Taxonogenomic description of four new Clostridium species isolated from human gut: 'Clostridium amazonitimonense', 'Clostridium merdae', 'Clostridium massilidielmoense' and 'Clostridium nigeriense'

Abstract

Culturomics investigates microbial diversity of the human microbiome by combining diversified culture conditions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene identification. The present study allowed identification of four putative new Clostridium sensu stricto species: 'Clostridium amazonitimonense' strain LF2^T, 'Clostridium massilidielmoense' strain MT26^T, 'Clostridium nigeriense' strain Marseille-P2414^T and 'Clostridium merdae' strain Marseille-P2953^T, which we describe using the concept of taxonogenomics. We describe the main characteristics of each bacterium and present their complete genome sequence and annotation.

Keywords: culturomics; emerging bacteria; human microbiota; taxonogenomics; ‘Clostridium amazonitimonense’; ‘Clostridium massilidielmoense’; ‘Clostridium merdae’; ‘Clostridium nigeriense’.

Fig. 1

Phylogenetic tree highlighting position of ‘Clostridium amazonitimonense,’ ‘Clostridium merdae,’ ‘Clostridium massilidielmoense’ and ‘Clostridium nigeriense’ relative to other type strains within Clostridium genus. Strains and their corresponding GenBank accession numbers for 16S rRNA genes are indicated in brackets. Sequences were aligned using Clustal W (http://www.clustal.org/clustal2/), and phylogenetic inferences were obtained using maximum-likelihood method within MEGA6 software (http://www.megasoftware.net/mega.php). Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 1000 times to generate majority consensus tree. Butyricicoccus pullicaecorum (NR_044490) was used as outgroup. Scale bar = 0.02% nucleotide sequence divergence.

Fig. 2

Reference mass spectra from ‘Clostridium amazonitimonense,’ ‘Clostridium merdae,’ ‘Clostridium massilidielmoense’ and ‘Clostridium nigeriense.’ Spectra from 12 individual colonies were compared and each reference spectrum generated (A). Gel view comparing ‘Clostridium amazonitimonense,’ ‘Clostridium merdae,’ ‘Clostridium massilidielmoense’ and ‘Clostridium nigeriense’ to other species within genus Clostridium. Gel view displays raw spectra of loaded spectrum files arranged in pseudo–gellike look. x-axis records m/z value. Left y-axis displays running spectrum number originating from subsequent spectra loading. Peak intensity expressed by greyscale scheme code. Colour bar and right y-axis indicate relation between colour peaks, with peak intensity in arbitrary units. Displayed species are indicated at left (B).

Fig. 3

Electron micrographs of ‘Clostridium nigeriense’ (A), ‘Clostridium amazonitimonense’ (B), ‘Clostridium massilidielmoense’ (C) and ‘Clostridium merdae’ (D).

Fig. 4

Graphical circular map of genome of ‘Clostridium nigeriense’ (A), ‘Clostridium amazonitimonense’ (B), ‘Clostridium massilidielmoense’ (C) and ‘Clostridium merdae’ (D). From outside to center: Contigs (red/grey), COGs category of genes on forward strand (three circles), genes on forward strand (blue circle), genes on reverse strand (red circle), COGs category on reverse strand (three circles), G+C content. COGs, Clusters of Orthologous Groups database.