- Extended genome report
- Open Access
Genome sequence of Clostridium sporogenes DSM 795T, an amino acid-degrading, nontoxic surrogate of neurotoxin-producing Clostridium botulinum
Standards in Genomic Sciences volume 10, Article number: 40 (2015)
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.
C. sporogenes was isolated from human faeces [1-3], but can also be found in soil and marine or fresh water sediments [4-7]. C. sporogenes strain DSM 795  serves as type strain for this species and as a consequence was chosen for whole genome sequencing.
Because C. sporogenes is closely related to C. botulinum group I strains, it is used as a non-toxic surrogate for this common food-borne pathogen. 16S rDNA sequencing revealed a 99.7% sequence similarity to proteolytic C. botulinum strains of serotypes A, B, and F . In this context, the genome of C. sporogenes strain PA 3679 was sequenced and a draft sequence published in 2012 . C. sporogenes can be isolated from infections, but does not play a prominent role as a pathogen. Only few clinical cases are reported, in which this species was found to participate. These cases include epynema, soft tissue abscesses, septic arthritis, or gas gangrene [11-16].
Requiring an anaerobic habitat, C. sporogenes is known to specifically colonize hypoxic areas of solid tumors. In 1964, Möse and co-workers demonstrated tumor colonization resulting in tumor lysis after intravenous application of C. butyricum M-55 in mice carrying Ehrlich carcinomas . The respective strain was subsequently reclassified as C. oncolyticum and finally as C. sporogenes ATCC 13732. They also demonstrated that C. sporogenes spores are immunologically inert by injecting them into themselves . As an excellent tumor colonizer, C. sporogenes bears promising therapeutic potential for cancer therapy . With CFU numbers up to 2 × 108 per gram of tumor tissue, C. sporogenes outperforms saccharolytic clostridia such as C. beijerinckii and C. acetobutylicum by far, as the latter reach only CFU numbers of 105-106 per gram of tumor tissue [20-22].
Restricted to the inner core of the tumor, clostridia cannot lyse the well-oxygenated outer rim of tumor cells, which remains viable and unaffected. Therefore, treatment with clostridial spores alone is not sufficient for complete tumor eradication. Several attempts have been made to genetically modify C. sporogenes for production of therapeutic proteins or pro-drug converting enzymes. The latter catalyze the conversion of innocuous pro-drug molecules into their active, cytotoxic form. This reaction takes place directly in the tumor allowing systemic application of higher drug concentrations and reduction of side effects . Among others, cytosine deaminase and nitroreductase were used for this purpose. A recombinant C. sporogenes DSM 795 mutant expressing cytosine deaminase induced growth delay of tumors in a mouse model after application of spores and the pro-drug 5-fluorouracil . Also, C. sporogenes mutants heterologously expressing nitroreductase exhibited a significant antitumor efficacy in different in vivo tumor models [23-25].
Classification and features
C. sporogenes belongs to the proteolytic branch of clostridia capable of amino acid fermentation. No carbohydrates are required for growth, although addition can have a stimulating effect . Amino acids are degraded via the Stickland reaction for energy conservation [40-43]. Required media composition and further nutritional demands have already been elucidated [48-55]. Growth can be obtained anaerobically in complex medium, but also several minimal media supplemented with amino acids are described [52,53].
A scanning electron microscopy image of C. sporogenes DSM 795 cell culture is shown in Figure 2. Several cell stages are depicted: vegetative dividing and sporulating cells and a mature spore (upper left part of the image).
C. sporogenes is considered as the non-toxic surrogate of neurotoxin producer Clostridium botulinum . Additional file 1: Table S1 provides an overview of all C. botulinum strains mentioned in this study. Generally, they are assigned to four groups (I-IV) based on their physiologic characteristics . Strains belonging to group I are proteolytic . They are further classified into serotypes A-F due to different types of the produced botulinum neurotoxin with several subtypes existing .
Phylogenetic relation of C. sporogenes DSM 795 to C. botulinum strains and other clostridia was investigated by calculation of a phylogenetic tree using 16S rDNA sequences (Figure 4). C. sporogenes DSM 795 positions itself in close relationship to proteolytic C. botulinum strains of types A, B, and F, confirming previous studies . Clostridial 16S rRNA reference sequences were retrieved from GenBank (NCBI database). At first, these sequences were aligned with MAFFT version 7 using default settings except for “globalpair” in fast Fourier transform . Then, based on the multiple sequence alignment, a phylogenetic tree was inferred with the program MrBayes 3.1.2  using the default settings.
C. sporogenes DSM 795 exhibits β-hemolysis on sheep and human erythrocytes (data not shown) due to production of clostridiolysin S . Further enzymes produced are desoxyribonuclease, thiaminase, chitinase, kininase, L-methioninase, hyaluronate lyase, and superoxide dismutase .
In general, C. sporogenes is resistant to streptomycin, neomycin, kanamycin, tobramycin, and amikamycin and susceptible to penicillin G, metronidazole, tinidazole, chloramphenicol, tetracycline, and doxycycline . C. sporogenes strain DSM 795 is additionally susceptible to thiamphenicol and erythromycin or clarithromycin in working concentrations of 15 μg/ml and 2.5 μg/ml, respectively.
Genome sequencing information
Genome project history
C. sporogenes DSM 795 was chosen for whole genome sequencing as it is the type strain of this species. Furthermore, it attracts special interest because of its potential use in tumor therapy and it is known as nontoxic surrogate of the food-borne and neurotoxin-producing pathogen C. botulinum . The sequencing of C. sporogenes DSM 795 genomic DNA delivered a high-quality draft genome sequence comprising 1 scaffold and 16 contigs. The sequence is deposited in GenBank database under the accession JFBQ00000000. A summary of the project information is listed in Table 2.
Growth conditions and genomic DNA preparation
For genomic DNA preparation via the hot phenol method, an overnight culture incubated at 37°C was used. The procedure was carried out as described previously . Redistilled and in TE buffer equilibrated phenol (pH 7.5-8) was used for the extraction.
Electron microscopic images were taken from an overnight and a sporulating culture (10 d, 30 °C) of C. sporogenes . SEM and TEM cell samples were washed 3 times with PBS and fixed with 1 vol 5% (v/v) glutaraldehyde in 0.2 M phosphate buffer pH 7.3 containing 2% (w/v) sucrose. Further treatment and visualization were conducted by the Central Facility for Electron Microscopy, University of Ulm.
Genome sequencing and assembly
Whole-genome sequencing of C. sporogenes was performed with a combined approach using the 454 GS-FLX Titanium XL system (Titanium GS70 chemistry, Roche Life Science, Mannheim, Germany), the Genome Analyzer II, and the MiSeq (Illumina, San Diego, CA). Shotgun libraries were prepared according to the manufacturer’s protocols, resulting in 126,343 reads for 454 shotgun sequencing (11.53 × coverage) and 1,445,024 112-bp and 5,654,920 150-bp paired-end Illumina reads (263.72 × coverage). For the initial hybrid de novo assembly with MIRA 3.4  and Newbler 2.8 (Roche Life Science, Mannheim, Germany), we used all of the 454 shotgun reads, 1,445,024 112-bp and 554,976 150-bp paired-end Illumina reads. The final assembly was composed of 298 contigs with an average coverage of 62.78. For scaffolding we used the Move Contigs tool of the Mauve Genome Alignment Software . Additionally, contigs that could not be ordered with Mauve were examined via Gene Ortholog Neighborhoods based on bidirectional best hits implemented at the IMG-ER (Integrated Microbial Genomes-Expert Review) system [65,66]. For contig ordering tasks, the genomes of C. sporogenes ATCC 15579 (ABKW00000000), C. botulinum ATCC 3502 (AM412317, AM412318), and C. botulinum BoNT/B1 Okra (CP000939) were used as references. Sequence gaps were closed in the Gap4 (v.4.11) software of the Staden Package  by PCR-based techniques and primer walking with conventional Sanger sequencing, using BigDye 3.0 chemistry on an ABI3730XL capillary sequencer (Applied Biosystems, Life Technologies GmbH, Darmstadt, Germany). The resulting draft genome is composed of 16 contigs in 1 scaffold.
The software tools YACOP and Glimmer  were used for automatic gene prediction, while identification of rRNA and tRNA genes was performed with RNAmmer and tRNAscan, respectively [69,70]. Automatic annotation was carried out with the IMG-ER (Integrated Microbial Genomes-Expert Review) system [65,66], but annotation was afterwards manually curated by employing BLASTP and the Swiss-Prot, TrEMBL, and InterPro databases .
The draft genome of C. sporogenes DSM 795 consists of one scaffold containing 16 contigs representing one circular chromosome with a size of 4.1 Mb and with an overall GC content of 27.81 mol%. 3,832 genes are encoded, from which 3,744 were putative protein coding, 8 were pseudo and 80 RNAs (10 rRNA and 70 tRNA genes). 77.51% of encoding genes could be assigned to a putative function while the remaining 843 genes were annotated as hypothetical proteins. The genome harbors 6 different selenocysteine-containing proteins, even SelD, the selenide water dikinase (CSPO_9c05010), necessary for incorporation of selenocysteine into proteins, contains one selenocysteine. 4 of the remaining gaps represent rRNA gene clusters and there are some indications in the draft genome that at least 4 of these clusters are double, triple, or even fivefold clusters. Statistics and genome properties are listed in Table 3.
Insights from the genome sequence
Of all protein coding genes 2,456 (64.09%) could be assigned to at least one COG category ; Table 4 shows the distribution into these functional groups. The two most abundant categories were “general function prediction” and “transcription”, to which 11.75% and 10.55%, respectively, could be assigned to, followed by “amino acid transport and metabolism”, “function unknown”, “signal transduction and mechanisms”, and “energy production and conversion” with 9.42%, 8.51%, 7.05% and 6.07%, respectively, of all protein coding genes.
9.42% of all protein coding genes were assigned to the COG category “amino acid transport and metabolism”, which indicates that utilization of amino acids plays an important role in the metabolism of C. sporogenes . Supporting this assumption, we identified several clusters coding for proteins involved in anaerobic amino acid degradation by the Stickland reaction [40-43]. One example is a gene cluster coding for several subunits of a proline reductase (CSPO_9c00030-CSPO_9c00180). This cluster contains two selenocysteine-containing proteins, the gamma subunit PrdB (CSPO_9c00070), and PrdC, a protein with high sequence homology to the C-subunit of Rnf-complex (CSPO_9c00100). The cluster shows a similar organization as in C. sticklandii [73,74], except for the additional presence of a transposase (CSPO_9c00150), a second copy of PrdH2 (CSPO_9c00160) and a non-selenocysteine-containing version of PrdC (CSPO_9c00170). Furthermore, we identified gene clusters for the degradation of glycine, another one for its derivative betaine (N,N,N-trimethylglycine), and a cluster involved in selenocysteine incorporation into proteins (Figure 5).
In contrast to Sporomusa ovata , in which all above mentioned gene are organized in one large cluster, the clusters identified in the genomes of C. sporogenes , C. botulinum ATCC 3502 and Eubacterium acidaminophilum al-2 DSM 3953 are localized in separate regions of the genomes. This is also true for the selenocysteine-incorporation genes (selABC [lilac tones]) as well as the Sec-specific tRNA (trnU [dark lilac]) as shown in Figure 5. The gene clusters of C. sporogenes show identical organization to those identified in C. botulinum and show only slightly differences to those found in E. acidaminophilum , whereas genes coding for the betaine-specific reductase is missing in the genome of C. botulinum . The glycine reductase cluster of C. sporogenes lacks the second copy of grdA [orange] in comparison to E. acidaminophilum , but contains two paralogs for thioredoxin reductases of the thioredoxin system [blue tones]. These two paralogs could also be identified in the cluster found in C. botulinum . In C. sporogenes the gene cluster coding for the betaine reductase (grdRIH [brown tones]) is much shorter than E. acidaminophilum ’s cluster, as genes coding for thioredoxin (trxA [light blue]) and thioredoxin reductase (trxB [dark blue]) as well as the two genes coding for the C-subunit of the reductase (grdCD [green tones]) are not present. The 32.5 kb comprising cluster of S. ovata includes genes coding for the glycine-specific subunit (grdEB [red tones]), genes coding for the betaine-specific subunit (grdIH [brown tones]), two copies of genes coding for the substrate-unspecific subunit C (grdCD [green tones]), and two copies coding for the thioredoxin and a thioredoxin reductase (trxAB [blue tones]). All these genes show identical clustering as identified in the genome of C. sporogenes . The genes coding for proteins necessary for the selenocysteine-incorporation show a different arrangement as identified in C. sporogenes , C. botulinum and E. acidaminophilum , where these genes are organized in a selABCtrnU operon [lilac tones] . It is also visible in Figure 5 that genes coding for glycine-specific subunit (grdBE [red tones]) show high sequence homology to genes coding for betaine-specific subunit (grdIH brown tones]).
C. sporogenes is able to produce solvents such as ethanol and butanol [79,80]. The genome of C. sporogenes DSM 795 harbors the complete set of genes necessary for glycolysis (phosphoglucomutase, glucose-6-phophate isomerase, 6-phopsphofructokinase, 1-phosphofructokinase, fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, aldehyde:ferredoxin oxidoreductase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate dehydrogenase) as well as aldehyde dehydrogenase and several bifunctional aldehyde-alcohol dehydrogenases, essential for ethanol production. Genes coding for key enzymes of butanol fermentation, such as butyryl-CoA dehydrogenase, acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, several alcohol dehydrogenases, acetate kinase, phosphate acetyltransferase, two copies of butyrate kinase, two copies of phosphate butyryltransferase as well as three copies of formate acetyltransferase are also present (Additional file 2: Table S2). In contrast to other solventogenic clostridia, such as C. beijerinckii , C. saccharobutylicum , C. saccharoperbutylacetonicum , or C. acetobutylicum , C. sporogenes is not able to produce acetone. In solventogenic clostridia, CoA transferase and acetoacetate decarboxylase, key enzymes of acetone production, are organized in the sol operon [81-86]. In C. beijerinckii , C. saccharobutylicum , and C. saccharoperbutylacetonicum aldehyde dehydrogenase is also part of this operon, whereas in C. acetobutylicum this enzyme is replaced by alcohol/aldehyde dehydrogenase. We could not identify CoA transferase and acetoacetate decarboxylase in the genome of C. sporogenes DSM 795 and both, alcohol/aldehyde dehydrogenase and aldehyde dehydrogenase are present, but located in different regions of the genome.
C. sporogenes is renowned as a nontoxic surrogate for the proteolytic C. botulinum , an organism which produces the botulinum neurotoxin (BoNT). C. botulinum is classified into seven serotypes (A to G) according to the neurotoxin antigenic specificity [57,87]. Serotypes A, B, E, and F cause human botulism, C and D are mainly described in animal toxicity, and no botulism case has been reported for serotype G [88,89]. For genome comparisons, two C. sporogenes species (ATCC 15579 and PA3679) and available representatives of all serotypes of C. botulinum , except for serotype G, were chosen and retrieved from NCBI (Figure 6A). For this purpose and to prepare data for comparisons we used the scripts ncbi_ftp_download v0.2, cat_seq v0.1 and cds_extractor v0.6 . Proteinortho v5.04  was utilized to identify orthologs between the different organisms with an identity cutoff of 50% and an E-value of 1e-10. The core genome of all three C. sporogenes species consists of 2,920 CDS with a total pan genome of 4,754 CDS. C. sporogenes DSM 795 has 3,258 orthologous genes with C. sporogenes PA379 and 2,981 with C. sporogenes ATCC 15579 (Figure 6B). C. sporogenes DSM 795 has the least number of genome specific proteins of the three C. sporogenes strains with 270 singletons, as C. sporogenes ATCC 15579 contains 577 singletons and C. sporogenes PA3679 557. We identified 163, 178, and 189 paralogs in C. sporogenes DSM 795, ATCC 15579, and PA3679, respectively; but these genes were not included into analysis.
Phylogenetic analysis based on 16S rDNA revealed that C. sporogenes is closely related to serotypes A, B, and F C. botulinum strains, whereas it is distantly related to serotypes C, D, E, and G. These results were confirmed by gene content analyses as we identified 2,739 orthologous proteins between C. sporogenes DSM 795 and C. botulinum ATCC 3502 (serotype A) (Figure 6C). In contrast, there are only 1,094 orthologous genes between C. sporogenes and C. botulinum E3 str. Alaska E43 (serotype E). This number is nearly identical to the quantity of orthologs (1,093) found between C. botulinum ATCC 3502 and C. botulinum E3 str. Alaska E43. We identified 163, 148, and 125 paralogs in C. sporogenes DSM 795, C. botulinum ATCC 3502, and C. botulinum E3 str. Alaska E43, respectively; but these genes were not included into analysis.
A genome comparison between C. sporogenes DSM 795 and C. botulinum ATCC 3502 revealed that both organisms have 2,739 orthologs in common, with 818 singletons in C. sporogenes DSM 795 and 672 singletons in C. botulinum ATCC 3502. The most important difference between both strains is the presence of the botulinum neurotoxin (BoNT/A) gene cluster in C. botulinum ATCC 3502 and its absence in C. sporogenes DSM 795 (Figure 7).
The region of the neurotoxin gene cluster is flanked by genes coding for several hypothetical proteins, components of different ABC transporters, as well as a ferrous iron transport system and several regulatory proteins (data not shown). As shown in Figure 7, these flanking genes are present in the C. botulinum strains as well as in all C. sporogenes strains. This region might be an area of high genome plasticity, as in C. sporogenes ATCC 15579 a subtype I-B/TNEAP CRISPR/cas system  is inserted, which could not be identified in the other strains used for this comparative approach.
Members of the non-toxic species C. sporogenes are closely related to neurotoxin producer C. botulinum . This study presents an overview of physiological, morphological, and genomic characteristics of the type strain C. sporogenes DSM 795. Detailed insight into its proteolytic metabolism was gained on genomic level. Also, the ability of C. sporogenes to produce solvents such as ethanol and butanol was linked to a set of genes and compared to other solventogenic clostridia. Genome comparison of C. sporogenes DSM 795 with two other sequenced strains of this species revealed high similarity. C. sporogenes DSM 795 was also compared at the genomic level with two strains of the close relative C. botulinum .
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This work was supported by a personal grant (LGFG) to K.R. by the state of Baden-Württemberg, Germany and carried out in frame of the Cooperative Research Training Group “Pharmaceutical Biotechnology” (Kooperatives Promotionskolleg Pharmazeutische Biotechnologie) of the University of Ulm and the Biberach University of Applied Sciences.
We thank the “Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV)” for support (project REACTIF within program ERA-IB 3). We would like to thank Paul Walther (Central Facility for Electron Microscopy, University of Ulm) for generating the electron microscopic images. We thank Frauke-Dorothee Meyer and Kathleen Gollnow for technical support.
The authors declare that they have no competing interests.
AP and RD planned the genome sequencing, AP did the genome sequencing, AP did the genome annotations, AP and KR prepared the figures, KR generated all microscopic images and the phylogenetic tree. SMK carried out the genomic DNA preparation. AP, KR and AL wrote the manuscript. PD conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Anja Poehlein and Karin Riegel contributed equally to this work.