Mesorhizobium terrae sp. nov., a novel species isolated from soil in Jangsu, Korea.

A gram-negative, white-pigmented, aerobic, rod-shaped bacterium, designated as strain NIBRBAC000500504T, was isolated from soil in Jangsu, Korea. Optimal growth of this strain was observed at 25 °C, pH 7.0, and in the presence of 0% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain NIBRBAC000500504T belonged to the genus Mesorhizobium and was closely related to Mesorhizobium shangrilense LMG 24762T (98.3% sequence similarity), Mesorhizobium australicum LMG 24608T (98.2%), Mesorhizobium qingshengii LMG 26793T (98.1%), Mesorhizobium ciceri ATCC 51585T (98.0%), Mesorhizobium loti DSM 2626T (98.0%), Mesorhizobium sophorae LMG 28223T (97.9%), Mesorhizobium waitakense LMG 28227T (97.8%), and Mesorhizobium cantuariense LMG 28225T (97.8%). Next-generation sequencing analysis indicated that the genome of strain NIBRBAC000500504T comprised a circular chromosome (5,731,152 bp, G+C content: 63.26%) and a plasmid (293,638 bp, G+C content: 61.39%) with 5672 coding sequences, 50 tRNAs, and 6 rRNAs. The major respiratory isoprenoid quinone was Q10; the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine; the major fatty acids were summed feature 8 (comprising C18:1 ω7c/C18:1 ω6c), C19:0 cyclo ω8c, C16:0, and C18:1 ω7c 11-methyl; and the G+C content of the genomic DNA was 62.9 mol%. The DNA-DNA relatedness values between NIBRBAC000500504T and its closest type strains were low. On the basis of these polyphasic taxonomic data, it is proposed that strain NIBRBAC000500504T represents a novel species of the genus Mesorhizobium, with the type strain being NIBRBAC000500504T (= KCTC 72278T = JCM 33432T).

Lactobacillus terrae sp. nov., a novel species isolated from soil samples in the Republic of Korea.

A novel strain, designated NIBRBAC000499792T, was isolated from a soil sample collected at Jukgye, Dongnam, Cheonan, Republic of Korea. Cells were Gram-positive, non-motile, non-spore-forming, rod-shaped, oxidase-negative and catalase-negative. Colonies grown on de Man, Rogosa and Sharpe agar were white, circular, raised and entire. Analysis of the 16S rRNA gene sequence analysis revealed that strain NIBRBAC000499792T belongs to the genus Lactobacillus (family Lactobacillaceae) and is most closely related to Lactobacillus nodensis DSM 19682T (96.1 % similarity) and Lactobacillus tucceti KCTC 21005T (96.7 %). The results of DNA-DNA hybridization experiments demonstrated that strain NIBRBAC000499792T represents a novel species. Major fatty acids are C18 : 1ω9c, C16 : 0 and unidentified 18.846 and/or C19 : 1ω6c and/or C19 : 0cyclo. The predominant respiratory quinones are menaquinone-8 and menaquinone-9. The major polar lipids are phosphatidylglycerol and diphosphatidylglycerol. The minor polar lipids are one unidentified aminophospholipid, one unidentified phospholipid, and four unidentified lipids. Next-generation sequencing analysis of strain NIBRBAC000499792T indicated that the total genome size was 1 548 794 bp with a G+C content of 33.1 mol%, 1586 coding sequences, 50 tRNAs and nine rRNAs. The most closely related genomes belonged to Lactobacillus species. Most metabolic pathways were related to carbon metabolism and carbon fixation. Based on this polyphasic analysis, strain NIBRBAC000499792T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus terrae sp. nov. is proposed, with the type strain NIBRBAC000499792T (=KCTC 21093T=JCM 32269T).

Detection and Molecular Characterization of Cryptosporidium spp. from Wild Rodents and Insectivores in South Korea.

In order to examine the prevalence of Cryptosporidium infection in wild rodents and insectivores of South Korea and to assess their potential role as a source of human cryptosporidiosis, a total of 199 wild rodents and insectivore specimens were collected from 10 regions of South Korea and screened for Cryptosporidium infection over a period of 2 years (2012-2013). A nested-PCR amplification of Cryptosporidium oocyst wall protein (COWP) gene fragment revealed an overall prevalence of 34.2% (68/199). The sequence analysis of 18S rRNA gene locus of Cryptosporidium was performed from the fecal and cecum samples that tested positive by COWP amplification PCR. As a result, we identified 4 species/genotypes; chipmunk genotype I, cervine genotype I, C. muris, and a new genotype which is closely related to the bear genotype. The new genotype isolated from 12 Apodemus agrarius and 2 Apodemus chejuensis was not previously identified as known species or genotype, and therefore, it is supposed to be a novel genotype. In addition, the host spectrum of Cryptosporidium was extended to A. agrarius and Crosidura lasiura, which had not been reported before. In this study, we found that the Korean wild rodents and insectivores were infected with various Cryptosporidium spp. with large intra-genotypic variationa, indicating that they may function as potential reservoirs transmitting zoonotic Cryptosporidium to livestock and humans.

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources.

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5 g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40 g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

Isolation and characterization of novel 3,3'-iminodipropionitrile biodegrading Paracoccus communis, from an industrial wastewater treatment bioreactor.

Until now, bacteria able to degrade, 3,3'-iminodipropionitrile (IDPN), a neurotoxin that destroys vestibular hair cells, causing ototoxicity, culminating in irreversible movement disorders, had never been isolated. The aim of this study was to isolate a novel IDPN-biodegrading microorganism and characterize its metabolic pathway. Enrichment was performed by inoculating activated sludge from a wastewater treatment bioreactor that treated IDPN-contaminated wastewater in M9 salt medium, with IDPN as the sole carbon source. A bacterial strain with a spherical morphology that could grow at high concentrations was isolated on a solid medium. Growth of the isolated strain followed the Monod kinetic model. Based on the 16S rRNA gene, the isolate was Paracoccus communis. Whole-genome sequencing revealed that the isolated P. communis possessed the expected full metabolic pathway for IDPN biodegradation. Transcriptome analyses confirmed the overexpression of the gene encoding hydantoinase/oxoprolinase during the exponential growth phase under IDPN-fed conditions, suggesting that the enzyme involved in cleaving the imine bond of IDPN may promote IDPN biodegradation. Additionally, the newly discovered P. communis isolate seems to metabolize IDPN through cleavage of the imine bond in IDPN via nitrilase, nitrile hydratase, and amidase reactions. Overall, this study lays the foundation for the application of IDPN-metabolizing bacteria in the remediation of IDPN-contaminated environments.

Antiphotoaging and Skin-Protective Activities of Ardisia silvestris Ethanol Extract in Human Keratinocytes.

Ardisia silvestris is a traditional medicinal herb used in Vietnam and several other countries. However, the skin-protective properties of A. silvestris ethanol extract (As-EE) have not been evaluated. Human keratinocytes form the outermost barrier of the skin and are the main target of ultraviolet (UV) radiation. UV exposure causes skin photoaging via the production of reactive oxygen species. Protection from photoaging is thus a key component of dermatological and cosmetic products. In this research, we found that As-EE can prevent UV-induced skin aging and cell death as well as enhance the barrier effect of the skin. First, the radical-scavenging ability of As-EE was checked using DPPH, ABTS, TPC, CUPRAC, and FRAP assays, and a 3-(4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay was used to examine cytotoxicity. Reporter gene assays were used to determine the doses that affect skin-barrier-related genes. A luciferase assay was used to identify possible transcription factors. The anti-photoaging mechanism of As-EE was investigated by determining correlated signaling pathways using immunoblotting analyses. As-EE had no harmful effects on HaCaT cells, according to our findings, and As-EE revealed moderate radical-scavenging ability. With high-performance liquid chromatography (HPLC) analysis, rutin was found to be one of the major components. In addition, As-EE enhanced the expression levels of hyaluronic acid synthase-1 and occludin in HaCaT cells. Moreover, As-EE dose-dependently up-regulated the production of occludin and transglutaminase-1 after suppression caused by UVB blocking the activator protein-1 signaling pathway, in particular, the extracellular response kinase and c-Jun N-terminal kinase. Our findings suggest that As-EE may have anti-photoaging effects by regulating mitogen-activated protein kinase, which is good news for the cosmetics and dermatology sectors.

Enrichment of Ca. Jettenia in sequencing batch reactors operated with low nitrogen loading rate and high influent nitrogen concentration.

To apply the anammox processes into the mainstream of domestic wastewater treatment plants, two laboratory-scale sequence batch reactors have been developed and used with two different activated sludges seeded in each sequence batch reactors with gradually increases influent total nitrogen concentrations under low nitrogen loading rates. During 320 days of operation, both sequence batch reactors showed high specific anammox activity (0.68 - 0.75 kgN kg-1VSS d-1) and a nitrogen removal efficiency of 97.50%. To monitor changes in microbial community dynamics during enrichment, high-throughput sequencing analysis was performed. Members taxonomically affiliated with Candidatus Jettenia were markedly enriched and predominant in both sequence batch reactors in response to the increasing influent total nitrogen concentrations. These results suggest that Candidatus Jettenia might be a prominent anammox genus under low nitrogen loading rate with high total nitrogen concentration conditions and could be suitably applied to the mainstream process of domestic wastewater treatment systems.

Effect of genetically modified poplars on soil microbial communities during the phytoremediation of waste mine tailings.

The application of transgenic plants to clean up environmental pollution caused by the wastes of heavy metal mining is a promising method for removing metal pollutants from soils. However, the effect of using genetically modified organisms for phytoremediation is a poorly researched topic in terms of microbial community structures, despite the important role of microorganisms in the health of soil. In this study, a comparative analysis of the bacterial and archaeal communities found in the rhizosphere of genetically modified (GM) versus wild-type (WT) poplar was conducted on trees at different growth stages (i.e., the rhizospheres of 1.5-, 2.5-, and 3-year-old poplars) that were cultivated on contaminated soils together with nonplanted control soil. Based on the results of DNA pyrosequencing, poplar type and growth stages were associated with directional changes in the structure of the microbial community. The rate of change was faster in GM poplars than in WT poplars, but the microbial communities were identical in the 3-year-old poplars. This phenomenon may arise because of a higher rate and greater extent of metal accumulation in GM poplars than in naturally occurring plants, which resulted in greater changes in soil environments and hence the microbial habitat.

Microbiome degrading linear alkylbenzene sulfonate in activated sludge.

As the most widely used anionic surfactant, linear alkylbenzene sulfonate (LAS) requires biological alkane degradation when it is treated using an activated sludge (AS) process in a wastewater treatment plant because of its structural carboxylic unavailability. As consumption of LAS is gradually increasing, LAS loading into the WWTP is accordingly increasing. However, fewer studies have examined the involvement of the AS microbial community in the LAS degradation. In this study, metagenomic approaches were used to define microbiomes involved in LAS degradation in AS, with a particular focus on ω-hydroxylation. The abundance and diversity of alkane-degrading genes were investigated, and these genes were integrated with reconstructed metagenome-assembled genomes (MAGs). Additionally, the association of functional genes and MAGs with respect to LAS degradation was investigated. The results showed that alkB and cytochrome P450 genes were only shared within specific MAGs. Unique sets of genes with diverse abundances were detected in each sample. The MAGs with the alkB and cytochrome P450 genes were strongly associated with the other MAGs and involved in positive commensal interactions. The findings provided significant insights into how the AS microbiomes, which have continuously treated anionic surfactants for decades, potentially metabolize LAS and interact with commensal bacteria.

Identification of Microbial Profiles in Heavy-Metal-Contaminated Soil from Full-Length 16S rRNA Reads Sequenced by a PacBio System.

Heavy metal pollution is a serious environmental problem as it adversely affects crop production and human activity. In addition, the microbial community structure and composition are altered in heavy-metal-contaminated soils. In this study, using full-length 16S rRNA gene sequences obtained by a PacBio RS II system, we determined the microbial diversity and community structure in heavy-metal-contaminated soil. Furthermore, we investigated the microbial distribution, inferred their putative functional traits, and analyzed the environmental effects on the microbial compositions. The soil samples selected in this study were heavily and continuously contaminated with various heavy metals due to closed mines. We found that certain microorganisms (e.g., sulfur or iron oxidizers) play an important role in the biogeochemical cycle. Using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis, we predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) functional categories from abundances of microbial communities and revealed a high proportion belonging to transport, energy metabolism, and xenobiotic degradation in the studied sites. In addition, through full-length analysis, Conexibacter-like sequences, commonly identified by environmental metagenomics among the rare biosphere, were detected. In addition to microbial composition, we confirmed that environmental factors, including heavy metals, affect the microbial communities. Unexpectedly, among these environmental parameters, electrical conductivity (EC) might have more importance than other factors in a community description analysis.

Rhizocompartments and environmental factors affect microbial composition and variation in native plants.

Molecular analysis based on large-scale sequencing of the plant microbiota has revealed complex relationships between plants and microbial communities, and environmental factors such as soil type can influence these relationships. However, most studies on root-associated microbial communities have focused on model plants such as Arabidopsis, rice or crops. Herein, we examined the microbiota of rhizocompartments of two native plants, Sedum takesimense Nakai and Campanula takesimana Nakai, using archaeal and bacterial 16S rRNA gene amplicon profiling, and assessed relationships between environmental factors and microbial community composition. We identified 390 bacterial genera, including known plant-associated genera such as Pseudomonas, Flavobacterium, Bradyrhizobium and Rhizobium, and uncharacterized clades such as DA101 that might be important in root-associated microbial communities in bulk soil. Unexpectedly, Nitrososphaera clade members were abundant, indicating functional association with roots. Soil texture/type has a greater impact on microbial community composition in rhizocompartments than chemical factors. Our results provide fundamental knowledge on microbial diversity, community and correlations with environmental factors, and expand our understanding of the microbiota in rhizocompartments of native plants.

Construction and Evaluation of a Korean Native Microbial Consortium for the Bioremediation of Diesel Fuel-Contaminated Soil in Korea.

A native microbial consortium for the bioremediation of soil contaminated with diesel fuel in Korea was constructed and its biodegradation ability was assessed. Microbial strains isolated from Korean terrestrial environments, with the potential to biodegrade aliphatic hydrocarbons, PAHs, and resins, were investigated and among them, eventually seven microbial strains, Acinetobacter oleivorans DR1, Corynebacterium sp. KSS-2, Pseudomonas sp. AS1, Pseudomonas sp. Neph5, Rhodococcus sp. KOS-1, Micrococcus sp. KSS-8, and Yarrowia sp. KSS-1 were selected for the construction of a microbial consortium based on their biodegradation ability, hydrophobicity, and emulsifying activity. Laboratory- and bulk-scale biodegradation tests showed that in diesel fuel-contaminated soil supplemented with nutrients (nitrogen and phosphorus), the microbial consortium clearly improved the biodegradation of total petroleum hydrocarbons, and all microbial strains constituting the microbial consortium, except for Yarrowia survived and grew well, which suggests that the microbial consortium can be used for the bioremediation of diesel fuel-contaminated soil in Korea.

Diversity analysis of Saccharomyces cerevisiae isolated from natural sources by multilocus sequence typing (MLST).

We used multilocus sequence typing (MLST) to analyze the diversity of natural isolates of Saccharomyces cerevisiae, the most important microorganism in alcoholic fermentation. Six loci, ADP1, RPN2, GLN4, ACC1, MET4, and NUP116, in S. cerevisiae genome were selected as MLST markers. To investigate genetic diversity within S. cerevisiae, 42 S. cerevisiae isolated from natural sources in Korea as well as six S. cerevisiae obtained from Genbank and four industrial S. cerevisiae were examined using MLST. Twenty-six polymorphic sites were found in the six loci. Among them, ACC1 had the most genetic variation with eight polymorphic sites. MLST differentiated the 52 strains into three clades. Alcohol fermentation results revealed that S. cerevisiae in Clade III produced less alcohol than those in Clades I and II. These results suggested that MLST is a powerful tool to differentiate S. cerevisiae and can potentially be used to select S. cerevisiae suitable for industrial use.

Physiological and genomic insights into the lifestyle of arsenite-oxidizing Herminiimonas arsenitoxidans.

Arsenic, a representative toxic metalloid, is responsible for serious global health problems. Most organisms possess arsenic resistance strategies to mitigate this toxicity. Here, we reported a microorganism, strain AS8, from heavy metal/metalloid-contaminated soil that is able to oxidize arsenite, and investigated its physiological and genomic traits. Its cells were rod-shaped and Gram-negative, and formed small beige-pigmented colonies. 16S rRNA-based phylogenetic analysis indicated that the strain belongs to the genus Herminiimonas and is closely related to Herminiimonas glaciei UMB49T (98.7% of 16S rRNA gene sequence similarity), Herminiimonas arsenicoxydans ULPAs1T (98.4%), and Herminiimonas saxobsidens NS11T (98.4%). Under chemolithoheterotrophic conditions, the strain utilized some organic acids and amino acids as carbon and/or nitrogen sources but not electron sources. Further, the strain grew as a sulfur oxidizer in a complex medium (trypticase soy agar). Unexpectedly, most carbohydrates failed to support its growth as sole carbon sources. Genome sequencing supported these observations, and very few ABC transporters capable of oligo/monosaccharide uptake were identified in the AS8 genome. The genome harbored genes required for the colonization, flagella biosynthesis, urea degradation, and heavy metal and antibiotic resistance. Based on these polyphasic and genomic analyses, we propose that the strain AS8 be named Herminiimonas arsenitoxidans.

First detection of West Nile virus in domestic pigeon in Korea.

West Nile virus (WNV) is a mosquito-borne zoonotic pathogen that has spread throughout Europe and the United States. Recently, WNV spread to East and Southeast Asia, and great efforts have been made in South Korea to prevent the spread of WNV from neighboring countries. In this study, we diagnosed the first case of WNV in pigeons (Columba livia domestica) residing in cities using a competitive enzyme-linked immunosorbent assay and confirmed it with nested reverse transcription polymerase chain reaction analysis and sequencing. This is the first report to provide convincing evidence that WNV is present within South Korea.

Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Wild Animals and Ixodidae Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic to central-northeastern China, southern Japan, and the Republic of Korea (ROK). To investigate SFTSV infections, we collected serum samples and ticks from wild animals. Using serum samples and ticks, SFTSV-specific genes were amplified by one-step RT-PCR and nested PCR and sequenced. Indirect immunofluorescence assay (IFA) was performed to analyze virus-specific antibody levels in wild animals. Serum samples were collected from a total of 91 animals: 21 Korean water deer (KWD), 3 Siberian roe deer, 5 gorals, 7 raccoon dogs, 54 wild boars (WBs), and 1 carrion crow. The SFTSV infection rate in wild animals was 3.30% (3 of 91 animals: 1 KWD and 2 WBs). The seropositive rate was 6.59% (6 of 91 animals: 5 KWD and 1 WB). A total of 891 ticks (3 species) were collected from 65 wild animals (9 species). Of the attached tick species, Haemaphysalis longicornis (74.86%) was the most abundant, followed by Haemaphysalis flava (20.20%) and Ixodes nipponensis (4.94%). The average minimum infection rate (MIR) of SFTSV in ticks was 4.98%. The MIRs of H. longicornis, H. flava, and I. nipponensis were 4.51%, 2.22%, and 22.73%, respectively. The MIRs of larvae, nymphs, and adult ticks were 0.68%, 6.88%, and 5.53%, respectively. In addition, the MIRs of fed and unfed ticks were 4.67% and 4.96%, respectively. We detected a low SFTSV infection rate in wild animals, no differences in SFTSV infection rate with respect to bloodsucking in ticks, and SFTSV infection for all developmental stages of ticks. This is the first report describing the detection of SFTSV in wild animals in the ROK.

Molecular characterization of highly pathogenic avian influenza H5N8 viruses isolated from Baikal teals found dead during a 2014 outbreak in Korea.

Nineteen highly pathogenic avian influenza (HPAI) H5N8 viruses were isolated from wild birds in the Donglim reservoir in Gochang, Jeonbuk province, Korea, which was first reported to be an outbreak site on January 17, 2014. Most genes from the nineteen viruses shared high nucleotide sequence identities (i.e., 99.7% to 100%). Phylogenetic analysis showed that these viruses were reassortants of the HPAI H5 subtype and the H4N2 strain and that their hemagglutinin clade was 2.3.4.4, which originated from Eastern China. The hemagglutinin protein contained Q222 and G224 at the receptor-binding site. Although the neuraminidase protein contained I314V and the matrix 2 protein contained an S31N substitution, other mutations resulting in oseltamivir and amantadine resistance were not detected. No substitutions associated with increased virulence and enhanced transmission in mammals were detected in the polymerase basic protein 2 (627E and 701D). Non-structural-1 was 237 amino acids long and had an ESEV motif with additional RGNKMAD amino acids in the C terminal region. These viruses caused deaths in the Baikal teal, which was unusual, and outbreaks occurred at the same time in both poultry and wild birds. These data are helpful for epidemiological understanding of HPAI and the design of prevention strategies.

Temporal shifts in cyanobacterial communities at different sites on the Nakdong River in Korea.

The studies of cyanobacterial blooms resulting from eutrophication or climate change and investigation of changes in the cyanobacterial community in freshwater environments are critical for the management of drinking water. Therefore, we investigated the cyanobacterial communities at 6 sites along the Nakdong River in South Korea from May 2012 to October 2012 by using high-throughput sequencing techniques and studied their relationship with various geochemical factors at sampling sites. Diverse genera (total of 175 genera) were detected within the cyanobacteria, and changes in their compositions were analyzed. The genus Prochlorococcus predominated in the May samples, especially in those obtained from the upstream part of the river, whereas the relative abundance of Microcystis and Anabaena increased with increase in water temperature. The relationship between the cyanobacterial community and environmental factors was analyzed by canonical correlation analysis, and the correlation between harmful cyanobacteria and chemical factors was analyzed by nonmetric multidimensional scaling ordination. Various environmental factors such as dissolved oxygen, pH, electric conductivity, temperature were found to affect the cyanobacterial communities in the river. The results of this study could help in the management of freshwater environments and in maintenance of drinking water quality.

Fungal community associated with genetically modified poplar during metal phytoremediation.

Due to the increasing demand for phytoremediation, many transgenic poplars have been developed to enhance the bioremediation of heavy metals. However, structural changes to indigenous fungal communities by genetically modified organisms (GMO) presents a major ecological issue, due to the important role of fungi for plant growth in natural environments. To evaluate the effect of GM plant use on environmental fungal soil communities, extensive sequencing-based community analysis was conducted, while controlling the influence of plant clonality, plant age, soil condition, and harvesting season. The rhizosphere soils of GM and wild type (WT) poplars at a range of growth stages were sampled together with unplanted, contaminated soil, and the fungal community structures were investigated by pyrosequencing the D1/D2 region of the 28S rRNA gene. The results show that the overall structure of the rhizosphere fungal community was not significantly influenced by GM poplars. However, the presence of GM specific taxa, and faster rate of community change during poplar growth, appeared to be characteristic of the GM plant-induced effects on soil-born fungal communities. The results of this study provide additional information about the potential effects of GM poplar trees aged 1.5-3 years, on the soil fungal community.