Draft Genome Sequence of Megasphaera sp. Strain DJF_B143, an Isolate from Pig Hindgut Unable to Produce Skatole

Abstract

The butyrate-producing Megasphaera spp. predominate in the pig hindgut and may play important roles in gut health. Moreover, one Megasphaera isolate has been reported to produce the boar taint compound, skatole. Here, we provide a 2.58-Mbp draft genome of a pig hindgut isolate, Megasphaera sp. DJF_B143, unable to produce skatole.

GENOME ANNOUNCEMENT

Bacteria of the genus Megasphaera are normal inhabitants of the gastrointestinal tract of mammals (1–4) and can constitute a considerable proportion of the microbial community in intestinal contents of pigs (5, 6). Due to its capability to convert lactate to butyrate, the type species M. elsdenii is a probiotic candidate for animals (7). Additionally, Megasphaera sp. TrE9262, isolated from sheep, has been reported to produce skatole (3-methylindole) (8), a main contributing compound of boar taint (9). We have isolated strain DJF_B143 belonging to the genus Megasphaera from pig hindgut content. According to 16S rRNA gene sequence analysis, strain DJF_B143 (EU728714) is only distantly related to the described Megasphaera spp., like M. indica (HM990964, 94.0% identity) and M. micronuciformis (GU470904, 93.9% identity), and its closest phylogenetic relative is Megasphaera sp. TrE9262 (DQ278866, 99.9% identity). We confirmed the production of butyrate, but observed no skatole production by DJF_B143 either in modified peptone-yeast-glucose or in colon fluid-glucose-cellobiose-agar (CGCA) media. We sequenced strain DJF_B143 to explore the genomic basis for these metabolic observations.

Megasphaera. sp. DJF_B143 was grown anaerobically in CGCA at 37°C. Genomic DNA was isolated using a Maxwell 16S DNA purification kit (Promega, USA), and automated DNA purification was performed on a Maxwell 16 Instrument (Promega). A sequencing library was prepared using the Nextera XT kit (Illumina, USA). Genome sequencing was performed using the Illumina MiSeq platform with a paired-end 300-bp MiSeq reagent kit version 3. The resulting sequence reads (ca. 1.7 million read pairs; ≈1 Gbp) were inspected for data quality using FastQC version 0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were trimmed using Trimmomatic version 0.32 (10) with the following parameters: CROP:250, HEADCROP:20, SLIDINGWINDOW:4:20, and ILLUMINACLIP:adapters.fasta:2:40:15 MINLEN:100. Trimmed Reads were assembled using SPAdes version 3.6.1 (11) with the following parameters: ‑k 21,33,55,77,99,127 –careful. Contigs most likely originating from contamination were removed using MetaWatt version 3.5 (12). The decontaminated reads mapping to the retained Megasphaera contigs (ca. 1.38 million read pairs; ≈0.53 Gbp) were extracted using BBMap version 34.94 (http://sourceforge.net/projects/bbmap/files) with the “minid = 0.98” option. The extracted reads were assembled using SPAdes as before, generating 32 contigs (>200 bp) with ca. 204× coverage. The draft genome sequence of DJF_B143 has a total length of 2,581,251 bp, an average G+C content of 49.5%, and an N₅₀ length of 161,321 bp. CheckM version 1.0.3 (13) estimated the genome to be 99.9% complete when using the gene marker set for the genus Megasphaera.

Prokka version 1.1 (14) identified 2,297 protein-coding sequences, 9 rRNAs (including 7 5S, 1 16S, and 1 23S) and 56 tRNAs. The absence of an aliphatic amidase (EC 3.5.1.4) gene in DJF_B143 suggests that this strain lacks the metabolic pathway from tryptophan to skatole via indole-3-acetate (15). Strain DJF_B143 possesses the genomic potential to produce butyrate from acetate via butyryl coenzyme A (CoA):acetate-CoA transferase (But) (EC 2.8.3.8).

Nucleotide sequence accession numbers.

The Megasphaera sp. DJF_B143 genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession number LODR00000000. The version described in this paper is the first version, LODR01000000.

Funding Statement

The work was supported by the Danish AgriFish Agency, Ministry of Food, Agriculture and Fisheries (Project 3405-10-OP-00134). Sequencing was funded by Aarhus University's Graduate School of Science and Technology and the Section for Microbiology. The first author was supported by a scholarship from China Scholarship Council (CSC).