Draft genome sequences of a strain of Clostridium neuense and four Candidatus Clostridium species

TL;DR

This paper presents draft genome sequences of five Clostridium strains, including four proposed new species, to better understand their potential uses in industry.

Contribution

The study provides new draft genome sequences for four proposed Candidatus Clostridium species and one Clostridium neuense strain.

Findings

Draft genomes of five Clostridium isolates were sequenced.

Four isolates are proposed as new Candidatus species.

The genomes may help explore the industrial potential of these bacteria.

Abstract

We report the draft genomes of five Clostridium isolates from soil and agricultural by-products, four of which are proposed as Candidatus species. Members of the genus Clostridium are of significant industrial interest, and the availability of their genome sequences facilitates the understanding and exploration of their functional potential.

Figures

Phylogenomic tree showing the relationship between five Clostridium strains (in bold) and closely related taxa of the genus Clostridium. The maximum-likelihood tree was reconstructed as described previously (8, 9), based on the protein sequences of 361 single-copy core genes annotated using Prokka version 1.14.6 (default parameters) (10), identified and aligned using SCARAP version 0.4.0 (parameters: -p 100 -f 100) (11), and trimmed using trimAl version 1.4 (parameter: -automated1) (12). The tree was inferred with IQ-TREE version 2.1.2 (13) using the LG + F + I + G4 protein substitution model, rooted by midpoint-rooting, and visualized using Interactive Tree Of Life version 6.9.1 (14). Lachnospira multipara ATCC 19207T from the neighboring family Lachnospiraceae was used as an outgroup. Ultrafast bootstrap values based on 1,000 replications are indicated at branching points. The scale bar denotes 0.1 substitutions per amino acid position.

Tables

TABLE 1. Proposed taxonomic assignments and genome characteristics of five Clostridium strainsa

Strain	WILCCON 0112	WILCCON 0114	WILCCON 0185	WILCCON 0202	WILCCON 0269
Isolation source	Sunflower meal	Okara	Soil	Soil	Soil
Taxonomic assignment	Candidatus Clostridium helianthi sp. nov.	Clostridium neuense	Candidatus Clostridium stratigraminis sp. nov.	Candidatus Clostridium radicumherbarum sp. nov.	Candidatus Clostridium eludens sp. nov.
Closest type strain	Clostridium beijerinckiiDSM 791T	Clostridium neuenseG1T	Clostridium rhizosphaeraeYIM B02515T	Clostridium rhizosphaeraeYIM B02515T	Clostridium kluyveriNBRC 12016T
16S rRNA GSS (%)	99.9	99.9	97.5	96.9	98.5
ANI (%)	96.2	NA	84.3	84.3	86.2
dDDH (%)	67.6	NA	20.1	20.8	28.7
BioProject acc. no.	PRJNA1187785	PRJNA1187786	PRJNA1187788	PRJNA1187789	PRJNA1187791
BioSample acc. no.	SAMN44804681	SAMN44804735	SAMN44804748	SAMN44804761	SAMN44804762
SRA acc. no. for raw reads	SRR31403109	SRR31393018	SRR31403192	SRR31403272	SRR31403754
No. of raw reads	11,427,098	11,875,844	10,488,504	10,231,748	10,825,438
Total bases of raw reads (bp)	1,714,064,700	1,781,376,600	1,573,275,600	1,534,762,200	1,623,815,700
No. of clean reads	11,302,930	11,765,546	10,406,918	10,091,090	10,731,566
Total bases of clean reads (bp)	1,681,815,945	1,751,505,498	1,552,151,258	1,501,551,961	1,599,543,825
WGS project acc. no.	JBJIAB000000000	JBJIAA000000000	JBJHZZ000000000	JBJHZY000000000	JBJHZX000000000
GenBank assembly acc. no.	GCA_045310795	GCA_045310695	GCA_045310675	GCA_045310525	GCA_045310515
Total genome size (bp)	6,059,073	5,021,087	3,657,445	4,053,182	5,093,834
Coverage	278×	349×	424×	370×	314×
DNA G + C content (%)	29.83	30.73	32.10	32.13	31.06
Completeness (%)	99.2	98.6	97.9	99.2	99.3
Contamination (%)	1.6	0.9	3.4	2.2	2.9
No. contigs	282	75	60	23	243
Largest contig	413,988	513,683	899,332	2,020,665	294,286
N50 (bp)	110,355	219,200	226,322	847,816	104,729
L 50	16	9	4	2	17
Gene content
No. coding sequences	5,474	4,626	3,474	3,859	5,118
No. tRNAs	72	64	71	72	64
No. ncRNAs	7	5	4	4	6
No. rRNAs	16	8	10	8	11
16S rRNA GenBank acc. no.	PQ625462	PQ614278	PQ625463	PQ625464	PQ625461
16S rRNA length (bp)	1,273	1,315	1,498	1,422	1,293

Full text

ANNOUNCEMENT

The taxonomy and systematics of the genus Clostridium remain complex and incompletely resolved. As of December 2024, according to the List of Prokaryotic names with Standing in Nomenclature, this genus comprises 162 validly published species (1). However, not all species have available genome sequences, including Clostridium neuense, whose type strain was isolated from lake sediment (2). Genome sequence availability not only improves the resolution of Clostridium systematics but also provides deeper insights into their physiology and metabolism. Here, we present the draft genome sequences of five Clostridium strains. Strains WILCCON 0112 and WILCCON 0114 were isolated from dried and pelleted sunflower meal after oil extraction (obtained from Russia in November 2019) and from okara, a byproduct of soybean processing (obtained from China in August 2019), respectively. Strains WILCCON 0185, WILCCON 0202, and WILCCON 0269 were isolated from a soil sample collected with a sterile spatula from the topsoil layer below a cannonball tree (Couroupita guianensis) at Jurong Lake Gardens, Singapore (1.3359°N, 103.7262°E). For the isolation of strains WILCCON 0112 and WILCCON 0114, an aliquot of the sample was resuspended via vortexing and 10-fold serially diluted in phosphate-buffered saline (NaCl 137 mM, KCl 2.7 mM, Na_2_HPO_4_ 10 mM, and KH_2_PO_4_ 1.8 mM; pH 7.4) in four steps. A total of 100 µL of each dilution was spread on an MRS agar plate (1.6% agar) and incubated at 37°C under anoxic conditions (4% hydrogen, 5% carbon dioxide, and 91% nitrogen) for 48 h. Strains WILCCON 0185, WILCCON 0202, and WILCCON 0269 were obtained in a similar way but using ATCC 207, TS, or RCM agar plates, respectively, and incubating at 30°C. All strains underwent three rounds of purification on fresh agar plates.

Single colonies were inoculated into the respective liquid medium in 2 mL Eppendorf tubes and propagated stationary under the respective conditions as specified above. Nucleic acids were extracted from cell pellets using the Maxwell 16 FFS nucleic acid extraction system (Promega). Short-read, paired-end sequencing was conducted using the Illumina NovaSeq 6000 system (2 × 150 bp reads; NovogeneAIT Genomics Singapore) with DNA libraries prepared using the NEBNext Ultra DNA library preparation kit. The 10,231,748–11,875,844 raw reads generated were quality-trimmed using Trimmomatic version 0.39 (parameters: ILLUMINACLIP:NovaSeq-adaptor-sequences.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36) (3). De novo assembly of the 10,091,090–11,765,546 clean reads obtained was performed using SPAdes version 3.15.5 (parameters: --careful --cov-cutoff auto; minimum contig length: 200 bp) (4). The resulting genome assemblies were evaluated for basic statistics using QUAST version 5.0.2 (5), assessed for completeness and contamination using CheckM version 1.2.2 (mode: lineage_wf) (6), and annotated using PGAP version 6.9 (default parameters) (7).

Detailed assembly statistics and gene content information of the five draft genomes are summarized in Table 1. The taxonomic identities of the five Clostridium strains were validated by overall genomic relatedness indices and phylogenomic analyses (Table 1; Fig. 1). Strain WILCCON 0114 was assigned to Clostridium neuense based on 16S rRNA gene sequence similarity; this identification requires confirmation upon the availability of the type genome. The remaining four strains were proposed as Candidatus Clostridium species, namely, Candidatus Clostridium helianthi (WILCCON 0112), Candidatus Clostridium stratigraminis (WILCCON 0185), Candidatus Clostridium radicumherbarum (WILCCON 0202), and Candidatus Clostridium eludens (WILCCON 0269).

Phylogenomic tree showing the relationship between five Clostridium strains (in bold) and closely related taxa of the genus Clostridium. The maximum-likelihood tree was reconstructed as described previously (8, 9), based on the protein sequences of 361 single-copy core genes annotated using Prokka version 1.14.6 (default parameters) (10), identified and aligned using SCARAP version 0.4.0 (parameters: -p 100 -f 100) (11), and trimmed using trimAl version 1.4 (parameter: -automated1) (12). The tree was inferred with IQ-TREE version 2.1.2 (13) using the LG + F + I + G4 protein substitution model, rooted by midpoint-rooting, and visualized using Interactive Tree Of Life version 6.9.1 (14). Lachnospira multipara ATCC 19207T from the neighboring family Lachnospiraceae was used as an outgroup. Ultrafast bootstrap values based on 1,000 replications are indicated at branching points. The scale bar denotes 0.1 substitutions per amino acid position.