Mouse samples for cultivation

The use of mice was approved by the local authorities in charge (animal welfare authorization 32–568, Freising District Office; Regierungspräsidium Freiburg T05-28). Laboratory mice were housed in conventional or specific pathogen-free facilities at the WZW School of Life Sciences (TU München), the Institute for Medical Microbiology and Hygiene (Universitätsklinikum Freiburg) or the Rodent Center (ETH Zurich). Samples from mice captured in the wild were obtained as described previously42. Starting materials for bacteria isolation included fresh faeces collected from living mice, mucosal samples, as well as small intestinal, caecal or colonic content collected from mice that had been euthanized by CO 2 inhalation or neck dislocation. The origins of bacteria (those eventually included in the collection) in terms of mouse genotype and gut location are provided in Supplementary Table 1. The working area and dissection set were cleaned and mice were copiously sprayed with 80% (vol/vol) ethanol before dissection in order to avoid contamination. In some instances, mice were dissected inside the anaerobic workstation to prevent any contact of gut samples with oxygen.

Culture media

All quantities are per litre of medium.

A II: Brain–heart–infusion (BHI), 18.5 g; yeast extract, 5 g; trypticase soy broth, 15 g; K 2 HPO 4 , 2.5 g; hemin, 10 µg; glucose, 0.5 g; palladium chloride, 0.33 g; agar, 15 g. After autoclaving, add Na 2 CO 3 , 42 mg; cysteine, 50 mg; menadione, 5 µg; fetal calf serum (complement-inactivated), 3% (vol/vol). Adapted from Aranki and Freter43.

BAC: WCA (Oxoid; CM0643; see manufacturer's instructions); Agar, 1.5 g; PdCl, 1 mg, dissolve first in HCl; phenosafranine, 2.5 mg; set pH to 7.0. After autoclaving (121 °C, 15 min), add kanamycin, 50 µg ml–1; cysteine (0.05% wt/vol), dithiothreitol (DTT) (0.02%), sheep blood (5% vol/vol), 1 vial of G-N Anaerobe Supplement (Oxoid SR0108) per 475 ml medium.

BARN: 1× Sigma salt (M6030); yeast extract, 0.5 g; maltose, 1 g; agar, 15 g; PdCl 2 2 mg; phenosafranin; set pH to 7.6. After autoclaving (121 °C, 15 min), add penicillin (25 µg ml–1), vancomycin (20 µg ml–1), kanamycin (50 µg ml–1), cysteine (0.05% wt/vol), trace element and vitamin solutions (see DSMZ medium 104 PYG).

BHI: DSMZ medium 215c (supplemented or not with 15 g agar).

BIL: 1× Sigma salt (M6030); peptone, 2 g; yeast extract, 200 mg; agar, 1.5 g; PdCl (1 mg; dissolve first in HCl); phenosafranine (2.5 mg). After autoclaving (121 °C, 15 min), add Oxgall (1.5% vol/vol; Fluka ref. B3883), taurine (200 µM), cysteine (0.05% wt/vol), TUDCA (100 µg l–1; Santa Cruz Biotechnol, ref. sc296449).

BLS: Blood agar with 10 mg l–1 colistin sulfate and 5 mg l–1 oxolinic acid.

COR: 1× Sigma salt (M6030); Tween 80 (1 ml); pig mucin type III (Sigma) (250 mg; Sigma M1778); agar (1.5% wt/vol); PdCl (1 mg; dissolve first in HCl); phenosafranine (2.5 mg). After autoclaving (121 °C, 15 min), add arginine, glycine, histidine and lysine (each 2.5 g), bile salts (0.5% wt/vol; Fluka ref. 48305), cysteine (0.05%), DTT (0.02%), sheep blood (5% vol/vol). For COR2 agar, add ampicillin and neomycin (50 µg ml–1 each) after autoclaving (121 °C, 15 min).

ECSA: 17.0 g pancreatic digested casein; 3.0 g peptic digested casein; 5.0 g yeast extract; 10.0 g ox gall; 5.0 g sodium chloride; 1.0 g aesculin; 0.5 g ammonium iron (iii) citrate; 0.25 g sozium azide; 1.0 g sodium citrate; 13.5 g agar.

mOs-SRB: 11.28 g M9 minimal salt medium (Sigma 6030); 20.0 g agar; 2.0 g Na 2 SO 4 anhydr.; 75 mg yeast extract; 75 mg Li-lactate. After autoclaving, add cysteine (0.05% wt/vol), DTT (0.02%).

MT10: 1× Sigma salt (M6030); pig mucin type III (Sigma) (250 mg; Sigma M1778); PdCl 2 2 mg; thioglycolic acid, 0.1 g; phenosafranin; 50 ml rumen fluid; agar, 15 g; set pH to 7. After autoclaving (121 °C, 15 min), add rifampicin (20 µg ml–1), cysteine (0.05% wt/vol), 2-mercaptoethanol (50 µM), trace element and vitamin solutions (see DSMZ medium 104 PYG).

Postgate (after Postgate, Appl Microbiol 1963, 11:265): KH 2 PO4, 0.5 g; NH 4 Cl, 1 g; Na 2 SO 4 , 2 g; CaCl 2 .6H 2 O, 0.1 g; MgSO 4 .7H 2 O, 1 g; Na-lactate, 3.5 g; yeast extract, 1 g; thioglycolic acid, 0.1 g; FeSO 4 .7H 2 O, 0.5 g; agar, 15 g; NaHCO 3 , 2 g; PdCl 2 2 mg; phenosafranin; set pH to 7.6. After autoclaving (121 °C, 15 min), add penicillin (25 µg ml–1), trace element and vitamin solutions (see DSMZ medium 104 PYG).

PYF: Peptone-yeast extract broth with 4% (vol/vol) Fildes' digest (peptic digestive of horse blood)19.

PYG: DSMZ medium 104; 15 g agar.

TA-BHI: Tannic acid-treated BHI agar, prepared as described in ref. 44.

TGB: Thioglycollate medium brewer modified (Becton, Dickinson and Company, cat. no. 5211716); 15 g agar; 0.5 ml l–1 thioglycolic acid (AppliChem; cat. no. A3533).

WCA: Wilkins–Chalgren Anaerobe (Oxoid, ref. CM0643).

WHC: WCA (oxoid) + 25 µg ml–1 penicillin (sodium salt).

WSB: WCA (Oxoid), 33 g; glucose, 4 g; hemin, 10 µg; l-cystine, 0.4 g; agar, 15 g. After autoclaving, add defibrinated sheep blood (Oxoid) 5% (vol/vol).

Bacterial strains isolation

Gut samples were re-suspended (1:10 wt/vol) in reduced buffered solutions or broth media (see composition above in the section ‘Culture media’). Mucosal samples were prepared as described previously10. Tenfold dilution series of gut suspensions were plated on agar media (see composition above) and bacteria were allowed to grow for 2–30 days under aerobic conditions, or in an anaerobic chamber containing a mixture of hydrogen, nitrogen and carbon dioxide (5:10:85) or hydrogen and nitrogen only (10:90). Single colonies were streaked at least three times onto fresh agar plates before transfer into broth media. Bacterial smears formed after growth of low dilutions were also re-streaked to obtain single colonies of low-abundant bacteria. Culture purity was ensured by observing colony morphology as well as cell morphology by light microscopy after Gram-staining. For identification and phylogenetic analysis of isolates, DNA was extracted from pure cultures and 16S rRNA genes were amplified and sequenced as described previously15. Electropherogram quality was checked and contigs were built using Bioedit45. Sequences were identified using EzTaxon46. The value of 97% 16S rRNA gene sequence identity, which is a widely used and generally accepted though rather conservative threshold, was chosen to delineate novel taxa in a consistent manner across bacterial phyla23. Further characterization of the strains is detailed in the next section. Routine media used for subculturing strains included reduced Wilkins Chalgren anaerobic (WCA) broth (Oxoid) supplemented with cysteine (0.05% wt/vol) and DTT (0.02%) as reducing agents. Cryo-stocks were prepared from freshly grown cultures by mixing bacterial suspensions 1:1 with filter-sterilized glycerol in culture medium (40% vol/vol) before freezing at −80 °C. Live and cryo-cultures of isolates were shipped to the German Collection of Microorganisms and Cell Cultures (DSMZ), where stocks for long-term storage were prepared and detailed taxonomic characterization was carried out. Strain information (including culture conditions) is available online at www.dsmz.de/miBC.

Strain characterization

Analyses included cell morphology by microscopy, enzymatic testing, cellular fatty acids, diaminopimelic acid, automated ribotyping and mass spectrometry analysis using a MALDI-biotyper (Bruker). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) sample preparation was performed according to protocol 3 as described by Schumann and Maier47. Ribotyping was carried out using the automated Riboprinter microbial characterization system (Dupont, Qualicon). Sample preparation and analysis were performed according to the manufacturer's instructions and EcoRI restriction enzyme was used to generate the DNA fragments. Protocol 1, published by Schumann48 was applied to screen Gram-positive bacterial cells for the presence of diaminopimelic acid. Two loops of wet biomass were added to 200 µl of 4 N HCl in 2 ml glass ampoules, which were heat-sealed and kept for 16 h at 100 °C. After cooling, hydrolysates were pressed through a charcoal bed using a pipette bulb and dried in a gentle air stream at 35 °C until all traces of acid were eliminated. Hydrolysates were dissolved in 100 µl distilled water and a volume of 2 µl was spotted on the baseline of cellulose thin-layer chromatography (TLC) plates (Merck, P/N 1.05577) using a glass capillary. For separation of the diamino acid, a solvent system was used according to ref. 49 (32 ml methanol, 4 ml pyridine, 7 ml distilled water and 1 ml 12 N HCl; all from Sigma-Aldrich). TLC plates were sprayed with ninhydrin reagent after development. Spots became visible after heating (100 °C, 5 min). Cellular fatty acids were determined from cells grown on blood agar plates or collected from pre-reduced liquid media. Fatty acids were analysed as methyl ester derivatives obtained from 10 mg dry cell material by saponication, methylation and extraction using the modifications by Kuykendall et al.50 of the method by Miller51. The fatty acid methyl esters were separated and identified using the Microbial Identification System (MIDI) Sherlock version 6.1 (TSBA40 database). Enzymatic testing was done according to the manufacturer's instructions (Biomérieux). Cell morphology was examined using phase-contrast microscopy (Zeiss Axioscope A.1, ×100 Plan-Neofluar oil-immersion objective, Ph3; Zeiss Axiocam MRc; Axiovision software). Slides were coated with a layer of highly purified agar (2%).

High-throughput 16S ribosomal RNA gene analysis

For analysis of mouse gut microbiota based on 16S rRNA gene amplicon sequencing with ensuing determination of core mouse gut bacteria, caecal samples were obtained from a total of 93 mice housed in eight different facilities in Europe and America (Supplementary Table 6). The aim was to obtain a data set that was not specific to one facility, and thus was as representative as possible of bacterial communities in the distal mouse intestine.

Mice were killed and caeca were immediately removed, placed in cryo-vials and snap-frozen in liquid nitrogen. Samples were stored at −80 °C until shipped on dry ice to the University of Nebraska at Lincoln and stored again at −80 °C until further processed. Once all samples were collected, they were thawed under anaerobic conditions and diluted 1:10 in sterile phosphate-buffered saline with 10% glycerol. Aliquots (200 µl) of each sample were used for DNA extraction following procedures described elsewhere52. 16S rRNA gene tag sequencing (MiSeq, Illumina) of V5–V6 regions was performed at the University of Minnesota Genomics Center using primers 784F (5′-RGGATTAGATACCC′) and 1064R (5′-CGACRRCCATGCANCACCT)53.

Raw sequence reads were processed using IMNGS (www.imngs.org) with a pipeline developed in-house based on UPARSE54. Details about the analysis have already been described elsewhere55. OTUs clustered at 97% sequence identity and occurring at a relative abundance of ≥0.1% total reads in at least one sample were analysed further. The analysis delivered 14,906,121 quality- and chimera-checked reads (160,281 ± 60,686 per sample) that clustered in 772 OTUs (344 ± 45 OTUs per sample) (Supplementary Table 7 and Supplementary Sequence File).

For phylogenetic analysis, the evolutionary history was inferred by using the maximum likelihood method based on the ‘General Time Reversible model’ and using 200 bootstrap replications in MEGA6 (ref. 56). The tree with the highest log-likelihood (representing the most probable tree topology) was selected. The percentage of trees in which the associated taxa clustered together was shown next to the branches (the tree was condensed to values ≥50%). All positions with <70% site coverage were eliminated. That is, fewer than 30% alignment gaps, missing data and ambiguous bases were allowed at any position. There were a total of 261 positions in the final data set.

Integrated 16S rRNA gene amplicon studies

To evaluate the prevalence and abundance of miBC members in gut microbiota, other host-derived ecosystems and environmental samples, we used an integrated metagenomic platform developed in-house and used in previous work (www.imngs.org)16,57. All 16S rRNA gene amplicon studies available in the SRA58 were extracted and organized in sample-specific databases. The IMNGS build 1510 used in the present study contained a total of 55,073 samples, including 6,001 and 11,705 from the mouse and human gut, respectively. The corresponding databases were searched using UBLAST59 based on nearly full-length 16S rRNA gene sequences of the 76 miBC species as queries to ensure good coverage across all studies, independent of 16S rRNA gene regions. Results were filtered on length (>200 nt), coverage (>70% read length) and similarity (>97 or 99%).

To determine whether certain species were host-specific, we looked at the number of SRA-derived samples positive for each species included in the collection, that is, those mouse or human samples in which sequences matched the corresponding 16S rRNA gene sequence at the level of 97% similarity. Of note, each OTU with a miBC match collected via IMNGS was subsequently assigned to one single closest sequence in the miBC reference set in order to avoid redundancy issues in the case of genera with closely related species (for example, for lactobacilli, enterococci, staphylococci). Prevalence had to be at least 5% in mouse gut samples so that species were considered for discriminative analysis between human and mice. The parameters used to categorize species were as follows: (1) rare: the species had a prevalence of <1% of mouse samples; (2) low prevalence: <5% mouse samples; (3) shared: the percentage of positive samples from human gut was at least as high as those from mice; (4) shared and dominant: definition in category 3 held true also when considering only SRA-derived OTUs occurring at a relative sequence abundance of >1% total reads (dominant OTUs); (5) mouse-enriched: the percentage of positive gut samples from mice was at least twice that from human subjects; (6) mouse-enriched and dominant: the given species was also enriched in mice when considering only dominant SRA-derived OTUs (as defined in category (4)).

For the interpretation of results, it is important to remember that the analysis described above is based on short 16S rRNA gene amplicons, which can affect similarity-based results when compared to full-length gene analysis. Moreover, although it is reasonable to assume that the large-scale analysis (including thousands of samples from many different studies) guarantees a certain degree of representativeness, sample descriptions in SRA can be imprecise and the analysis certainly includes outlier samples for a given ecosystem (for example, from hosts with peculiar pathophysiological conditions).

Genome sequencing and processing

A total of 53 draft genomes were obtained in the present study (EBI project accession no. PRJEB10572). The 53 strains were selected following three criteria: (1) novelty; (2) relevance according to the IMNGS and MGS analysis (mouse-enriched, prevalent or dominant); and (3) need to fill diversity gaps (species with no genome yet available). The miBC collection also includes 12 strains, for which novel genomes have already been deposited and will be published in a separate study (SRA accessions are provided in Supplementary Table 1). Genomes from 11 additional species in the collection were retrieved from NCBI (Supplementary Table 1). The corresponding raw sequence files were processed as for the genome sequences generated in-house.

Genomic DNA was obtained from pure cultures by precipitation after mechanical lysis following a protocol published previously60. DNA libraries were prepared using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina). The protocol was optimized (DNA shearing and fragment size selection) to improve assembly quality61. Libraries were sequenced using the Illumina MiSeq system according to the manufacturer's instructions.

Reads were assembled using Spades v3.6.1 with activated BayesHammer tool for error correction and MismatchCorrector module for post-assembly mismatch and indel corrections62. Assemblies were evaluated using Quast v3.1 (ref. 63). Prediction and annotation of open reading frames (ORFs) on contigs >1,000 nt were performed with prokka v1.11 (ref. 64). Genome sequences were submitted to the European Nucleotide Archive and are available under the accession numbers provided in Supplementary Table 1.

Genome-based analyses

16S rRNA gene sequences were extracted from both the mouse isolate genomes and reference genomes using RNAmmer (ref. 65). All pairwise similarities among these sequences were calculated from exact pairwise sequence alignments using recommended settings23.

For digital DNA:DNA hybridization (dDDH), the Genome-to-Genome Distance Calculator 2.0 (GGDC), a web service freely available at http://ggdc.dsmz.de, provided a genome sequence-based delineation of (sub-)species by reporting dDDH estimates as well as their confidence intervals66. This approach was shown to have several advantages over alternative species delineation approaches66,67, without mimicking the pitfalls of conventional DDH.

A whole-genome phylogeny (based on the proteome data) was inferred using the latest version of the Genome-BLAST Distance Phylogeny (GBDP) method66,68. Here, pairwise proteome comparisons (including pseudo-bootstrap replicates) were performed under the greedy-with-trimming algorithm and further recommended settings69. The final tree was inferred using FastME v2.07 with TBR post-processing70. GBDP settings were trimming algorithm, formula d5 and an e-value threshold of 10e-8. The tree was rooted at the mid-point71 and numbers above branches are greedy-with-trimming pseudo-bootstrap support values from 100 replicates. Only support above 60% is shown.

The difference in genomic G+C content was used to delineate species. When computed from genome sequences, the G+C content differences vary no more than 1% within species67.

Similarity search against the mouse gene catalogue

The mouse gene catalogue3 was searched against each of the 15 genome databases separately, using BLASTn (ref. 72). The gene matched criteria were percent identity ≥95% over 100 bp or more and e-value ≤10×10-5 (very similar results were obtained using 80% coverage threshold with ≥95% identity. The number of gene matches assigned to metagenomic species (MGS), as defined previously3,24, was counted (see Supplementary Table 5 for details). The occurrences of the five MGS through the mouse cohort sampled in ref. 3, with 85% of the genes matching the novel isolates, were used to generate Supplementary Fig. 1b.

Metagenome analysis

Metagenomic DNA was extracted from mouse intestinal contents as described above for pure cultures60. Libraries of 300 bp insert sizes were prepared from 500 ng fragmented DNA (Covaris S220 AFA System; duty factor 10%, peak incident power 175 W, 200 cycles per burst, 40 s) for each sample using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370S, New England Biolabs) according to the manufacturer's instructions. Samples were barcoded and sequenced on a TruSeq Rapid PE flowcell (PE-402-4001; Illumina) using an Illumina HiSeq 2500 sequencer with TruSeq Rapid SBS chemistry (FC-402-4001) and the 2 × 100 bp paired-end read module. Real-time analysis (RTA) software (1.17.20) was used for image analysis and base calling. Sequence files (.fastq) were generated with the CASAVA BCL2FASTQ Conversion Software (1.8.3). Paired-end sequencing reads were assembled into metagenomics contigs using Megahit73. Contigs with a length of >500 nt were classified with Kraken74 and those classified as bacteria were kept for further analysis.

Three complementary approaches were followed to determine metagenomic coverage by cultivated bacteria. First, a local database was created of all miBC genomes and the metagenomes in a presence/absence binary code (1/0) of protein families (PFAM). Deterministic incremental selection of best fitted genomes was performed to assess coverage of the metagenomes by all miBC species (n = 76), or by species included in the minimal bacteriome MIBAC-1 (n = 18; this study), SIHUMI (n = 8)27 and ASF (n = 8)26. Second, ORFs in metagenomic contigs were selected and initial annotation was carried out with prokka64. The predicted metaproteome of miBC and the three minimal bacteriomes were used for creation of protein BLAST databases. Sequence coverage of metagenomes was calculated based on a BLASTp search. ORFs that significantly (expected value E < 10−5) aligned with a database entry protein at minimum length coverage ≥80% were used for the calculation of functional coverage (that is, at the level of homologous proteins). Third, from the ORFs retained in step (2), only those with ≥80% sequence similarity were used for calculation of close match sequence coverage. Of note, all newly generated or database-derived metagenomes used in the present analysis were completely independent of the samples used for strain isolation.

Description of novel bacteria

The descriptions are based on genome sequence analysis, enzymatic testing and chemotaxonomy (cellular fatty acids and detection of meso-diaminopimelic acid). Genome-based analyses included whole proteome-based phylogenomic GBDP analysis, dDDH, 16S rRNA gene sequence analysis and differences in G+C content of DNA. First, the relative subtree height (RSH) of any given putative novel bacteria within the phylogenomic tree relative to the RSH of related species allowed reliable conclusions on the taxonomic properties of novel strains. Second, a dDDH value of <70% indicated affiliation of an isolate to a novel species. Third, a 16S rRNA gene sequence identity of ≤94.5% was considered strong evidence for different genera and ≤86.5% for distinct families75. Fourth, because within-species differences in the genome-based G+C content of DNA are almost exclusively <1%, larger differences strongly supported the status of distinct species. Finally, percentage of conserved proteins (POCP) analysis was done using the IMG software tool Genus definition76,77, considering genus delineation at a POCP value of <50%.

Novel bacterial taxa included (1) four species: ‘Bacteroides caecimuris’ strain I48T (=DSM 26085T = KCTC 15547T), ‘Blautia caecimuris’ strain SJ18T (=DSM 29492T = KCTC 15541T), ‘Enterorhabdus muris’ strain WAC-131-CoC-2T (=DSM 29508T = KCTC 15543T) and ‘Pasteurella caecimuris’ strain AA-424-CC-1T (=DSM 28627T = KCTC 52216T); (2) ten genera: ‘Longicatena caecimuris’ strain PG-426-CC-2T (=DSM 29481T = KCTC 15535T), ‘Longibaculum muris’ strain MT10-315-CC-1.2-2T (=DSM 29487T = KCTC 15536T), ‘Extibacter muris’ strain JM-40T (=DSM 28560T = KCTC 15546T), ‘Muricomes intestini’ strain 2-PG-424-CC-1T (=DSM 29489T = KCTC 15545T), ‘Turicimonas muris’ strain YL45T (=DSM 26109T = KCTC 15542T), ‘Frisingicoccus caecimuris’ strain PG-426-CC-1T (=DSM 28559T = KCTC 15538T), ‘Flintibacter butyricus’ strain BLS21T (=DSM 27579T = KCTC 15544T), ‘Cuneatibacter caecimuris’ strain BARN-424-CC-10T (=DSM 29486T = KCTC 15539T), ‘Acutalibacter muris’ strain KB18T (=DSM 26090T = KCTC 15540T), ‘Irregularibacter muris’ 2PG-426-CC-4.2T (=DSM 28593T = KCTC 15548T); and (3) one member of a novel family: ‘Muribaculum intestinale’ strain YL27T (=DSM 28989T = KCTC 15537T) within the ‘Muribaculaceae’.

Description of Acutalibacter gen. nov

(A.cu.ta.li.bac'ter. L. adj. acutalis tapered, pointed; N.L. n. bacter rod; N.L. masc. n. Acutalibacter a rod-shaped bacterium with tapered ends, pertaining to the cell morphology of the type strain of the type species).

The closest phylogenomic neighbour is Clostridium sporosphaeroides. Both genomes cluster distantly from Clostridium leptum. They do not represent members within the Clostridium sensu stricto cluster. The dDDH is 23% and POCP is 41%, indicating the status of a separate genus, for which the name Acutalibacter is proposed. The dDDH value between the genome of strain KB18T and Ruminococcus bromii L2-63 is 25.6%. The G+C content of genomic DNA of the type strain is 54.6 mol%. The type species is Acutalibacter muris.

Description of Acutalibacter muris sp. nov

Acutalibacter muris (mu'ris L. gen. n. muris of a mouse). Its cells are long rods with tapered ends. It is strictly anaerobic. It is positive for α-galactosidase, β-galactosidase, β-galactosidase-6-phosphate, β-glucosidase, α-arabinosidase, β-glucuronidase and α-fucosidase (Rapid ID32A) and esculin hydrolysis (Api 20A).

D-xylopyranose can be formed from xylose, which is converted to d-xylulose by xylose isomerase activity. D-xylulose is subsequently phosphorylated to d-xylose 5-phosphate (E.C. 2.7.1.17, xylokinase, 01104), an intermediate of the pentose phosphate pathway. Hemicellulose consists of xylan, which is linked with sugar side chains (arabinose, galactose). Arabinoxylan can be converted to xylan by an arabinofructofuranose (E.C. 3.2.1.55, 00023). Xylan can be hydrolysed by E.C. 3.2.1.37, xylosidase (00237, 02775) or E.C. 3.2.1.54, dextrinase (03478). Several esterases are detectable within the genome.

The main cellular fatty acids comprise C 16:0 (22.1%), C 16:0 DMA (21.7%), iso-C 15:0 (16.8%), anteiso-C 15:0 (9.8%) and C 18:1 ω9c (12.2%). The diagnostic amino acid of the peptidoglycan type is meso-diaminopimelic acid. The type strain is KB18T (=DSM 26090T = KCTC 15540T).

Description of Bacteroides caecimuris sp. nov

Bacteroides caecimuris (cae.ci.mu'ris. L. n. caecum caecum; L. gen. n. muris of a mouse; N. L. gen. n. caecimuris from the caecum of a mouse). The closest phylogenomic neighbour is Bacteroides acidifaciens and the dDDH value between both genomes is 40.2%.

It is strictly anaerobic, Gram-negative, and in the form of short rods. It is positive for α- and β-galactosidase, β-galactosidase 6-phosphate, α- and β-glucosidase, α-arabinosidase, β-glucuronidase, α-fucosidase, alkaline phosphatase, leucine arylamidase and alanine arylamidase (Rapid ID 32A). It is positive for esculin hydrolysis and for acidification from d-glucose, d-lactose, saccharose, d-maltose, salicin, d-xylose, l-arabinose, d-cellobiose, d-mannose, d-raffinose, l-rhamnose and d-trehalose (Api 20A).

The strain is able to degrade pectin consisting of polygalacturonan and rhamnogalacturonan. The end products are d-galactopyranuronate and 4-deoxy-l-threo-hex-4-enopyranuronate. Pectin can be depolymerized by esterase activity. Enzymes include E.C. 3.1.1.11, Prokka_03269; E.C. 3.2.1.82, galacturonisidase, Prokka_03292; E.C. 4.2.2.23, rhamnogalacturonan endolyase, Prokka_03307; E.C. 4.2.2.24, rhamnogalacturonan exolyase, Prokka_03311; E.C. 3.2.1.172, rhamnogalacturonyl hydrolase, Prokka_03310.

β-l-rhamnopyranose can be converted to l-lactaldehyde and dihydroxyacetone phosphate, which can enter glycolysis (E.C. 5.3.1.14, L-rhamnose isomerase, Prokka_01322; E.C. 2.7.1.5, L-rhamnulokinase, Prokka_01323; E.C. 4.1.2.19, phosphate-aldolase, Prokka_01320). Xylose can be converted to d-xylulose 5-phosphate, which can enter the pentose phosphate pathway (E.C. 5.3.1.5, d-xylose epimerase, Prokka_03917; E.C. 2.7.1.17, kinase, Prokka_03916). The pentose phosphate pathway includes E.C. 5.3.1.6, Prokka_01016; E.C. 5.1.3.1, Prokka_00323; E.C. 2.2.1.1, Prokka_01617; E.C. 2.2.1.2, Prokka_03873; E.C. 2.2.1.1, Prokka_01617). The end product is d-glyceraldehyde 3-phosphate.

Trehalose can be converted to α-d-glucose and β-d-glucopyranose. The latter can be used to form β-d-glucose 6-phosphate, which can enter glycolysis (E.C. 3.2.1.28, Prokka_00582; E.C. 5.1.3.3, Prokka_01268; E.C. 2.7.1.2, Prokka_01994). Melibiose can be converted to α-d-galactose and α-d-glucose. Starch can be degraded to form β-d-glucose 6-phosphate and β-d-glucopyranose, which may enter glycolysis (E.C. 3.2.1, α-amylase, Prokka_03907; E.C. 2.4.1.1, Prokka_00503; E.C. 5.4.2.2, Prokka_01849; E.C. 2.4.1.25, Prokka_00656; E.C. 5.1.3.3, Prokka_01994).

L-glutamine can be converted to l-glutamate (E.C. 3.5.1.2, Prokka_01943), which can be dehydrogenized to 2-oxoglutatarate (E.C. 1.4.1.2, Prokka_02968) or converted to ammonium and fumarate (E.C. 2.6.1.1, Prokka_01814; E.C. 4.3.1.1, Prokka_02293).

Major cellular fatty acids are anteiso-C 15:0 (33.7%), iso-C 17:0 3-OH (15.9%), iso-C 15:0 (13.3%), C 16:0 3-OH (7.5%) and C 16:0 (4.8%). The G+C content of genomic DNA is 42.6%. The type strain is I48T (=DSM 26085T = KCTC 15547T).

Description of Blautia caecimuris sp. nov

Blautia caecimuris (cae.ci.mu'ris. L. n. caecum caecum; L. gen. n. muris of a mouse; N. L. gen. n. caecimuris from the caecum of a mouse). The closest phylogenomic neighbour is Blautia wexlerae and the dDDH value between both genomes is 25.6%. The G+C content difference between strain SJ18T and Blautia wexlerae is 1.6%.

The cells are coccoid and oval-shaped. It is a Gram-positive, strictly anaerobic bacterium. It is positive for α- and β-galactosidase, α-glucosidase, α-fucosidase, arginine arylamidase and leucine arylamidase (Rapid ID32A). It produces acid from d-glucose, d-lactose, d-saccharose, d-maltose, salicin, d-xylose, l-arabinose, d-raffinose, d-trehalose and l-rhamnose (Api 20A).

Nine alpha-amylases, which can be assigned to glycoside hydrolase family GH13 and catalyse the glycosidic linkage of glycosides, are detectable within the genome. All enzymes responsible for degradation of glycogen/starch are present: GlgX, a glycogen branching enzyme (E.C. 2.4.1.18; several copies), glycogenphosphorylase (E.C. 2.4.1.1, Prokka_03262), maltodextrinphosphorylase (E.C. 2.4.1.1, Prokka_02645) and phospoglucomutase (E.C. 5.4.2.2, Prokka_01686). A second pathway for glycogen degradation to d-glycopyranose 6-phosphate is also present: the first two steps are identical, but then maltotetraose is converted to maltotriose (E.C. 2.4.1.1, Prokka_02645), which is degraded to d-glycopyranose 6-phosphate: amylomaltase (E.C. 2.1.4.25, Prokka_02646); glucokinase (E.C. 2.7.1.2, Prokka_03476). The glucokinase is also involved in degradation of trehalose. Trehalose uptake is carried out by a trehalose-specific phosphotransferase system (PTS; Prokka_03240). The strain also possesses trehalose import proteins (Prokka_02649, 00179), transport system permease (Prokka_0062, 0067) and the operon repressor TreR (03239).

Chitobiose can also be transported and is subsequently phosphorylated to N,N′-diacetylchitobiose 6′-phosphate (E.C. 2.4.1.280; several copies), which is deacetylated (Prokka_03287) and hydrolysed by a glycosyl hydrolase to d-glucosamine and N-acetyl-d-glucosamine 6-phosphate (E.C. 3.2.1.86, Prokka_00121). N-acetyl-d-glucosamine 6-phosphate can enter the N-acetylglucosamine degradation I pathway for conversion to β-d-fructofuranose 6-phosphate, which may enter glycolysis (E.C. 3.5.1.25, N-acetylglucosamine 6-phosphate deacetylase, Prokka_00134; glucosamine 6-phosphate deaminase, Prokka_03782).

Xylose can be bound and imported by XylF and XylG proteins (Prokka_01553, 01554) in combination with the transport permease XylH (Prokka_01554). The xylose operon is regulated by XylR (Prokka_03489). D-xylopyranose is converted to d-xylulose by an isomerase (Prokka_02659) and phosphorylated to d-xylulose-5-phosphate by a kinase (Prokka_01743). The latter compound may enter the pentose phosphate pathway to form d-glyceraldehyde-3-phosphate (epimerase E.C. 5.1.3.1, Prokka_00665; isomerase E.C. 5.3.1.6, Prokka_02662; transketolase E.C. 2.2.1.1, several copies; transaldolase E.C. 2.2.1.2, Prokka_01937).

Lactose can be hydrolysed by a β-d-galactosidase (E.C. 3.2.1.23, Prokka_03707). Several amino acids can be degraded: arginine decarboxylase (E.C. 4.1.1.19, Prokka_03783), l-serine ammonia lyase (E.C. 4.3.1.17, Prokka_02259), threonine ammonia lyase (E.C. 4.3.1.19, Prokka_03091), methionine γ-lyase (E.C. 4.4.1.11, Prokka_00824) and d-cysteine (E.C. 4.4.1.15, Prokka_02595).

Major cellular fatty acids comprise C 18:0 (26.9%), C 12:0 (22.5%), iso I-C 19:1 (15.5%), C 16:0 (13.1%), iso -C 17:1 /anteiso-C 17:1 (8.8%) and C 14:0 (4.8%). The diagnostic amino acid of the peptidoglycan type is meso-diaminopimelic acid. The G+C content of genomic DNA is 43.0 mol%. The type strain is SJ18T (=DSM 29492T = KCTC 15541T).

Description of Cuneatibacter gen. nov

Cuneatibacter (Cu.ne.a.ti.bac'ter. L adj cuneatus wedge-shaped; L. masc. n. bacter rod; N.L. masc. n. Cuneatibacter a rod-shaped bacterium with wedge-shaped ends). The nearest phylogenomic neighbours are Clostridium clostridioforme and Clostridium symbosium. It is placed into the Lachnospiraceae cluster, apart from the type species of the genus Clostridium, Clostridium butyricum. POCP between C. butyricum and C. clostridioforme or C. symbosium is 39.9% and 39.7%, respectively. These low values confirm that both are not members of the Clostridium sensu stricto cluster. POCP between the genome of C. clostridioforme and C. symbosium is 36.5% and dDDH is 22.2%. The dDDH values between strain BARN-424-CC-10T and C. clostridioforme or C. symbosium are 21.6% or 22.3%, respectively. The difference in G+C content of DNA is 1.4% for both comparisons. All these values confirm the separate genus status of the isolate. The G+C content of genomic DNA of the type strain is 49.1 mol%. The type species is Cuneatibacter caecimuris.

Description of Cuneatibacter caecimuris sp. nov

Cuneatibacter caecimuris (cae.ci.mu'ris. L. n. caecum caecum; L. gen. n. muris of a mouse; N. L. gen. n. caecimuris from the caecum of a mouse). Its cells are small rods with tapered ends, 2–3 µm in length. It is Gram-positive and strictly anaerobic. It is positive for α- and β-galactosidase, β-glucoronidase, α- and β-glucosidase and α-arabinosidase (Rapid ID32A), esculin hydrolysis and acidification from l-rhamnose (Api 20A).

β-d-galactose can be degraded to form UDP-glucose. In a first step, the aldose-1-epimerase converts β-d-galactose to α-d-galactose (E.C. 5.1.3.3, Prokka_02857), which is subsequently phosphorylated by a galactokinase (E.C. 2.7.1.6, Prokka_00204). As a result of galactose 1-phosphate uridinyltransferase activity (E.C. 2.7.7.12, Prokka_00203), UDP-α-galactose is formed and rearranged to UDP-glucose (E.C. 00996; Prokka_0096).

N-acetyl-d-glucosamine can be degraded to form d-fructose 6-phosphate: N-acetyl-glucosamine 6-phosphate deacetylase (E.C. 3.5.1.25, Prokka_01387); glucosamine 6-phosphate isomerase (E.C. 3.5.99.6, Prokka_01388).

L-arabinose can be degraded to d-xylulose 5-phosphate due to l-arabinose isomerase activity (E.C. 5.3.1.4, Prokka_00574) and l-ribulose-5-phosphate 4-epimerase (E.C. 5.1.3.4; Prokka_01532). D-xylulose 5-phosphate may also be formed via degradation of d-xylose (E.C. 5.1.3.1.5, Prokka_01741; E.C. 2.7.1.17, Prokka_01860), which may enter the pentose phosphate pathway to generate d-fructose 6-phosphate (E.C. 2.2.1.1, transketolase, Prokka_00573; E.C. 2.2.1.2 transaldolase, Prokka_00768) and d-erythrose 4-phosphate. The latter can be converted to d-glyceraldehyde 3-phosphate by a transketolase activity, which may be converted to phosphoenolpyruvate (E.C. 1.2.1.12, Prokka_02031; E.C. 4.4.1.12, Prokka_02034; E.C. 2.7.2.3, Prokka_02032; E.C.5.4.2.12, Prokka_02345; E.C. 4.2.1.11, Prokka_02345). Phosphoenolpyruvate can be transformed to l-lactate (E.C. 2.7.1.40, pyruvate kinase, Prokka_01764; E.C. 1.1.1.27, dehydrogenase, Prokka_00578). D-erythrose 4-phosphate can also enter the chorismate biosynthesis pathway (all enzymes of this pathway are present in the genome of strain BARN-424-CC-10): E.C. 2.5.1.54, Prokka_00306; E.C. 4.2.3.4, Prokka_0226; E.C. 4.2.1.10, Prokka_00678; E.C. 1.1.24, Prokka_00680; E.C. 2.7.1.71, Prokka_03116; E.C. 2.5.1.19, Prokka_00308; E.C. 4.2.3.5, Prokka_00530.

The glycosyl hydrolases of family GH 28 (E.C. 3.2.1.15, polygalacturoronase), GH 78 (α-l-rhamnosidase) and GH105 (E.C. 4.2.2.23, rhamnogalacturonyl hydrolase) are involved in the degradation of pectin at the beginning of the pathway followed by activity of YesY (E.C. 3.1.1, Prokka_02705) and YesR (E.C. 3.2.1.172, Prokka_00382). The resulting 4-deoxy-l-threo-hex-4-enopyranuronate can be degraded by action of E.C. 5.3.1.17, isomerase, Prokka_01284; E.C. 1.1.1.67, dehydrogenase, Prokka_00822; E.C. 2.7.1.45, kinase, Prokka_00327 and aldolase (Prokka_00328). The end products are d-glyceraldehyde 3-phosphate and pyruvate.

A pathway for degradation of glycogen is detectable and includes the following reactions: phosphorylase (E.C. 2.4.1.1, Prokka_02446); 4-α-glucanotransferase (E.C. 2.4.1.25, Prokka_03092); α-glucosidase (E.C. 3.2.1.33; Prokka_02446); phosphorylase (E.C. 2.4.1.1, Prokka_02446) and phosphoglucomutase (E.C. 5.4.2.2, Prokka_02715). The end product is d-glucose 6-phosphate, which may enter glycolysis.

The predominant cellular fatty acid is C 16:0 (43.4%). Other fatty acids detectable with the MIDI system are iso-C 17:1 /anteiso-C 17:1 (10.1%), iso-C 15:1 /C 13:0 OH (9.0%), C 17:1 ω8c (8.3%) and C 14:0 (6.2%). Meso-diaminopimelic acid is absent. The type strain is BARN-424-CC-10T (=DSM29486T = KCTC 15539T).

Description of Enterorhabdus muris sp. nov

Enterorhabdus muris (mu'ris L. gen. n. muris of a mouse). Phylogenomic analysis places strain WCA-131-CoC-2T into the Enterorhabdus cluster. The dDDH value between the genome of E. mucosicola and E. caecimuris is 35.3% and between strain WCA-131-CoC-2T, E. mucosicola and E. caecimuris is 38.3 and 43.1%, respectively. These values clearly confirm the separate species status of strain WCA-131-CoC-2T.

The cells are rods, up to 5 µm in length, and are strictly anaerobic. The substrate profile in Api 20A kit is negative. Arginine hydrolase activity is detected with Rapid ID 32A. Arginine can de degraded to CO 2 by activity of the following enzymes: E.C. 3.5.3.6, deaminase, Prokka_01275; E.C. 2.1.3.3, carbamoyltransferase, Prokka_01150; E.C. 2.7.2.2, carbamate kinase, Prokka_00063. L-serine can be degraded to pyruvate: E.C. 4.3.1.17, Prokka_02086. Formate can be converted to 5,10-methylenetetrahydrofolate or to CO 2 (E.C. 6.3.4.3, ligase, Prokka_01739; E.C. 3.5.4.9, cyclohydrolase, Prokka_01741; E.C. 1.5.1.5, reductase; E.C. 1.2.1.2, Prokka_00906). Alkylnitronate can be metabolized to nitrite (E.C. 1.13.12.16, Prokka_00873). L-rhamnose can be degraded to form l-lactaldehyde dihydroxyacetone phosphate (E.C. 5.3.1.14, isomerase, Prokka_00371; E.C. 2.7.1.5, rhamnulokinase, Prokka_00372; E.C. 4.1.2.19, aldolase, Prokka_00370). All enzymes of the pentose phosphate pathway are present: E.C. 5.3.1.6, Prokka_01108; E.C. 5.3.1.1, Prokka_01187; E.C. 2.2.1.1, Prokka_02151; E.C. 2.2.1.2, Prokka_00123; E.C. 2.1.1, Prokka_02151.

The major cellular fatty acids are C 18:1 ω9c (14.3%), iso-C 17:1 /anteiso-C 17:1 (12.1%), anteiso-C 15:0 (12.3%), iso-C 15:0 (7.9%), C 16:0 (13.8%), C 18:0 (7.5%), iso-C 14:0 (5.1%) and C 14:0 (3.7%). The diagnostic amino acid of the peptidoglycan type is meso-diaminopimelic acid. The G+C content of genomic DNA is 65.1 mol%. The type strain is strain WCA-131-CoC-2T (=DSM 29508T = KCTC 15543T).

Description of Extibacter gen. nov

Extibacter (Ex.ti.bac'ter. L. neut. n. extum bowels or entrails of an animal; N.L. masc. n. bacter rod; N.L. masc. n. Extibacter a rod isolated from the intestine). The nearest phylogenetic neighbour is Clostridium hylemonae. POCP between the genome of C. hylemonae and Clostridium butyricum, the type species of the genus Clostridium, is only 14.3%, indicating the separate genus status of C. hylemonae. The dDDH value between both Clostridium genomes is 28.4% and between C. hylemonae and strain 40cc-B5824-ARET 23.8%. The G+C content of genomic DNA of the type strain is 47.9 mol%. Type species is Extibacter muris.

Description of Extibacter muris sp. nov

Extibacter muris (mu'ris, L. gen. n. muris of a mouse). Cells stain Gram-positive, and are strictly anaerobic rods up to 3 µm in length. It is positive for β-galactosidase and proline arylamidase (Rapid ID32A) and acidification from d-glucose, d-lactose, l-rhamnose and xylose (Api20A).

Fructose 6-phosphate can be converted to acetyl-CoA via phosphoenolpyruvate. ABC transporters for Na+, phosphate, cobalt, biotin, raffinose and melibiose are present. Trehalose can be converted via phosphoenolpyruvate to acetyl-CoA (E.C. 2.4.1.64, Prokka_03664). A β-galactosidase (E.C. 3.2.1.23, Prokka_01478), which may hydrolyse lactose, and all components of the xylose operon are present: xylose isomerase (Prokka_02910); 12 copies of xylB (kinase); the transport binding proteins XylF (Prokka_02319) and XylG (Prokka_02978); and the membrane transporter XylH (Prokka_02977). The xylFGH operon is positively regulated by XylR (Prokka_03195).

Degradation of glycogen/starch starts with a glycogenphosphorylase (E.C. 2.4.1.1; Prokka_00499, 02735), which releases α-d-glucopyranose 1-phosphate. The 1,6-glucohydrolase (E.C. 3.2.1, Prokka_02220) produces a debranched α-limit dextrin and shorter maltotetraose units first. α-D-glucopyranose 1-phosphate is then converted to d-glucopryanose 6-phosphate due to the activity of a phosphoglucomutase (E.C. 5.4.2.2, Prokka_00906). A maltodextrin phosphorylase cleaves maltodextrin to α-d-glucose 1-phosphate after several steps (E.C. 2.4.1.25, amylomaltase; Prokka_03040) and glucokinase activity (E.C. 2.7.1.2, Prokka_01911).

Several amino acids can be degraded and the enzymes involved are highly specific to their substrates: arginine decarboxylase (E.C. 4.1.1.19, Prokka_00457), l-serine ammonia lyase (E.C. 4.3.1.17, Prokka_00547), d-serine ammonia lyase (E.C. 4.3.1.18, Prokka_01317), threonine ammonia lyase (E.C. 4.3.1.19, Prokka_03487), methionine γ-lyase (E.C. 4.4.1.11, Prokka_01328).

Predominant fatty acids are C 16:0 (25.3%), C 14:0 (23.7%), iso-C 17:1 /anteiso-C 17:1 (14.0%) and iso-C 15:1 /C 13:0 OH (10.0%). Meso-diaminopimelic acid is absent. The type strain is 40ccB-5824-ARET (=DSM 28560T = KCTC 15546T).

Description of Flintibacter gen. nov

Flintibacter (Flin.ti.bac'ter. N.L. n. Flint name Flint; L. masc. n. bacter rod; N.L. masc. n. Flintibacter rod-shaped bacterium named after Professor Harry J. Flint, a microbiologist from Aberdeen, UK, who dedicated his career to the investigation of gut bacteria, especially butyrate producers and their role in health and disease). The nearest phylogenomic neighbours are Intestimonas butyriciproducens, Pseudoflavonifractor capillosus and Flavonifractor plautii. Corresponding dDDH values are 24.4, 24.7 and 24.7%, respectively. The respective differences in G+C content of DNA are 1.4, 1.1 and 3.0%. The dDDH value between P. capillosus and F. plautii is 26.6% (and the POCP is 42.7%), and the dDDH between the genomes of I. butyriciproducens and F. plautii is 23.3%. The G+C content of genomic DNA of the type strain is 58 mol%. The type species is Flintibacter butyricus.

Description of Flintibacter butyricus sp. nov

Flintibacter butyricus (bu.ty'ri.cus. N.L. masc. adj. butyricus related to butter, butyric, referring to the production of the short-chain fatty acid butyrate). It is strictly anaerobic, non-motile, non-spore forming, and catalase and oxidase negative, and stains Gram-negative. It grows at temperatures between 30 and 40 °C and in RCM broth containing up to 1% NaCl. It forms long filaments that separate into small irregular needle-like rods. It is negative for all reactions in Api 20A, and is positive for α-galactosidase in Rapid ID32A. It produces butyrate and acetate when grown in the presence of amino acids glutamine and glutamate, but there is no growth on leucine, lysine, arginine, glutamic and aspartic acid.

L-glutamate can be fermented by the hydroxyglutarate pathway to crotonyl-CoA, and further to butanoate (butyrate) and acetate. The following reactions are included in this pathway: E.C. 1.4.1.2, dehydrogenase, Prokka_00142; E.C. 2.8.3.12, transferase; Prokka_01566, 01567; E.C. 4.1.1.70, decarboxylase, Prokka_02670; E.C.1.1.1.157, 3-hydroxybutyryl-CoA dehydrogenase, Prokka_00188, 01591, 01835. Pyruvate can be converted to lactate (E.C. 1.1.1.27, Prokka_00634) or dehydrogenized (Prokka_00041) to form acetyl-CoA, which can be used to form acetate (E.C. 2.3.1.8, acetyl transferase, Prokka_00599).

Raffinose may be hydrolysed (E.C. 3.2.1.22, α-galactosidase) to form α-d-galactose and β-d-glucose. Melibiose can also be hydrolysed to α-d-galactose to form β-d-glucose 6-phosphate: E.C. 5.1.3.3, aldose epimerase, Prokka_02811; E.C. 2.7.1.6, galactokinase, Prokka_0072; E.C. 2.7.7.12, uridyltransferase, Prokka_0773; E.C. 1.3.2, UDP-glucose-4-epimerase, Prokka_01687; E.C. 5.4.2.2, phosphoglucomutase, Prokka_00685, 00705.

N-acetyl-glucosamine can be deacetylated by E.C. 3.5.1.25 (Prokka_01697) and converted to β-d-fructofuranose 6-phosphate by E.C. 3.5.99.6 (Prokka_01696). Fructofuranose 6-phosphate can also be formed from d-mannose, which is picked up by a PTS system. D-mannose 6-phosphate is converted to fructofuranose 6-phosphate via isomerase E.C. 5.3.1.8 (Prokka_01350).

A cellulase that forms cellobiose is detectable in the genome (E.C. 3.2.1.4, Prokka_02188), and cellobiose can be converted to a glucopyranose by glucosidase activity (E.C. 3.2.1.21, Prokka_02301).

Glycogen/starch can be degraded to form α-d-glucose 6-phosphate from maltotetraose or β-d-glucose 6-phosphate. The first reactions include E.C. 2.4.1.1 (Prokka_00610, 02033) and E.C. 3.2.1 (Prokka_03259), followed by E.C. 2.4.1.1 again and phosphoglucomutase activity (E.C. 5.4.2.2, Prokka_00685). Maltotriose can be converted to β-d-glucose 6-phosphate by glucosidase activity (E.C. 3.2.1.20, Prokka_03810), 4-α-glucotransferase (E.C. 2.4.1.25, Prokka _02266, 00609) and glucokinase K activity (E.C. 2.7.1.2, Prokka_01053, 01439).

Main cellular fatty acids are iso C 17:1 /anteiso C 17:1 (16.1%), iso-C 19:1 (14.8%), C 18:1 ω9c (13.5%), C 12:0 (12.2%), C 14:0 (11.4%), C 16:0 (11.2%) and C 18:0 (8.8%). The G+C content of the genomic DNA is 58 mol%. The type strain is BLS21T (=DSM 27579T = KCTC 15544T).

Description of Frisingicoccus gen. nov

Frisingicoccus (Fri.sing.i.coc'cus. L. fem. n. Frisinga the Roman name for the city of Freising; N.L. masc. n. coccus coccus; N.L. masc. n. Frisingicoccus; the type strain of the type species grows as coccobacilli and was originally isolated in the German city Freising). In the phylogenomic tree, strain PG-426-CC-1T branches before the group composed of Cuneatibacter muris DSM 29486T, Clostridium clostridioforme and Clostridium symbosium. The dDDH between both Clostridium genomes is 22.2%. dDDH of strain PG-426-CC-1T against Cun. muris is 21.9% and against C. clostridioforme and C. symbosium 23.3 and 22.5%, respectively. The respective differences in genomic G+C content are 5.4, 5.4 and 4.0%, respectively. The G+C content of genomic DNA of the type strain is 43.7 mol%. The type species is Frisingicoccus caecimuris.

Description of Frisingicoccus caecimuris sp. nov

Frisingicoccus caecimuris (cae.ci.mu'ris. L. n. caecum caecum; L. gen. n. muris of a mouse; N. L. gen. n. caecimuris from the caecum of a mouse). The cells are rods that separate into coccoid forms, and are strictly anaerobic. All Api 20A reactions are negative. It is positive for arginine and leucine arylamidase in Rapid ID 32A. It is able to utilize various amino acids by decarboxylation or non-oxidative deamination: arginine (E.C. 4.1.1.19, Prokka_01494); aspartate (E.C. 4.1.1.11, Prokka_00650); serine (E.C. 4.3.1.17, Prokka_00515); and threonine (E.C. 4.1.3.19, Prokka_02055). L-glutamate can be degraded to l-aspartate by transaminase activity (E. 2.6.1.1, Prokka-02564), which is metabolized to fumarate and NH 3 (E.C. 4.3.1.1, aspartate ammonia lyase, Prokka_00708).

A total of 50 ABC transporters can be detected in the genome of strain PG-426-CC-1, including those for phosphate, iron, branched chain amino acids, oligopeptides and d-methionine. Allose can be converted to β-d-fructofuranose 6-phosphate, which enters glycolysis (E.C. 2.7.1.55, d-allose kinase, Prokka_00524; E.C. 5.3.1.–, isomerase Prokka_00367). N-acetylglucosamine 6-phosphate may enter the cell by a PTS system and can subsequently be converted to d-glucosamine-6-phosphate by deacetylation and to β-d-fructofuranose 6-phosphate by deamination (E.C. 3.1.5.25, Prokka_00143; PTS phosphor carrier protein, Prokka_00706; PTS phosphorylation component, Prokka_00564; E.C. 3.5.99.6, Prokka_00155). β-d-fructofuranose 6-phosphate can then enter glycolysis.

The cellular fatty acids are C 18:1 ω9c (22.3%), C 16:0 (22.1%), iso-C 17:1 /anteiso-C 17:1 (10.3%) and C 18:0 (10.0%), with low amounts of C 14:0 (7.5%), iso-C 15:1 /C 13:0 3-OH (4.6%) and an unknown component (4.7%; RT 14.949). Meso-diaminopimelic acid is absent. The G+C content of genomic DNA is 43.7 mol%. The type strain is PG-426-CC-1T (=DSM 28559T = KCTC 15538T).

Description of Irregularibacter gen. nov

Irregularibacter (Ir.re.gu.la.ri.bac'ter. L. adj. irrgeularis irregular; N.L. masc. n. bacter rod; N.L. masc. n. Irregularibacter an irregular rod-shaped bacterium). Phylogenomic analysis places strain 2PG-426-CC-4.2T into a separate lineage within the Firmicutes. dDDH values in comparison to the three closest phylogenomic neighbours (Anaerofustis stercorihominis, Eubacterium limosum and Pseudoramibacter alactolyticus) are 25.5, 28.5 and 31.5%, respectively. The respective differences in G+C content of DNA are 2.4, 11.7 and 15.9%. The RSH is larger than within the neighboured group that already contains three distinct genera. Hence, we propose to place the strain into a novel genus. The G+C content of genomic DNA of the type strain is 35.8 mol%. The type species is Irregularibacter muris.

Description of Irregularibacter muris sp. nov

Irregularibacter muris (mu'ris L. gen. n. muris of a mouse). Cells are rods that can form long filaments of up to 10 µm. It is positive for indole production (Rapid ID 32A) and esculin hydrolysis (Api 20A). All other reactions are negative.

Glutamate can be degraded to n-butanoate including the following reactions: E.C. 1.4.1.2, dehydrogenase, Prokka_00372; E.C.2.8.3.12, CoA-transferase, Prokka_02420; CoA-dehydratase, Prokka_0065; E.C. 1.1.1.157, butyryl-CoA-dehydrogenase, Prokka_02002, 02766; E.C. 2.8.3.8, acetate CoA-transferase, Prokka_02840, 00642. Glycerol can be converted to glycerol-3-phosphate (E.C. 2.7.1.30, kinase, Prokka_02919, 00840) and further metabolized to form glycerone phosphate, which may enter glycolysis (E.C. 1.1.1.94, glycerol 3-phosphate dehydrogenase, Prokka_00228).

Glycogen biosynthesis from ADP-d-glucose is possible, including the following enzyme activities: E.C. 2.4.1.21, Prokka_02946; E.C. 2.4.1.18, Prokka_02943; E.C. 2.7.7.27, Prokka_02944; E.C. 5.4.2.2, phosphoglucomutase, Prokka_02192.

It is able to ferment amino acids: arginine (E.C. 4.1.1.19, Prokka_00095); threonine (E.C. 4.3.1.19, Prokka_00046); l-serine (E.C. 4.3.1.17, Prokka_00048). L-cysteine may be converted to pyruvate, NH 3 and H 2 S (E.C. 4.4.1.1, Prokka_01832). Tryptophan may also be metabolized to indole, pyruvate and ammonium (E.C. 4.1.99.1; Prokka_00116).

The cellular fatty acids profile comprises C 18:1 ω9c (23.0%), C 16:0 (19.6%), C 14:0 (18.5%) and C 18:0 (12.8%) as major components; C 12:0 (8.7%), C 16:1 w7c /iso-C 15:0 2-OH (5.3%) and C 10:0 (4.9%) also occur. Meso-diaminopimelic acid is absent. The type strain is 2PG-426-CC-4.2T (=DSM 28593T = KCTC 15548T).

Description of Longicatena gen. nov

Longicatena (Lon.gi.ca.te'na. L. adj. longus long; L. fem. n. catena chain, N.L. fem. n. Longicatena the type strain of the type species grows as long chains). The closest phylogenomic neighbour of strain PG-426-CC-2T is Clostridium innocuum and the corresponding dDDH is 21%. The dDDH between the isolate and the type species of the genus Clostridium, Clostridium butyricum, is 27.8%. The respective difference in genomic G+C content is 9.3%. Eubacterium dolichum is distantly related to strain PG-426-CC-2T, as is the type species of the genus Eubacterium, Eubacterium limosum, with which strain PG-426-CC-2T shares a dDDH of only 30.7%. The corresponding difference in genomic G+C content is 9.7%. This value supports the creation of a novel genus to accommodate strain PG-426-CC-2T. The G+C content of genomic DNA of the type strain is 37.8 mol%. The type species is Longicatena muris.

Description of Longicatena caecimuris sp. nov

Longicatena caecimuris (cae.ci.mu'ris. L. n. caecum caecum; L. gen. n. muris of a mouse; N. L. gen. n. caecimuris from the caecum of a mouse). The cells are rods, forming long filaments. All reactions in Rapid ID 32A are negative, but it is positive for esculin hydrolysis in Api 20A.

The complete pathway for formaldehyde oxidation is present, starting with d-ribulose 5-phosphate and formaldehyde to form hexulose 6-phosphate: E.C. 4.1.2.43, hexulose 6-phosphate synthase, Prokka_00565; E.C. 5.3.1.27, hexuloisomerase, Prokka_00566; E.C. 5.3.1.9, glucose 6-phosphate isomerase, Prokka_01514; E.C. 1.1.1.49, glucose 6-phosphate dehydrogenase, Prokka_02255; E.C. 1.1.31 phosphoglucanolactonase, Prokka_02253; E.C. 1.1.1.343, phosphogluconate dehydrogenase, Prokka_02254.

Pyruvate can be converted to lactate (E.C. 1.1.1.27, Prokka 01579). L-serine and l-threonine can be fermented (E.C. 43, 1.17, Prokka_02192; E.C. 4.3.1.19, Prokka_01200). Several enzymes involved in the degradation of glycogen are available: E.C. 2.4.1, phosphorylase, Prokka_01627, 02537; E.C. 2.4.1.25, transferase, Prokka_01628; E.C. 5.4.2.2, phosphoglucomutase, Prokka_01780.

The predominant fatty acid is C 18:1 ω9c (77.4%), and minor fatty acids are C 18:1 ω7c (5.7%), C 16:0 (4.6%), iso-C 19:0 (2.9%), C 18:1-2OH (2.0%) and C 18:0 (1.9%). Meso- diaminopimelic acid is absent. The type strain is PG-426-CC-2T (=DSM 29481T = KCTC 15535T).

Description of Longibaculum gen. nov

Longibaculum (Lon.gi.ba'cu.lum. L. adj. longus long; L. neut. n. baculum rod, stick; N.L. neut. n. Longibaculum a long rod, pertaining to the ability to grow as long cells, filaments). Phylogenomic analysis places strain MT10-315-CC-1.2-2T into the Erysipelotrichaceae cluster, distantly related to all other members of this cluster. The RSH of strain MT10-315-CC-1.2-2T is larger than within the neighboured group, which includes four distinct genera (Catenibacterium mitsuokai, Kandleria vitulina, Sharpea azabuensis and Eggerthia catenaformis). The nearest neighbour is ‘Stoquefichus massiliensis’, which currently has no standing in nomenclature and was therefore not included in the analysis. We thus propose that strain MT10-315-CC-1.2-2T represents a novel genus. The G+C content of genomic DNA is 30.8 mol%. The type species is Longibaculum muris.

Description of Longibaculum muris sp. nov

Longibaculum muris (mu'ris L. gen. n. muris of a mouse). It is able to form long filaments >20 µm in length, and is strictly anaerobic. It is positive for β-galactosidase, β-glucoronidase, N-acetyl glucosamine and pyroglutamic acid arylamidase (Rapid ID 32A). All reactions for acidification of carbohydrates are negative (Api 20A).

The genome contains 30 glucosidases of family GH1. Arbutin and salicin can be converted to β-d-glucose-6-phosphate (E.C. 2.7.1.69, phosphotransferase, Prokka_00070; 3.2.1.86, glucosidase, Prokka_00223). Sucrose can be degraded to form β-d-fructofuranose-6-phosphate (E.C. 3.2.1.26, hydrolase, Prokka_01036; E.C. 2.7.1.2, glucokinase, Prokka_00474; E.C. 5.3.1.9, Prokka_00116; or directly by fructokinase activity (E.C. 2.7.1.4; Prokka_00908). Rhamnose can be converted to l-lactaldehyde dihydroacetone, which may enter glycolysis (E.C. 5.3.1.14, isomerase, Prokka_00519; E.C. 2.7.1.5, kinase, Prokka_00518; E.C. 4.1.2.19, aldolase, Prokka_00520). β-d-galactose can be converted to glucose 6-phopshate, which can also enter glycolysis (E.C. 5.1.3.3, epimerase, Prokka_02823; E.C. 2.7.1.6, galactokinase, Prokka_02824; E.C. 2.7.7.12, transferase, Prokka_02822). Lactose can be used to form β-d-galactose and β-d-glucose (E.C. 3.2.1.23, Prokka_00851).

A type II secretion pathway including eps E, F, H is present. The following enzymes for degradation of glycogen are also present: E.C. 2.4.1.1, phosphorylase, Prokka_02190; E.C. 2.4.1.25, glucotransferase, Prokka_01371; E.C. 3.2.1.10, 1,6-glucosidase Prokka_02259, 03135; E.C.5.4.2.2, phosphoglucomutase, Prokka_02540.

The main fatty acids are C 16:0 (30.1%), C 18:1 ω9c (15.4%), C 18:1 ω7c (9.9%), C 16:1 ω7c /iso-C 15:0-OH (8.9%), C 18:0 (6.6%) and C 16:1 ω11c (5.4%). The diagnostic amino acid of the peptidoglycan type is meso-diaminopimelic acid. The type strain is MT10-315-CC-1.2-2T (=DSM 29487 T = KCTC 15536T).

Description of Muribaculum gen. nov

Muribaculum (Mu.ri.ba'cu.lum. L. masc. n. mus, muris mouse; L. neut. n. baculum rod, stick; N.L. neut. n. Muribaculum rod-shaped bacterium from a mouse).

Phylogenomic analysis places strain YL27T into a separate lineage nearby the genera Coprobacter and Barnesiella. The nearest phylogenetic neighbour as determined by 16S rRNA gene sequence analysis is Barnesiella intestinihomis, showing only 86.2% sequence similarity, which is below the proposed threshold of 86.5% for families75. dDDH values between strain YL27T and the genomes of Coprobacter fastidiosus, B. intestinihominis and Barnesiella viscericola are 23.8, 23.4 and 23.4%, respectively. Corresponding differences in genomic G+C content are 11.6, 6.0 and 1.7%. The G+C content of genomic DNA of the type strain is 49.9 mol%. The type species is Muribaculum intestinale.

Description of Muribaculum intestinale sp. nov

Muribaculum intestinale (in.tes.ti.na'le. N.L. neut. adj. intestinale pertaining to the intestines). It is strictly anaerobic. It is positive for α-galactosidase, β-galactosidase, N-acetyl-β-glucosamidase, alkaline phosphatase, leucyl glycine arylamidase and alanine arylamidase (Rapid ID 32A), and is negative for all reactions in Api 20A.

Galactose can be degraded to form β-d-glucose 6-phosphate (E.C. 5.1.3.3, epimerase, Prokka_01653; E.C. 2.7.1.6, kinase, Prokka_01654; E.C. 5.1.3.2, epimerase, Prokka_02048; E.C. 5.4.2.2, phosphoglucomutase, Prokka_00248).

Pectin can be depolymerized to d-galactopyranuronate (E.C. 3.1.1.11, pectin esterase, Prokka_02543; E.C. 3.2.1.82, galacturonidase, Prokka_02522; E.C. 4.2.2.2, pectic acid lyase PL10, Prokka_00090).

The main cellular fatty acid is anteiso-C 15:0 (59.9%); C 16:0-30H (8.3%), iso-C 15:0 (5.6%), C 14:0 (3.9%) and C 15:0 3-OH (3.3%) are detected in minor amounts. Meso-diaminopimelic acid is absent. The type strain is YL27T (=DSM 28989T = KCTC 15537T).

Description of Muricomes gen. nov

Muricomes (Mu.ri.co'mes, L.n. mus, muris mouse; L.n. comes a companion; N.L. masc. n., Muricomes a companion of a mouse). Phylogenomic analysis places strain 2-PG-424-CC-1T into a separate lineage with a RSH clearly larger than within the neighboured group holding species from two distinct genera: Clostridium oroticum, Eubacterium contortum and Eubacterium fissicatena. These species share dDDH values of 20.1, 20.0 and 20.1% with strain 2-PG-424-CC-1T, respectively. These data indicate that strain 2-PG-424-CC-1T represents a novel genus. C. oroticum is characterized by dDDH values of 21.0 and 21.2% with E. contortum and E. fissicatena, respectively. The two Eubacterium species show a dDDH of 27.6%. They are distantly related to the type species of the genus Eubacterium (Eubacterium limosum) and must be reclassified in future. The G+C content of genomic DNA of the type strain is 43.1 mol%. The type species is Muricomes intestini.

Description of Muricomes intestini sp. nov

Muricomes intestini (in.tes'ti.ni. L. gen. n. intestini of the gut, referring to the ecosystem of origin of the type strain).

Its cells are Gram-positive rods able to grow under anaerobic conditions, and it is positive for α-glucosidase and proline arylamidase (Rapid ID32A). It is able to utilize l-arabinose, d-glucose, d-mannitol, d-maltose, d-mannose, l-rhamnose, d-sorbitol and d-xylose (Api 20A).

Carbohydrate binding modules (CBM) with carbohydrate-binding activities are detectable within the genome for xylan/mannan (CBM 35 family; Prokka_00901) and chitin (CBM 50 family, Prokka_01736). Chitin can be degraded to N-acetylglucosamine by a diacetylchitobiase (E.C. 2.4.1.280, Prokka_01771). Subsequently, N-acetyl-glucosamine 6-phosphate is formed by the activity of a phosphotransferase system and can be transformed to d-glucosamine 6-phosphate due to deacetylase activity (E.C. 3.5.1.25, Prokka_02773). Deamination (E.C. 3.5.99.6, Prokka_01084) leads to formation of d-fructofuranose 6-phosphate. Cellulose can be degraded (E.C. 3.2.1.4, Prokka_00599) to form more soluble components. An arabinofuranidase (E.C. 3.2.1.55, Prokka_02409) can extract arabinose and xylan to produce xylodextrin and subsequently xylotriose and xylobiose (E.C. 3.2.1.54, dextrinase, Prokka_02684).

All enzymes for degradation of glycogen to α-glucose 6-phosphate are detectable within the genome: glycogenphosphorylase (E.C. 2.4.1.1, Prokka_01745); α-1,6 glucohydrolase (E.C. 3.2.1.111, Prokka_00827); maltodextrinphosphorylase (E.C. 2.4.1.1, Prokka_02991); and a phosphoglucomutase (E.C. 5.4.2.2, Prokka_01606). A second pathway for glycogen degradation to d-glycopyranose 6-phosphate is also present: the first two steps are as above, but maltotetraose is then converted to maltotriose (E.C. 2.4.1.1, phosphorylase; Prokka_02991), which is degraded to d-glycopyranose 6-phosphate (E.C. 2.1.4.25, amylomaltase, Prokka_02987; E.C. 2.7.1.1, glucokinase, Prokka_0362).

β-d-glucose 6-phosphate and β-d-fructofuranose 6-phosphate can be converted to phosphoenolpyruvate and pyruvate via glycolysis. Phosphoenolpyruvate and pyruvate can be utilized within a mixed acid fermentation with acetate, CO 2 and H 2 being the end products: pyruvate kinase (E.C. 2.7.1.40, Prokka_03100); pyruvate formate-lyase (E.C. 2.3.1.54, Prokka_00010); formate hydrogenlyase E.C. 1.2.1.2, Prokka_03349); phosphate acetyltransferase (E.C. 2.3.1.8, Prokka_00287); acetate kinase (E.C. 2.7.2.1, Prokka_00288).

The fatty acid profile comprises C 16:0 (40.7%), C 14:0 (15.7%), iso-C 15:1 /C 13:0 3-OH (12.2%), C 19:0 10-methyl (7.6%), iso-C 17:1 (5.2%) and C 18:1 ω7c (5.2%). The diagnostic amino acid of the peptidoglycan type is meso-diaminopimelic acid. The type strain is 2-PG-424-CC-1T (=DSM 29489T = KCTC 15545T).

Description of Pasteurella caecimuris sp. nov

Pasteurella caecimuris (cae.ci.mu'ris. L. n. caecum caecum; L. gen. n. muris of a mouse; N. L. gen. n. caecimuris from the caecum of a mouse). The nearest phylogenomic neighbour is Pasteurella pneumotropica. dDDH between the genome of strain AA-424-CC-1T and P. pneumotropica is 30.8%, confirming the separate species status of strain AA-424-CC-1T. It is Gram-negative. It is positive for resorufin-β-d-galactopyranoside, β-galactosidase, α-galactosidase, alkaline phosphatase, trehalose, maltose, urease, potassium nitrate, glucose and cytochrome oxidase (Rapid ID32 Strep and Api 20NE). It has weak reactions for d-ribose, d-lactose, d-raffinose, sodium pyruvate, melibiose and sucrose.

N-acetyl d-glucosamine 6-phosphate can be converted to β-d-fructofuranose 6-phosphate (E.C. 3.5.1.25, deacetylase, Prokka_02486; E.C. 3.5.99.6, deaminase, Prokka_02485). Glycogen debranching enzymes are present: E.C. 3.2.1.33, α-glucosidase, Prokka_02499; E.C. 2.4.1.1, glycogen phosphorylase, Prokka_02298; E.C. 5.4.2.8, mannomutase, Prokka_02405; E.C. 2.4.1.25, glucanotransferase, Prokka_02299; E.C. 2.4.1.60, kinase, Prokka_02482. Sucrose can be converted to β-d-fructofuranose, including the following reactions: E.C. 2.7.7.9, transferase, Prokka_00902; E.C. 2.7.1.4, fructokinase, Prokka_01004; E.C. 5.4.2.8, mannomutase, Prokka_02405; E.C. 5.3.1.9, isomerase, Prokka_01476. Xylose can be converted to d-xylulose 5-phosphate, which may enter the pentose phosphate pathway: E.C. 5.3.1.5, xylulose isomerase, Prokka_01918; E.C. 2.7.1.17, xylulose kinase, Prokka_01919.

Enzymes for a mixed acid fermentation are detectable. Phosphoenolpyruvate can be metabolized to form succinate, but also to form lactate from pyruvate. Pyruvate can also be converted to formate and CO 2 or to acetate via acetyl-CoA: E.C. 2.7.1.40, kinase, Prokka_01479; E.C. 1.1.1.28, dehydrogenase, Prokka_00386; E.C. 2.3.1.54, transferase, Prokka_02383; E.C. 2.3.1.8 phosphotransferase, Prokka_02157; E.C. 2.7.2.1, acetate kinase, Prokka_02158; E.C. 4.1.1.31, PEP-carboxylase, Prokka_01077; E.C. 1.1.1.37, dehydrogenase, Prokka_01374; E.C. 4.2.1.2, fumarate dehydrogenase, Prokka_02131; E.C. 1.3.5.4, fumarate reductase, Prokka_01183.

The fatty acid profile comprises C 16:0 (31.0%), C 14:0 (29.4%), C 16:1 ω7c /iso-C 15:0 2-OH (24.4%) and iso-C 16:1 /C 14:0 3-OH (8.2%). The G+C content of genomic DNA is 40.2 mol%. The type strain is AA-424-CC-1T (=DSM 28627T = KCTC 52216T).

Description of Turicimonas gen. nov

Turicimonas (Tu.ri.ci.mo'nas. L. neut. n Turicum the Roman name for the city of Zurich; Gr. fem. n. monas a unit, a monad; N.L. fem. n. Turicimonas a unit of Turicum/Zurich; the type strain of the type species was isolated in the Swiss city Zurich). The closest phylogenomic neighbour is Parasutterella excrementihominis. The dDDH value and 16S rRNA gene sequence identity between the two bacteria are 21.4 and 93.9%, respectively. The amino acid identity (AAI) value between the two bacteria is 66.1% and the difference in genomic G+C content is 4.1%. The G+C content of genomic DNA of the type strain is 44.0 mol%. The type species is Turicimonas muris.

Description of Turicimonas muris sp. nov

Turicimonas muris (mu’.ris. L. gen. n. muris of a mouse). It grows as single, straight, rod-shaped cells, 1–2 µm in length. All reactions are negative in Api 20A and Rapid ID 32A. It is able to degrade l-asparagine (E.C.5.1.1, Prokka_02303) and l-serine (E.C. 4.3.1.17, Prokka_01696). The type strain is YL45T (= DSM 26109T = KCTC 15542T).

Accession codes

The genome and metagenome sequences obtained in the present study are available at the European Nucleotide Archive under project accession no. PRJEB10572 and at the Sequence Read Archives under accession nos. SRX1092347, SRX109234, SRX1092352 to 55 and SRX1092357 to 62. These numbers and the GenBank IDs of 16S rRNA genes of all miBC isolates are listed in Supplementary Table 1.