative phenotypes are considered central to the functioning of microbial communities in many contexts, including communication via quorum sensing, biofilm formation, antibiotic resistance, and pathogenesis 1-5 . The human intestine houses a dense and diverse microbial community critical to health 1,2,4-9 , yet we know little about cooperation within this important ecosystem. Here we experimentally test for evolved cooperation within the Bacteroidales, the dominant Gram-negative bacteria of the human intestine. We show that during growth on certain dietary polysaccharides, the model member Bacteroides thetaiotaomicron exhibits only limited cooperation. Although this organism digests these polysaccharides extracellularly, mutants lacking this ability are outcompeted. In contrast, we discovered a dedicated cross-feeding enzyme system in the prominent gut symbiont Bacteroides ovatus, which digests polysaccharide at a cost to itself but at a benefit to another species. Using in vitro systems and gnotobiotic mouse colonization models, we find that extracellular digestion of inulin increases the fitness of B.ovatus due to reciprocal benefits when it feeds other gut species such as Bacteroides vulgatus. This is a rare example of naturally-evolved cooperation between microbial species. Our study reveals both the complexity and importance of cooperative phenotypes within the mammalian intestinal microbiota.
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              Complex Glycan Catabolism by the Human Gut Microbiota: The Bacteroidetes Sus-like Paradigm*

              Trillions of microbes inhabit the distal gut of adult humans. They have evolved to compete efficiently for nutrients, including a wide array of chemically diverse, complex glycans present in our diets, secreted by our intestinal mucosa, and displayed on the surfaces of other gut microbes. Here, we review how members of the Bacteroidetes, one of two dominant gut-associated bacterial phyla, process complex glycans using a series of similarly patterned, cell envelope-associated multiprotein systems. These systems provide insights into how gut, as well as terrestrial and aquatic, Bacteroidetes survive in highly competitive ecosystems.
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                Author and article information

                Contributors
                Role: Editor
                Role: Solicited external reviewer
                Role: Solicited external reviewer
                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                13 August 2019
                Jul-Aug 2019
                : 10
                : 4
                : e01762-19
                Affiliations
                [a ]Division of Infectious Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
                The Forsyth Institute
                Institut Pasteur
                University of Chicago
                Author notes
                Address correspondence to Laurie E. Comstock, lcomstock@ 123456rics.bwh.harvard.edu .
                Article
                mBio01762-19
                10.1128/mBio.01762-19
                6692515
                31409684
                e4b567a7-6029-48f7-bd97-a120e4354920
                Copyright © 2019 García-Bayona and Comstock.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                History
                : 3 July 2019
                : 12 July 2019
                Page count
                supplementary-material: 3, Figures: 4, Tables: 0, Equations: 0, References: 65, Pages: 13, Words: 7983
                Funding
                Funded by: HHS | National Institutes of Health (NIH), https://doi.org/10.13039/100000002;
                Award ID: AI120633
                Award Recipient :
                Categories
                Research Article
                Applied and Environmental Science
                Custom metadata
                July/August 2019

                Life sciences
                bacteroides,microbiota,mutant construction,vectors
                Life sciences
                bacteroides, microbiota, mutant construction, vectors

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