bioRxiv preprint doi: https://doi.org/10.1101/2019.12.21.885665; this version posted December 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Taxonomic description 2 Sporaefaciens musculi gen. nov., sp. nov., a novel bacterium 3 isolated from the caecum of an obese mouse 4 Torben Sølbeck Rasmussen1*, Theresa Streidl2, Thomas C.A. Hitch2, Esther Wortmann2, 1 4 1 2,3 5 Paulina Deptula , Michael Hansen , Dennis Sandris Nielsen , Thomas Clavel , Finn Kvist 6 Vogensen1* 7 1 Section of Microbiology and Fermentation, Dept. of Food Science, Faculty of Science, University of 8 Copenhagen, Denmark 9 2 Functional Microbiome Research Group, Institute of Medical Microbiology, RWTH University Hospital, 10 Aachen, Germany 11 3 ZIEL Core Facility Microbiome, Technical University of Munich, Freising, Germany 12 4 Center for Advanced Bioimaging, Department of Plant and Environmental Sciences, University of 13 Copenhagen, Frederiksberg, Denmark 14 Keywords: Firmicutes, Lachnospiraceae, mouse faeces, diet-induced obesity, spore-forming, 15 succinate, acetate 16 *Correspondence: Torben S. Rasmussen – [email protected] & Finn K. Vogensen - 17 [email protected] 18 Abbreviations: WCA, Wilkens-Chalgren-Anaerobe; GAM, Gifu Anaerobic Medium; RT, Room 19 Temperature; ONT, Oxford Nanopore Technologies; ANI, Average Nucleotide Identity; SEM, 20 Scanning Electron Microscopy; SIM, Sulphide Indole Motility; HPLC-RI, High Performance Liquid 21 Chromatography – Refractive Index; SCFA, Short Chain Fatty Acid; CRISPR, Clustered Regularly 22 Interspaced Short Palindromic Repeats; VLPs, virus-like-particles; TUM, Technical University of 23 Munich; UPCH, University of Copenhagen; POCP, Percentage of conserved proteins. 24 Repositories: The GenBank accession number for the 16S rRNA gene sequence of strain WCA- 25 9-b2T is MN756014, and the accession number for the genome assembly is PRJNA592877. Raw 26 sequencing Illumina NextSeq (PRJEB35655) and ONT MinION (PRJEB35656) data can be 27 accessed at EMBL-EBI. 28 Four supplementary figures are available with the online version of this article. Page 1 of 17 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.21.885665; this version posted December 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 29 Abstract 30 A bacterial strain, designated WCA-9-b2, was isolated from the caecal content of an 18-week-old 31 obese C57BL/6NTac male mouse. According to phenotypic analyses, the isolate is rod-shaped, 32 Gram-positive, strictly anaerobic, spore-forming and non-motile under the conditions tested. 33 Bacterial colonies were irregular and non-pigmented. Analysis of the 16S rRNA gene indicated that 34 the isolate belonged to the family Lachnospiraceae with Clostridium scindens ATTC 35704 (94.9% 35 sequence identity) and Dorea formicigenerans ATCC 27755 (94.8%) being the closest relatives. 36 Whole genome sequencing showed average nucleotide identity (ANI) ranging from 69.80–74.23% 37 and percentage of conserved proteins (POCP) values < 50% against the nine closest relatives. The 38 genome-based G+C content of genomic DNA was 44.4%. The predominant metabolic end products 39 of glucose fermentation were acetate and succinate. Based on these data, we propose that strain 40 WCA-9-b2 represents a novel species within a novel genus, for which the name Sporaefaciens 41 musculi gen. nov., sp. nov. is proposed. The type strain is WCA-9-b2T (=DSM 106039T = CCUG 42 pending IDT). 43 Page 2 of 17 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.21.885665; this version posted December 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 44 Introduction 45 The mammalian gut is inhabited by a high diversity of strictly anaerobic bacteria predominated by 46 Gram-positive species from the phylum Firmicutes [1], of which many have not yet been cultured. 47 Metagenome sequencing has been an efficient tool to perform in silico characterisation of the 48 unculturable gut bacteria [2], although metagenome sequencing often misses spore-forming 49 bacteria due to the difficulty of DNA extraction from the robust structure of the spores [3]. As the 50 mammalian gut microbiota (GM) influences host health [4–6], combining in silico characterisation 51 of gut bacteria with culturing novel isolates in vitro opens new research avenues by investigating 52 their functional properties. A large diversity of bacteria belonging to the class Clostridia contribute 53 in maintaining gut health, amongst other through the production of short chain fatty acids (SCFAs) 54 [7, 8]. Especially, butyrate has been associated with beneficial health effects [7], even though 55 many butyrate producers are dependent on cross-feeding from other bacteria producing 56 intermediate products such as acetate or lactate [9]. Whereas succinate, an intermediate product 57 of propionate, is a double-edge sword that in some conditions sustain the stability of the GM 58 component by cross-feeding, whilst in other conditions elevates the pathogenic potential of 59 infectious bacteria such as Clostridioides difficile or Clostridium rodentium. In the current study we 60 report the cultivation and detailed characterisation of a spore-forming, succinate and acetate 61 producing bacterial strain WCA-9-b2T isolated from the gut of an obese male mouse, which we 62 propose represents a novel genus within family Lachnospiraceae, phylum Firmicutes. 63 Isolation and ecology 64 Strain WCA-9-b2T originated from the caecum of a C57BL/6NTac mouse fed a high-fat diet (HF, 65 Research Diets D12492, USA) ad libitum for 13 weeks as part of a previously published study [10]. 66 The initial isolation of strain WCA-9-b2 took place at the ZIEL Core Facility Microbiome of the 67 Technical University of Munich (TUM), Germany. Downstream analyses were conducted at the 68 University of Copenhagen (UCPH), Denmark. Anaerobic handling and agar plate incubation during 69 isolation at TUM was performed in an anaerobic chamber (VA500 workstation, Don Whitley 70 Scientific) containing an atmosphere of 90 % (v/v) N2 and 10 % H2 a at a temperature of 37°C and 71 a humidity of 75 %. All materials were brought into the anaerobic chamber at least 24 hours before 72 use to ensure anaerobic conditions. Liquid and solid media were autoclaved and contained 0.02% 73 1,4-Dithiothreitol (Sigma, cat. No. DTT-RO) and 0.05% L-cysteine (Sigma, cat. no. 168149) as 74 reducing agents and 1 mg/L resazurin as a redox potential indicator. Broth media were heated 75 prior to flushing with 100% N2 by an anaerobic gassing unit (AGU, QCAL Messtechnik GmbH) for Page 3 of 17 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.21.885665; this version posted December 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 76 at least 3 min. pr. 10 mL. Agar media in Petri dishes contained 1.5% agar (Oxoid™, cat. no. 77 LP0011). Incubations took place at 37°C unless otherwise stated. 78 Strain WCA-9-b2T was isolated on Wilkens-Chalgren-Anaerobe agar (WCA, Sigma, cat. no. 79 W1886) supplemented with 0.1% bile salt (WCA-BS) (sodium taurocholate hydrate, Sigma, cat. no. 80 86339) to enhance spore germination. Approx. ~20 mg thawed caecum content was transferred to 81 a 0.20 µm sterile filtered 1:1 mixture of PBS (NaCl 137mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 82 1.8mM) and 70% ethanol to kill vegetative cells. The suspension was incubated at RT (room 83 temperature) under aerobic conditions for four hours and vortexed every 30 min., followed by 3x 84 centrifugation (11000 x g, 5 min.) and resuspension in aerobic PBS. Ten-fold serial dilutions were 85 prepared with anoxic PBS containing 0.02 % peptone (Sigma, cat. no. 91249). Each dilution (10 µl) 86 was deposited on WCA-BS agar and immediately tilted for vertical migration on the plate, followed 87 by incubation at 37°C for 4 days. Bacterial colonies from a representative area were picked and 88 further re-streaked on WCA agar 3x to obtain pure cultures. WCA-9-b2T was deposited at the 89 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM 106039) and the Culture 90 Collection University of Gothenburg (CCUG pending IDT). Liquid nitrogen was used to snap-freeze 91 cryo-aliquots (pivotal for survival of the strain), i.e., cultures diluted 1:1 in 50% glycerol, and then 92 stored at -80°C. 93 Gifu Anaerobic Medium (GAM, HyServe, cat. no. 5422) was used for downstream analyses, since 94 strain WCA-9-b2T showed improved growth in GAM vs. WCA. GAM agar was supplemented with 2 95 mL/L titanium(III) chloride (GAM-TC) (Sigma, cat. no. 1107890001) solution [11, 12] as an 96 additional oxygen scavenger to obtain clear colonies on Petri dishes. The anaerobic handling of 97 downstream analyses at UCPH was performed in another anaerobic chamber (Model AALC, Coy 98 Laboratory Products) containing ~93% (v/v) N2, ~2% H2, ~5% CO2 and an atmosphere kept at RT. 99 Agar plates were incubated at 37C in an anaerobic jar (Cat. No. HP0011A, Thermo Scientific) 100 containing an anaerobic sachet (Cat.