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      α-Galactosidase and Sucrose-Kinase Relationships in a Bi-functional AgaSK Enzyme Produced by the Human Gut Symbiont Ruminococcus gnavus E1

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          Abstract

          Plant α-galactosides belonging to the raffinose family oligosaccharides (RFOs) and considered as prebiotics, are commonly degraded by α-galactosidases produced by the human gut microbiome. In this environment, the Ruminococcus gnavus E1 symbiont–well-known for various benefit–is able to produce an original RgAgaSK bifunctional enzyme. This enzyme contains an hydrolytic α-galactosidase domain linked to an ATP dependent extra-domain, specifically involved in the α-galactoside hydrolysis and the phosphorylation of the glucose, respectively. However, the multi-modular relationships between both catalytic domains remained hitherto unexplored and has been, consequently, herein investigated. Biochemical characterization of heterologously expressed enzymes either in full-form or in separated domains revealed similar kinetic parameters. These results were supported by molecular modeling studies performed on the whole enzyme in complex with different RFOs. Further enzymatic analysis associated with kinetic degradation of various substrates followed by high pressure anionic exchange chromatography revealed that catalytic efficiency decreased as the number of D-galactosyl moieties branched onto the oligosaccharide increased, suggesting a preference of RgAgaSK for RFO’s short chains. A wide prevalence and abundance study on a human metagenomic library showed a high prevalence of the RgAgaSK encoding gene whatever the health status of the individuals. Finally, phylogeny and synteny studies suggested a limited spread by horizontal transfer of the clusters’ containing RgAgaSK to only few species of Firmicutes, highlighting the importance of these undispersed tandem activities in the human gut microbiome.

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          MUSCLE: multiple sequence alignment with high accuracy and high throughput.

          We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.
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            Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4

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              A human gut microbial gene catalogue established by metagenomic sequencing.

              To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                12 November 2020
                2020
                : 11
                : 579521
                Affiliations
                [1] 1Aix-Marseille Université, CNRS, Centrale Marseille, iSm2 , Marseille, France
                [2] 2TBI, Université de Toulouse, CNRS, INRAE, INSA , Toulouse, France
                [3] 3CNRS, Aix-Marseille Université, AFMB , Marseille, France
                [4] 4Yelen Analytics, Aix-Marseille Université, ICR , Marseille, France
                [5] 5Aix-Marseille Université, CNRS, IMM, LCB , Marseille, France
                [6] 6Aix-Marseille Université, CNRS, IMM, BIP , Marseille, France
                Author notes

                Edited by: Hauke Smidt, Wageningen University & Research, Netherlands

                Reviewed by: Diego Fabian Gomez-Casati, National University of Rosario, Argentina; Evelyne Forano, INRAE Clermont-Auvergne-Rhône-Alpes, France

                *Correspondence: Thierry Giardina, thierry.giardina@ 123456univ-amu.fr

                These authors have contributed equally to this work

                This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2020.579521
                7688924
                33281771
                7bb72c06-b5dc-4622-8e6b-3ee74b3511b7
                Copyright © 2020 Lafond, Tauzin, Bruel, Laville, Lombard, Esque, André, Vidal, Pompeo, Quinson, Perrier, Fons, Potocki-Veronese and Giardina.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 02 July 2020
                : 19 October 2020
                Page count
                Figures: 12, Tables: 2, Equations: 0, References: 56, Pages: 16, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                raffinose oligosaccharide family,sucrose,α-galactosidase,sucrose-kinase,human gut microbiome,gh36 family

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