Microorganisms have historically played an important role in the livestock industry. Researchers are particularly interested in identifying functional microorganisms that are capable of hydrolyzing non-degradable materials, such as xylan and cellulose, in grass feed to develop cattle feed rich in nutrients. Furthermore, xylanase application stimulates fiber usage, reduces intestinal viscosity, and increases nutrient absorption (Romero et al., 2016; He et al., 2020). The same principle can be used to increase productivity in the livestock industry.

Recently, microbial research has been extended to the livestock industry with a focus on elucidating the relationship between microbial communities and animal phenotypes. Among these, studies on microbial populations in reproductive organs have been related to productivity. Most studies on the microbiome of reproductive organs were first conducted in humans. Microbial communities in the mother’s reproductive organs are being investigated because microbes have an impact on babies (Dobbler et al., 2019). Studies on the characteristics of microorganisms in reproductive organs are also being carried out in cattle. Recent studies have been conducted on the microbial communities in the uterus and vagina of cattle (Appiah et al., 2020). Most microbiological studies on the reproductive organs of cattle have focused on the decrease in fertility caused by diseases and infections (Sheldon and Dobson, 2004). A recent study on dairy cows revealed that vaginal microbial populations differ depending on the success rate of artificial insemination (Chen et al., 2020). Therefore, it is necessary to understand the microorganisms present in the reproductive organs. Research has been conducted on bacteria that cause uterine diseases in Hanwoo (Korean native cattle) (Woo et al., 2015). However, studies on healthy vaginal microorganisms during artificial insemination and functional genetic analysis of such vaginally derived microorganisms have not been carried out. In this study, we assembled and annotated the complete genome sequence of Lacrimispora xylanolytica sy1 isolated from Hanwoo vagina.

Lacrimispora xylanolytica sy1 was isolated from the vaginal discharge of a 25-month-old, clinically healthy Hanwoo, from Cheongju Province, South Korea. For sample collection, a long-handled sterile swab (15 cm) was used to rub the middle of the cow’s vagina during artificial insemination. The collected sample was stored at -20°C. The study was approved by the Ethics Committee of the National Institute of Animal Science (approval no: NIAS20201979). The strain sy1 was subcultured at 37°C for 72 h using NX-A media (peptone 5 g/L, beef extract 1.5 g/L, yeast extract 10.5 g/L, xylan 5g/L, L-cysteine 0.5 g/L, CaCl₂·H₂O 0.1 g/L, MgSO₄·7H₂O 0.2 g/L, K₂HPO₄ 0.4 g/L, KH₂PO₄ 0.4 g/L, NaHCO₃ 2 g/L, NaCl 5 g/L, agar 15 g/L) in anaerobic condition. The lactic acid production ability of the strain was confirmed by the formation of transparent rings in 1.5% CaCO₃ (w/v) in MRS agar medium (Kim et al., 2011). To evaluate the xylan resolution, we performed a reference study using trypan blue (Park et al., 2012). As the xylan bound to trypan blue was decomposed, the diameters of the transparent ring were measured every 24, 48, and 72 h with a digital caliper (repeated five times).

Microbial DNA was extracted using the QIAmp PowerFecal DNA kit (Qiagen) from the cultured L. xylanolytica sy1 in NX-A media and stored at -20°C. The PacBio Sequel (Pacific Bioscience) and Illumina NovaSeq 6000 (Illumina) platforms were used to sequence the microbial genomes. For PacBio Sequel sequencing, the SMRTbell template was used to generate a single SMRT cell using SMRT Link pipeline version 10.1. To increase accuracy, NovaSeq reads were used to correct erroneous base pairs. The Hierarchical Genome Assembly Process (HGAP4, version 4.0, Pacific Bioscience) was used to perform de novo assembly of sequencing reads. Each CDS was annotated using a homology search against the Swiss-Prot (The UniProt Consortium, 2014), EggNOG ver. 5.0 (Powell et al., 2013), Kyoto Encyclopedia of Genes and Genomes (Kanehisa et al., 2013), and Clusters of Orthologous Groups (Tatusov et al., 2000) databases. The Orthologous Average Nucleotide Identity (OrthoANI) value was calculated using OrthoANI version 0.5.0 (Lee et al., 2016).

On the PacBio Sequel platform, 1,025,134 subreads (N50 value, 11,066) were generated, and 5,679,432 qualified filtered reads (Q20, 99.2%; mapped reads, 5,677,707; genome coverage, 184.15X) were generated using the Illumina NovaSeq. The complete genome of Lacrimipora xylanolytica sy1 consisted of a 4,516,221 bp single chromosome with a GC content of 42.32%. Plasmids were not present in the genome. Table 1 and Fig. 1 summarize the genomic characteristics. In total, 4,046 coding genes, 18 rRNAs (5S, 16S, and 23S), and 67 tRNAs were identified in the genome. Based on the EggNOG assignments, 3,733 genes were functionally classified.

Table 1

Feature	Lacrimispora xylanolytica sy1
GenBank accession No.	CP113524
Assemble method	SMRT Analysis version 10.1
Genome size (bp)	4,516,221
Contigs	1
GC content (%)	42.32
Total number of genes	4,163
Protein coding genes (CDS)	4,046
rRNA genes (5S, 16S, 23S)	6, 6, 6
tRNA genes	67

Fig. 1. Chromosome map of Lacrimispora xylanolytica sy1. Marked characteristics are shown from outside to center; coding DNA sequences (CDS) on forward strand, CDS on reverse strand, tRNA, rRNA, GC content, and GC skew.

We identified hydrolytic enzymes through gene annotation analysis. Among the annotated genes, seven xylan degradation genes (beta-xylosidase xynB, arabinoxylan arabinofuranohydrolase xynD, glucuronoxylanase xynC_1, endo-1,4-beta-xylanase Y xynY, xylan alpha-(1->2)-glucuronosidase aguA_1, endo-1,4-beta-xylanase Z xynZ_1, and endo-1,4-beta-xylanase Z xynZ_2) were identified. Additionally, a genome contained the lactic acid fermentation gene (L-lactate dehydrogenase, ldh) and a chitin degradation enzyme gene (chitinase A1). Xylose is the decomposition product of xylan and a component of glycosaminoglycan that promotes oocyte maturation (Götting et al., 2002). Furthermore, glycosaminoglycans can establish a reproductive environment that enhances sperm motility; hence, a female reproductive organ function enhancer containing xylose has been developed (Ellington and Dennis, 2018). Previously, Lachnobacterium bovis belonging to the same family was isolated from cattle; however, this species was unable to use xylan, and the orthologous average nucleotide identity with the sy1 strain was only 68.08% (Whitford et al., 2001). Lactic acid bacteria generate lactate, which is essential for sustaining a vaginal environment that is especially beneficial for females (Auriemma et al., 2021).

Based on the orthologous average nucleotide identity (orthoANI), strain sy1 was 98.77% (99.28% of the 16S rRNA gene) identical to L. xylanolytica DSM 6555. L. xylanolytica, previously named Clostridium xylanolyticum, was reclassified into the genus Lacrimispora in 2020 (Haas and Blanchard, 2020). In a previous study, L. xylanolytica DSM 6555 was isolated from the wood chips of pine trees (Pinus patula) (Rogers and Baecker, 1991).

To the best of our knowledge, this is the first study to discover an L. xylanolytica strain that produces xylanase and lactic acid in cattle. This study suggests that this strain may be advantageous for the livestock industry because it has antibacterial effects on pathogens and increases the bioavailability of feed that is difficult to decompose, such as straw, using xylan resolution and lactic acid generation capacity. This complete genome sequence provides a basic understanding of the strain Lacrimispora xylanolytica sy1, which can be used as a potential feed additive for ruminants.

Nucleotide sequence accession and deposit numbers

Lacrimispora xylanolytica sy1 was deposited in the Korean Agricultural Culture Collection under KACC 81220BP, and the complete genome sequence was deposited in GenBank under accession number CP113524.

xylan을 분해하는 한우 질에서 분리된 Lacrimispora xylanolytica sy1 의 유전체를 분석하였다. 이 균주는 원형 염색체(4,516,221 bp)로 이루어져 있으며, GC 함량은 42.32%이다. Lacrimispora xylanolytica sy1 균주의 유전체에는 xylanase 및 lactate dehydrogenase 등 가수분해효소 유전자 정보를 포함하여 4,163개의 유전자가 확인됐다. 이 균주는 소화율을 높이고 건강을 증진시킬 수 있어 향후 가축의 사료 첨가제로서 이용 가능성을 보인다.

This study was carried supported by the National Institute of Animal Sciences, Rural Development Administration, Republic of Korea (PJ014826).

The authors declare no conflicts of interest.