Genome sequence of Clostridium sporogenes DSM 795(T), an amino acid-degrading, nontoxic surrogate of neurotoxin-producing Clostridium botulinum

Abstract

Clostridium sporogenes DSM 795 is the type strain of the species Clostridium sporogenes, first described by Metchnikoff in 1908. It is a Gram-positive, rod-shaped, anaerobic bacterium isolated from human faeces and belongs to the proteolytic branch of clostridia. C. sporogenes attracts special interest because of its potential use in a bacterial therapy for certain cancer types. Genome sequencing and annotation revealed several gene clusters coding for proteins involved in anaerobic degradation of amino acids, such as glycine and betaine via Stickland reaction. Genome comparison showed that C. sporogenes is closely related to C. botulinum. The genome of C. sporogenes DSM 795 consists of a circular chromosome of 4.1 Mb with an overall GC content of 27.81 mol% harboring 3,744 protein-coding genes, and 80 RNAs.

Keywords: Anaerobic; Butanol; C. botulinum; C. sporogenes; Gram-positive; Stickland reaction.

Figure 1

Gram staining of Gram-positive C. sporogenes DSM 795. Scale bar represents 5 μm.

Figure 2

Scanning electron microscopy image of C. sporogenes DSM 795.

Figure 3

Transmission electron microscopy image of C. sporogenes DSM 795; A: dividing cell; B, C: sporulating cells; D: spore; scale bars represent 1 μm.

Figure 4

Phylogenetic tree of C. sporogenes DSM 795 based on 16S rDNA gene sequences. Estimation is based on Bayesian inference and MAFFT alignment.

Figure 5

Tblastx comparison of glycine-, betaine-reductase and the selABC gene cluster of C. sporogenes DSM 795 with C. botulinum ATCC 3502, E. acidaminophilum al-2 DSM 3953 and S. ovata H1 DSM 2662: An E-value cutoff of 1e-10 was used and visualization was done with the program Easyfig [75]. The analyzed gene clusters are localized in different regions in the genomes of C. sporogenes DSM 795, C. botulinum ATCC 3502 as well as E. acidaminophilum al-2 DSM 3953. In S. ovata one genomic region includes all three gene clusters. Borders between the glycine-reductase (CSPO_4c08160-CSPO_4c08230), betaine-reductase (CSPO_4c10340-CSPO_4c10360) and selABCtrnU (CSPO_9c04980-CSPO_9c05010) gene cluster are indicated with red vertical lines.

Figure 6

Genome comparison of C. sporogenes with different C. sporogenes and C. botulinum strains: A: Genes encoded by the leading and the lagging strand (circle 1 and 2) of C. sporogenes DSM 795 are marked in COG colors in the artificial chromosome map. The presence of orthologs (circle 3 to 10) is indicated for the genomes of C. sporogenes PA3679 (AGAH00000000), C. sporogenes ATCC 15579 (ABKW00000000) , C. botulinum ATCC 3502 (CP000727.1), C. botulinum B1 str. Okra (CP000939.1, CP000940.1), C. botulinum F str. Langeland (CP000728.1, CP000729.1), C. botulinum E3 str. Alaska E43 (CP001078.1), C. botulinum D str. 1873 (ACSJ01000001), C. botulinum C str. Eklund (ABDQ01000001) are illustrated in red to light yellow and singletons in grey (grey: >e⁻¹⁰-1; light yellow: <e⁻⁵⁰- > e⁻¹⁰; gold: <e⁻⁵⁰- > e⁻⁹⁰; light orange: <e⁻⁹⁰- > e⁻¹⁰⁰; orange: <e⁻¹⁰⁰- > e⁻¹²⁰; red: <e⁻¹²⁰-0). The two innermost plots represent the GC-content and the GC-skew. The artificial chromosome was built after scaffolding with Mauve alignment tool and concatenating the 16 contigs of the draft genome. Venn diagrams showing orthologs genes between the three sequenced C. sporogenes species (B) and between C. sporogenes DSM 795, the phylogenetic closely related C. botulinum ATCC 3502 and distantly related C. botulinum E3 str. Alaska E43 (C). Ortholog detection was done with the Proteinortho software (blastp) with an identity cutoff of 50% and an E-value of 1e-10. The total number of genes and paralogs, respectively, were depicted under the corresponding species name.

Figure 7

Tblastx comparison of the botulinum neurotoxin cluster (BoNT/A) and the flanking regions between C. botulinum and C. sporogenes strains. For the tblastx comparison an E-value cutoff of 1e-10 was set. Visualization was done with Easyfig. The neurotoxin cluster was marked in red tones, haemagglutinin components of the neurotoxin complex in blue tones, transposases in bright yellow, genes identified only in the C. botulinum strains in light grey, the CRISPR/cas system of C. sporogenes ATCC 15579 in olive tones, and singletons identified for the latter strain in grey. Core genes are anthracite-colored.