The Description and Analysis of the Complete Genome of Dermacoccus barathri FBCC-B549 Strain.

Dermacoccus barathri is the first reported pathogen within the Dermacoccus genus to cause a catheter-related bloodstream infection, which occurred in 2015. In this study, the complete genome assembly of Dermacoccus barathri was constructed, and the complete genome of Dermacoccus barathri FBCC-B549 consists of a single chromosome (3,137,745 bp) without plasmids. The constructed genome of D. barathri was compared with those of two closely related species within the Dermacoccus genus. D. barathri exhibited a pattern similar to Dermacoccus abyssi in terms of gene clusters and synteny analysis. Contrary to previous studies, biosynthetic gene cluster (BGC) analysis for predicting secondary metabolites revealed the presence of the LAP biosynthesis pathway in the complete genome of D. barathri, predicting the potential synthesis of the secondary metabolite plantazolicin. Furthermore, an analysis to investigate the potential pathogenicity of D. barathri did not reveal any antibiotic resistance genes; however, nine virulence factors were identified in the Virulence Factor Database (VFDB). According to these matching results in the VFDB, despite identifying a few factors involved in biofilm formation, further research is required to determine the actual impact of D. barathri on pathogenicity. The complete genome of D. barathri is expected to serve as a valuable resource for future studies on D. barathri, which currently lack sufficient genomic sequence information.

Investigation of SARS-CoV-2 lineages and mutations circulating in a university-affiliated hospital in South Korea analyzed using Oxford Nanopore MinION sequencing.

OBJECTIVES: Despite the introduction of vaccines, treatments, and massive diagnostic testing, the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to overcome barriers that had slowed its previous spread. As the virus evolves towards increasing fitness, it is critical to continue monitoring the occurrence of new mutations that could evade human efforts to control them. METHODS: We performed whole-genome sequencing using Oxford Nanopore MinION sequencing on 58 SARS-CoV-2 isolates collected during the ongoing coronavirus disease 2019 pandemic at a tertiary hospital in South Korea and tracked the emergence of mutations responsible for massive spikes in South Korea. RESULTS: The differences among lineages were more pronounced in the spike gene, especially in the receptor-binding domain (RBD), than in other genes. Those RBD mutations could compromise neutralization by antibodies elicited by vaccination or previous infections. We also reported multiple incidences of Omicron variants carrying mutations that could impair the diagnostic sensitivity of reverse transcription-polymerase chain reaction-based testing. CONCLUSION: These results provide an understanding of the temporal changes of variants and mutations that have been circulating in South Korea and their potential impacts on antigenicity, therapeutics, and diagnostic escape of the virus. We also showed that the utilization of the nanopore sequencing platform and the ARTIC workf low can provide convenient and accurate SARS-CoV-2 genomic surveillance even at a single hospital.

Investigation of memory-enhancing effects of Streptococcus thermophilus EG007 in mice and elucidating molecular and metagenomic characteristics using nanopore sequencing.

Over the past decades, accumulating evidences have highlighted the gut microbiota as a key player in the brain functioning via microbiota-gut-brain axis, and accordingly, the beneficial role of several probiotic strains in cognitive ability also have been actively investigated. However, the majority of the research have demonstrated the effects against age-related cognitive decline or neurological disease. To this end, we aimed to investigate lactic acid bacteria strains having beneficial effects on the cognitive function of healthy young mice and elucidate underlying characteristics by carrying out nanopore sequencing-based genomics and metagenomics analysis. 8-week consumption of Streptococcus thermophilus EG007 demonstrated marked enhancements in behavior tests assessing short-term spatial and non-spatial learning and memory. It was revealed that EG007 possessed genes encoding various metabolites beneficial for a health condition in many aspects, including gamma-aminobutyric acid producing system, a neurotransmitter associated with mood and stress response. Also, by utilizing 16S-23S rRNA operon as a taxonomic marker, we identified more accurate species-level compositional changes in gut microbiota, which was increase of certain species, previously reported to have associations with mental health or down-regulation of inflammation or infection-related species. Moreover, correlation analysis revealed that the EG007-mediated altered microbiota had a significant correlation with the memory traits.

Positive Effect of Lactobacillus acidophilus EG004 on Cognitive Ability of Healthy Mice by Fecal Microbiome Analysis Using Full-Length 16S-23S rRNA Metagenome Sequencing.

Evidence for the concept of the "gut-brain axis" (GBA) has risen. Many types of research demonstrated the mechanism of the GBA and the effect of probiotic intake. Although many studies have been reported, most were focused on neurodegenerative disease and, it is still not clear what type of bacterial strains have positive effects. We designed an experiment to discover a strain that positively affects brain function, which can be recognized through changes in cognitive processes using healthy mice. The experimental group consisted of a control group and three probiotic consumption groups, namely, Lactobacillus acidophilus, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus. Three experimental groups fed probiotics showed an improved cognitive ability by cognitive-behavioral tests, and the group fed on L. acidophilus showed the highest score. To provide an understanding of the altered microbial composition effect on the brain, we performed full 16S-23S rRNA sequencing using Nanopore, and operational taxonomic units (OTUs) were identified at species level. In the group fed on L. acidophilus, the intestinal bacterial ratio of Firmicutes and Proteobacteria phyla increased, and the bacterial proportions of 16 species were significantly different from those of the control group. We estimated that the positive results on the cognitive behavioral tests were due to the increased proportion of the L. acidophilus EG004 strain in the subjects' intestines since the strain can produce butyrate and therefore modulate neurotransmitters and neurotrophic factors. We expect that this strain expands the industrial field of L. acidophilus and helps understand the mechanism of the gut-brain axis. IMPORTANCE Recently, the concept of the "gut-brain axis" has risen and suggested that microbes in the GI tract affect the brain by modulating signal molecules. Although many pieces of research were reported in a short period, a signaling mechanism and the effects of a specific bacterial strain are still unclear. Besides, since most of the research was focused on neurodegenerative disease, the study with a healthy animal model is still insufficient. In this study, we show using a healthy animal model that a bacterial strain (Lactobacillus acidophilus EG004) has a positive effect on mouse cognitive ability. We experimentally verified an improved cognitive ability by cognitive behavioral tests. We performed full 16S-23S rRNA sequencing using a Nanopore MinION instrument and provided the gut microbiome composition at the species level. This microbiome composition consisted of candidate microbial groups as a biomarker that shows positive effects on cognitive ability. Therefore, our study suggests a new perspective for probiotic strain use applicable for various industrialization processes.

Complete Genome Sequence of the Newly Developed Lactobacillus acidophilus Strain With Improved Thermal Adaptability.

Lactobacillus acidophilus (L. acidophilus) is a representative probiotic and is widely used in many industrial products for its beneficial effects on human and animal health. This bacterium is exposed to harsh environments such as high temperatures for manufacturing industrial products, but cell yield under high temperatures is relatively low. To resolve this issue, we developed a new L. acidophilus strain with improved heat resistance while retaining the existing beneficial properties through the adaptive laboratory evolution (ALE) method. The newly developed strain, L. acidophilus EG008, has improved the existing limit of thermal resistance from 65°C to 75°C. Furthermore, we performed whole-genome sequencing and comparative genome analysis of wild-type and EG008 strains to unravel the molecular mechanism of improved heat resistance. Interestingly, only two single-nucleotide polymorphisms (SNPs) were different compared to the L. acidophilus wild-type. We identified that one of these SNPs is a non-synonymous SNP capable of altering the structure of MurD protein through the 435th amino acid change from serine to threonine. We believe that these results will directly contribute to any industrial field where L. acidophilus is applied. In addition, these results make a step forward in understanding the molecular mechanisms of lactic acid bacteria evolution under extreme conditions.

Genotyping-by-Sequencing of the regional Pacific abalone (Haliotis discus) genomes reveals population structures and patterns of gene flow.

Continuous monitoring of the present genetic status is essential to preserve the genetic resource of wild populations. In this study, we sequenced regional Pacific abalone Haliotis discus samples from three different locations around the Korean peninsula to assess population structure, utilizing Genotyping-by-Sequencing (GBS) method. Using PstI enzyme for genome reduction, we demonstrated the resultant library represented the whole genome region with even spacing, and as a result 16,603 single nucleotide variants (SNVs) were produced. Genetic diversity and population structure were investigated using several methods, and a strong genetic heterogeneity was observed in the Korean abalone populations. Additionally, by comparison of the variant sets among population groups, we were able to discover 26 Korean abalone population-specific SNVs, potentially associated with phenotype differences. This is the first study demonstrating the feasibility of GBS for population genetic study on H. discus. Our results will provide valuable data for the genetic conservation and management of wild abalone populations in Korea and help future GBS studies on the marine mollusks.

Is Lactobacillus Gram-Positive? A Case Study of Lactobacillus iners.

Lactobacillus iners is the most prevalent bacterial species in the human vaginal microbiome, and there have been few reports of its Gram-negative stain appearances despite the fact that the genus Lactobacillus is universally described as Gram-positive. Here, using transmission electron microscopy, we reveal that the thinness of the cell wall (17.39 ± 0.8 nm) gives the Gram-negative stain appearance, which can lead to over-diagnosis of bacterial vaginosis. Moreover, comparative genome analysis identified four genes commonly absent in L. iners genomes that might contribute to this phenotypic difference. We suggest that, along with the several niche-specific attributes identified, this unique feature may contribute to the species' distinguished capability to thrive as the predominant species in the fluctuating vaginal environment as well.

Complete Genome of Lactobacillus iners KY Using Flongle Provides Insight Into the Genetic Background of Optimal Adaption to Vaginal Econiche.

Despite the importance of Lactobacillus iners and its unique characteristics for the study of vaginal adaption, its genome and genomic researches for identifying molecular backgrounds of these specific phenotypes are still limited. In this study, the first complete genome of L. iners was constructed using a cost-effective long-read sequencing platform, Flongle from Oxford Nanopore, and comparative genome analysis was conducted using a total of 1,046 strain genomes from 10 vaginal Lactobacillus species. Single-molecule sequencing using Flongle effectively resolved the limitation of the 2nd generation sequencing technologies in dealing with genomic regions of high GC contents, and comparative genome analysis identified three potential core genes (INY, ZnuA, and hsdR) of L. iners which was related to its specific adaption to the vaginal environment. In addition, we performed comparative prophage analysis for 1,046 strain genomes to further identify the species specificity. The number of prophages in L. iners genomes was significantly smaller than other vaginal Lactobacillus species, and one of the specific genes (hsdR) was suggested as the means for defense against bacteriophage. The first complete genome of L. iners and the three specific genes identified in this study will provide useful resources to further expand our knowledge of L. iners and its specific adaption to the vaginal econiche.

Expression and Purification of Extracellular Solute-Binding Protein (ESBP) in Escherichia coli, the Extracellular Protein Derived from Bifidobacterium longum KACC 91563.

Bifidobacterium longum KACC 91563 secretes family 5 extracellular solute-binding protein via extracellular vesicle. In our previous work, it was demonstrated that the protein effectively alleviated food allergy symptoms via mast cell specific apoptosis, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, we cloned the gene encoding extracellular solute-binding protein of the strain into the histidine-tagged pET-28a(+) vector and transformed the resulting plasmid into the Escherichia coli strain BL21 (DE3). The histidine-tagged extracellular solute-binding protein expressed in the transformed cells was purified using Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the process, two liters of transformed culture produced 7.15 mg of the recombinant proteins. This is the first study describing the production of extracellular solute-binding protein of probiotic bacteria. Establishment of large-scale production strategy for the protein will further contribute to the development of functional foods and potential alternative treatments for allergies.

Accurate and Strict Identification of Probiotic Species Based on Coverage of Whole-Metagenome Shotgun Sequencing Data.

Identifying the microbes present in probiotic products is an important issue in product quality control and public health. The most common methods used to identify genera containing species that produce lactic acid are matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA sequence analysis. However, the high cost of operation, difficulty in distinguishing between similar species, and limitations of the current sequencing technologies have made it difficult to obtain accurate results using these tools. To overcome these problems, a whole-genome shotgun sequencing approach has been developed along with various metagenomic classification tools. Widely used tools include the marker gene and k-mer methods, but their inevitable false-positives (FPs) hampered an accurate analysis. We therefore, designed a coverage-based pipeline to reduce the FP problem and to achieve a more reliable identification of species. The coverage-based pipeline described here not only shows higher accuracy for the detection of species and proportion analysis, based on mapping depth, but can be applied regardless of the sequencing platform. We believe that the coverage-based pipeline described in this study can provide appropriate support for probiotic quality control, addressing current labeling issues.

Horizontal gene transfer of Chlamydia: Novel insights from tree reconciliation.

Recent comparative genomics studies have suggested that horizontal gene transfer (HGT) is one of the major processes in bacterial evolution. In this study, HGT events of 64 Chlamydia strains were investigated based on the pipeline employed in HGTree database constructed in our recent study. Tree reconciliation method was applied in order to calculate feasible HGT events. Following initial detection and an evaluation procedure, evidence of the HGT was identified in 548 gene families including 42 gene families transferred from outside of Chlamydiae phylum with high reliability. The donor species of inter-phylum HGT consists of 12 different bacterial and archaeal phyla, suggesting that Chlamydia might have even more various host range than in previous reports. In addition, each species of Chlamydia showed varying preference towards HGT, and genes engaged in HGT within Chlamydia and between other species showed different functional distribution. Also, examination of individual gene flows of niche-specific genes suggested that many of such genes are transferred mainly within Chlamydia genus. Our results uncovered novel features of HGT acting on Chlamydia genome evolution, and it would be also strong evidence that HGT is an ongoing process for intracellular pathogens. We expect that the results provide more insight into lineage- and niche-specific adaptations regarding their infectivity and pathogenicity.

Genome sequence of the Japanese oak silk moth, Antheraea yamamai: the first draft genome in the family Saturniidae.

Background: Antheraea yamamai, also known as the Japanese oak silk moth, is a wild species of silk moth. Silk produced by A. yamamai, referred to as tensan silk, shows different characteristics such as thickness, compressive elasticity, and chemical resistance compared with common silk produced from the domesticated silkworm, Bombyx mori. Its unique characteristics have led to its use in many research fields including biotechnology and medical science, and the scientific as well as economic importance of the wild silk moth continues to gradually increase. However, no genomic information for the wild silk moth, including A. yamamai, is currently available. Findings: In order to construct the A. yamamai genome, a total of 147G base pairs using Illumina and Pacbio sequencing platforms were generated, providing 210-fold coverage based on the 700-Mb estimated genome size of A. yamamai. The assembled genome of A. yamamai was 656 Mb (>2 kb) with 3675 scaffolds, and the N50 length of assembly was 739 Kb with a 34.07% GC ratio. Identified repeat elements covered 37.33% of the total genome, and the completeness of the constructed genome assembly was estimated to be 96.7% by Benchmarking Universal Single-Copy Orthologs v2 analysis. A total of 15 481 genes were identified using Evidence Modeler based on the gene prediction results obtained from 3 different methods (ab initio, RNA-seq-based, known-gene-based) and manual curation. Conclusions: Here we present the genome sequence of A. yamamai, the first genome sequence of the wild silk moth. These results provide valuable genomic information, which will help enrich our understanding of the molecular mechanisms relating to not only specific phenotypes such as wild silk itself but also the genomic evolution of Saturniidae.

The Genetic Origin of Short Tail in Endangered Korean Dog, DongGyeongi.

The tail of many animal species is responsible for various physiological functions. The functional importance of tail may have brought tail-loss to attention in many evolutionary and developmental studies. To provide a better explanation for the loss of tail, the current study aims to identify the evolutionary history and putative causal variants for the short tail in DongGyeongi (DG), an endangered dog breed, which is also the only dog in Korea that possesses a short tail. Whole genome sequencing was conducted on 22 samples of DG, followed by an investigation of population stratification with 10 other dog breeds. The genotypes, selective sweep and demography of DG were also investigated. As a result, we discovered the unique genetic structure of DG and suggested two possible ways in which the short tail phenotype developed. Moreover, this study suggested that selective sweep genes, ANKRD11 and ACVR2B may contribute to the reduction in tail length, and non-synonymous variant in the coding sequence of T gene and the CpG island variant of SFRP2 gene are the candidate causal variants for the tail-loss.