ABSTRACT
Clostridia are common mammalian gut commensals with emerging roles in human health. Here, we describe 10 Clostridia genomes from a consortium of spore forming bacteria, shown to protect mice from metabolic syndrome. These genomes will provide valuable insight on the beneficial role of spore forming bacteria in the gut.
KEYWORDS: Clostridia, spore-former
ANNOUNCEMENT
Spore forming (SF) bacteria are an important part of a healthy microbiome. Loss of SF bacteria has been associated with diseases including obesity and type 2 diabetes (1–3). Recent literature has found that the class Clostridia provides protection from inflammatory bowel disease, metabolic syndrome, infections, and colorectal cancer (4–7). However, this class of bacteria is often fastidious to grow, which has limited the availability of quality genomes to study.
To advance our understanding of SF bacteria, genomes from 10 isolates were sequenced. To isolate SF bacteria, feces from C57BL/6 specific pathogen-free mice were incubated anaerobically with 0.1% cysteine and 3% chloroform at 37°C for 1 hour to kill off vegetative bacteria. Chloroform was removed by bubbling CO2 through the sample for 30 s. To propagate the enrichment of SF bacteria, the sample was gavaged into a breeder pair of germ-free C57BL/6 mice housed in gnotobiotic conditions and feces from resulting offspring were collected, homogenized, serial diluted, and plated on YCFA anaerobically. Individual colonies were picked, streaked to isolation, and liquid cultures started from individual colonies in YCFA; DNA was extracted using Purelink Microbiome DNA purification kit (Invitrogen). Mouse work was performed under IACUC Protocol 00001562.
Five of the isolates' genomes were hybrid assembled from Illumina NovaSeq paired-end 150 and Oxford Nanopore Technologies (ONT) minION reads. Illumina libraries were prepared with NEBNext Ultra II FS DNA kit (NEB, E7805S), and reads were adapter and quality-trimmed with cutadapt (v2.10) (8) in the trim_galore (v0.6.6) wrapper using default parameters. ONT libraries were prepared without DNA shearing or size selection using rapid barcoding kit R9.4.1 chemistry (ONT, SQK-RBK004). Reads were basecalled, demultiplexed, adapter, and barcode-trimmed with guppy (v6.0.1_gpu), then quality-filtered with NanoFilt (9) using a minimum average read quality of 10 and a minimum length of 200. Hybrid genomes were assembled with SPAdes v3.15.5 within Unicycler v0.5.0 pipeline “normal mode” and filtered contigs < 200 bp (10, 11). Five genomes were sequenced and assembled with PacBio reads using Flye v2.8.1 (12) with parameters “--plasmids --iterations 2”. SMRTbell libraries were prepared without shearing using PacBio Express Template Prep Kit 2.0, pooled, and size selected using Sage Sciences' BluePippin with 0.75% DF Marker S1 High-Pass 6–10 kb v3 run protocol, S1 marker, and a cutoff of 8,000 (BPstart value), and then libraries were bound per SMRT Link Setup and sequenced on a Sequel II. Assemblies were annotated by NCBI's PGAP v6.6 (13).
All isolates are domain Bacteria, phylum Bacillota, class Clostridia, and order Eubacteriales (see Table 1 for full NCBI-assigned taxonomy). While some of the genomes had published close matching reference genomes, others such as Lachnospiraceae KK002 and KK008 were as far away as 88% and 78% best match by average nucleotide identity (ANI), respectively (FastANI v0.1.3 via GTDB-tk toolkit), indicating that they are likely new and undescribed species (14). Each genome's features including GC% content, genome size, and the number of predicted genes are described in Table 1.
TABLE 1.
Taxonomy and genome characteristics of spore forming bacteriaa
| Isolate ID | GenBank accession | Taxon beyond class clostridia order eubacteriales | Closest ref genome | ANI | Sequencing | Contigs | N50 | Size | %GC | CDS | SRR | Illumina Paired-End read number | PacBio subread number | PacBio subread N50 | ONT read number | ONT read N50 | ONT flowcell |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| JLR.KK001 | JAYMNO000000000 |
f__Lachnospiraceae;
g__Sporofaciens sp. KK001 |
GCA_9105 74715.1 |
98.44 | Hybrid | 31 | 4,968,565 | 6,390,410 | 46% | 6,399 | SRR28014014, SRR28014007 | 4,717,052 | NA | NA | 26,547 | 7,327 | FLO-MIN106 |
| JLR.KK005 | JAYMNR000000000 |
f__Candidatus;
g__Ventrimonas sp. KK005 |
GCA_0099 11065.1 |
96.15 | Hybrid | 205 | 197,068 | 5,083,539 | 44% | 4,949 | SRR28014013, SRR28014006 | 3,632,234 | NA | NA | 15 | 4,054 | FLO-FLG001 |
| JLR.KK006 | JAYMNS000000000 |
f__Lachnospiraceae;
g__Candidatus Merdisoma sp. KK006 |
GCA_9105 74255.1 |
98.86 | Hybrid | 112 | 111,685 | 4,698,239 | 45% | 4,502 | SRR28014010, SRR28014005 | 5,151,028 | NA | NA | 77 | 3,513 | FLO-FLG001 |
| JLR.KK011 | JAYWSZ000000000 |
f__Lachnospiraceae;
g__Candidatus Merdisoma sp. KK011 |
GCA_9105 75725.1 |
98.57 | PacBio | 7 | 2,532,420 | 5,043,844 | 48% | 4,958 | SRR28516891 | NA | 92,862 | 11,648 | NA | NA | NA |
| JLR.KK002 | JAYMNP000000000 | f__Lachnospiraceae bacterium KK002 | GCF_0004 03845.2 |
88.4 | Hybrid | 5 | 4,212,295 | 4,276,026 | 46% | 4,102 | SRR28014009, SRR28014004 | 4,880,942 | NA | NA | 11,608 | 8,894 | FLO-MIN106 |
| JLR.KK008 | CP143548 | f__Lachnospiraceae bacterium KK008 | GCA_9105 85345.1 |
78.35 | PacBio | 1 | 3,188,748 | 3,188,748 | 48% | 3,078 | SRR28516890 | NA | 39,804 | 12,282 | NA | NA | NA |
| JLR.KK009 | CP143549 | f__Lachnospiraceae bacterium KK009 | GCA_0004 03315.2 |
99.31 | PacBio | 1 | 5,268,209 | 5,268,209 | 47% | 5,165 | SRR28516889 | NA | 81,055 | 12,085 | NA | NA | NA |
| JLR.KK004 | JAYMNQ000000000 |
f__Oscillospiraceae;
g__Acutalibacter sp. KK004 |
GCF_0099 36035.1 |
96.44 | Hybrid | 12 | 2,635,137 | 3,852,823 | 54% | 4,046 | SRR28014008, SRR28014003 | 3,640,502 | NA | NA | 15,286 | 8,990 | FLO-MIN106 |
| JLR.KK007 | JAYWSY000000000 |
f__Oscillospiraceae;
g__Lawsonibacter sp. KK007 |
GCA_9105 84605.1 |
98.48 | PacBio | 5 | 1,637,602 | 4,295,262 | 58% | 4,458 | SRR28516888 | NA | 109,452 | 12,756 | NA | NA | NA |
| JLR.KK010 | CP143550 | f__Eubacteriales Family XIII;g__Emergencia sp. KK010 | GCF_0099 36045.1 |
98.09 | PacBio | 1 | 3,076,573 | 3,076,573 | 44% | 2,874 | SRR28516887 | NA | 260,145 | 12,915 | NA | NA | NA |
NA = Not applicable.
ACKNOWLEDGMENTS
We thank the Round Lab for help with mouse work and bacteria isolation.
This work was funded by R01AT011423-03, a W.M. Keck Award, and a Burrough’s Welcome grant to J.L.R. K.A.K. was funded by NIH NRSA 30DK127846. A.M.W. was funded by NIH NCI NRSA F32CA243501.
Contributor Information
June L. Round, Email: june.round@path.utah.edu.
Vanja Klepac-Ceraj, Department of Biological Sciences, Wellesley College, Wellesley, Massachusetts, USA.
DATA AVAILABILITY
All sequences are available through NCBI Bioproject PRJNA1061597. GenBank and SRA accession numbers listed in Table 1. Isolates are available from the Round Lab.
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Associated Data
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Data Availability Statement
All sequences are available through NCBI Bioproject PRJNA1061597. GenBank and SRA accession numbers listed in Table 1. Isolates are available from the Round Lab.