Rhodoferax potami sp. nov. and Rhodoferax mekongensis sp. nov., isolated from the Mekong River in Thailand.

Four newly discovered Gram-stain-negative bacteria, designated as BL00010T, BL00058, D8-11T and BL00200, were isolated from water samples collected at three hydrological monitoring stations (namely Chiang Saen, Chiang Khan and Nong Khai) located along the Mekong River in Thailand. An investigation encompassing phenotypic, chemotaxonomic and genomic traits was conducted. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that all four isolates represented members of the genus Rhodoferax. These isolates were closely related to Rhodoferax bucti KCTC 62564T with a similarity of 99.59%. The major fatty acids of the four novel isolates included C16:0 and C16:1ω7c and/or C16 : 1ω6c, whereas the major respiratory quinone was identified as ubiquinone Q-8. In addition, phosphatidylethanolamine was identified as a major polar lipid in these bacteria. The genomes of BL00010T, BL00058, D8-11T and BL00200 were similar in size (3.88-4.01 Mbp) and DNA G+C contents (59.5, 59.3, 59.5 and 59.3 mol%, respectively). In contrast to R. bucti KCTC 62564T and Rhodoferax aquaticus KCTC 32394T, the newly discovered species possessed several genes involved in nitrite and nitrile metabolism, which may be related to their unique adaptation to nitrile-rich environments. From the results of the pairwise analysis of average nucleotide identity of the whole genome and digital DNA-DNA hybridisation, it was evident that BL00010T and D8-11T represented two novel species, for which we propose the nomenclature Rhodoferax potami sp. nov., with the type strain BL00010T (TBRC 17198T = NBRC 116413T), and Rhodoferax mekongensis sp. nov., with the type strain D8-11T (TBRC 17307T = NBRC 116415T).

Fodinisporobacter ferrooxydans gen. nov., sp. nov.-A Spore-Forming Ferrous-Oxidizing Bacterium Isolated from a Polymetallic Mine.

A novel acidophilic, aerobic bacterium strain, MYW30-H2T, was isolated from a heap of polymetallic mine. Cells of strain MYW30-H2T were Gram-stain-positive, endospore-forming, motile, and rod-shaped. Strain MYW30-H2T grew at a temperature range of 30-45 °C (optimum 40 °C) and a pH range of 3.5-6.0 (optimum 4.0) in the presence of 0-0.5% (w/v) NaCl. Strain MYW30-H2T could grow heterotrophically on yeast extract and glucose, and grow mixotrophically using ferrous iron as an electron donor with yeast extract. Menaquinone-7 (MK-7) was the sole respiratory quinone of the strain. Iso-C15:0 and anteiso-C15:0 were the major cellular fatty acids. The 16S rRNA gene sequence analysis showed that MYW30-H2T was phylogenetically affiliated with the family Alicyclobacillaceae, and the sequence similarity with other Alicyclobacillaceae genera species was below 91.51%. The average amino acid identity value of the strain with its phylogenetically related species was 52.3-62.1%, which fell into the genus boundary range. The DNA G+C content of the strain was 44.2%. Based on physiological and phylogenetic analyses, strain MYW30-H2T represents a novel species of a new genus of the family Alicyclobacillaceae, for which the name Fodinisporobacter ferrooxydans gen. nov., sp. nov. is proposed. The type strain is MYW30-H2T (=CGMCC 1.17422T = KCTC 43278T).

Fungal communities as dual indicators of river biodiversity and water quality assessment.

Given their ecological importance, bioindicators are used for the assessment of the health of river ecosystems. This study explored the fungal compositions and the potential of fungal taxa as bioindicators for indicating the water quality of the Mekong River, as the use of fungal indicators of the Mekong River was not previously well characterized. The Mekong River exhibited dynamic variations in both physicochemical/hydrochemical properties and fungal communities according to seasons and locations. The results revealed the dominance of alkaline earth metal ions and weak acids in the water. The magnesium-bicarbonate water type was found in the dry season, but the water became the chloride-calcium type or mixed type of magnesium-bicarbonate and chloride-calcium in the rainy season at downstream sites. Fungal composition analysis revealed the dominance of Chytridiomycota in the dry season and intermediate periods, and Ascomycota and Basidiomycota in the rainy season. The fungal communities were influenced by stochastic and deterministic assembly processes, mainly homogeneous selection, heterogeneous selection, and dispersal limitation. The extent of environmental filtering implied that some fungal taxa were affected by environmental conditions, suggesting the possibility of identifying certain fungal taxa suitable for being bioindicators of water quality. Subsequently, machine learning with recursive feature elimination identified specific fungal bins mostly consisting of Agaricomycetes (mainly Polyporales, Agaricales, and Auriculariales), Dothideomycetes (mainly Pleosporales), Saccharomycetes (mainly Saccharomycetales), Chytridiomycota, and Rozellomycota as bioindicators that could predict ambient and irrigation water quality with high selectivity and sensitivity. These results thus promote the use of fungal indicators to assess the health of the river.

Interplay of xenobiotic-degrading and antibiotic-resistant microorganisms among the microbiome found in the air, handrail, and floor of the subway station.

Investigating the quality of the subway environment, especially regarding antibiotic resistance genes (ARGs) and xenobiotics, conveys ecological and health impacts. In this study, compositions and relations of microorganisms harboring ARGs and xenobiotic degradation and metabolism genes (XDGs) in the Sukhumvit subway station (MRT-SKV) in Bangkok was assessed by analyzing the taxonomic and genetic diversity of the microbiome in the air and on the surfaces of floor and handrail. The major bacteria in the MRT-SKV (including Moraxella, which was abundant in the bioaerosol and handrail samples, and Staphylococcus, which was abundant in the bioaerosol samples) were found to contain both ARGs and XDGs. The co-abundance correlation network revealed notable relationships among bacteria harboring antibiotic resistance genes (ARGs) and xenobiotic degradation genes (XDGs). Significant associations were observed between ARGs linked to glycopeptide and fluoroquinolone resistance and genes associated with benzoate, styrene, and atrazine degradation pathways, as well as between ARGs related to cephamycin, cephalosporin, and MLS resistance and XDGs associated with the cytochrome P450-dependent drug metabolism pathway. These correlations suggested that selective pressure exerted by certain xenobiotics and antibiotics can simultaneously affect both ARGs and XDGs in the environment and should favor correlations and co-survival among ARG- and XDG-containing bacteria in the environments. The correlations may occur via shared mechanisms of resistance to both xenobiotics and antibiotics. Finally, different correlation pairs were seen in different niches (air, handrail, floor) of the subway environment or different geolocations. Thus, the relationship between ARG and XDG pairs most likely depends on the unique characteristics of the niches and on the prominent types of xenobiotics and antibiotics in the subway environment. The results indicated that interactions and connections between microbial communities can impact how they function. These microorganisms can have profound effects on accumulation of xenobiotics and ARGs in the MRT-SKV.

Elucidating potential bioindicators from insights in the diversity and assembly processes of prokaryotic and eukaryotic communities in the Mekong River.

Drivers for spatio-temporal distribution patterns of overall planktonic prokaryotes and eukaryotes in riverine ecosystems are generally not fully understood. This study employed amplicon metabarcoding to investigate the distributions and assembly mechanisms of bacterial and eukaryotic communities in the Mekong River. The prevailing bacteria taxa were found to be Betaproteobacteria, Actinobacteria, and Bacteroidetes, while the dominant eukaryotic organisms were cryptophytes, chlorophytes, and diatoms. The community assemblages were influenced by a combination of stochastic and deterministic processes. Drift (DR) and dispersal limitation (DL), signifying the stochastic mechanism, were the main processes shaping the overall prokaryotic and eukaryotic communities. However, homogeneous selection (HoS), indicating deterministic mechanism, played a major role in the assembly process of core prokaryotic communities, especially in the wet season. In contrast, the core eukaryotic communities including Opisthokonta, Sar, and Chlorophyta were dominated by stochastic processes. The significance of HoS within prokaryotic communities was also found to exhibit a decreasing trend from the upstream sampling sites (Chiang Saen and Chiang Khan, Nong Khai) towards the downstream sites (Mukdahan, and Khong Chiam) of the Mekong River. The environmental gradients resulting from the site-specific variations and the gradual decrease in elevation along the river may have a potential influence on the role of HoS in community assembly. Crucial environmental factors that shape the phylogenetic structure within distinct bins of the core prokaryotic communities including water depth, temperature, chloride, sodium, and sulphate were identified, as inferred by their correlation with the beta Net Relatedness Index (betaNRI) during the wet season. Overall, these findings enhance understanding of the complex mechanisms governing the spatio-temporal dynamics of prokaryotic and eukaryotic communities in the Mekong River. Finally, insights gained from this study could provide information on further use of specific core bacteria as microbial-based bioindicators that are effective for the assessment and conservation of the Mekong River ecosystem.

Genes controlling hydrolysate toxin tolerance identified by QTL analysis of the natural Saccharomyces cerevisiae BCC39850.

Lignocellulosic material can be converted to valorized products such as fuels. Pretreatment is an essential step in conversion, which is needed to increase the digestibility of the raw material for microbial fermentation. However, pretreatment generates by-products (hydrolysate toxins) that are detrimental to microbial growth. In this study, natural Saccharomyces strains isolated from habitats in Thailand were screened for their tolerance to synthetic hydrolysate toxins (synHTs). The Saccharomyces cerevisiae natural strain BCC39850 (toxin-tolerant) was crossed with the laboratory strain CEN.PK2-1C (toxin-sensitive), and quantitative trait locus (QTL) analysis was performed on the segregants using phenotypic scores of growth (OD600) and glucose consumption. VMS1, DET1, KCS1, MRH1, YOS9, SYO1, and YDR042C were identified from QTLs as candidate genes associated with the tolerance trait. CEN.PK2-1C knockouts of the VMS1, YOS9, KCS1, and MRH1 genes exhibited significantly greater hydrolysate toxin sensitivity to growth, whereas CEN.PK2-1C knock-ins with replacement of VMS1 and MRH1 genes from the BCC39850 alleles showed significant increased ethanol production titers compared with the CEN.PK2-1C parental strain in the presence of synHTs. The discovery of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin tolerance in S. cerevisiae indicates the roles of the endoplasmic-reticulum-associated protein degradation pathway, plasma membrane protein association, and the phosphatidylinositol signaling system in this trait. KEY POINTS: • QTL analysis was conducted using a hydrolysate toxin-tolerant S. cerevisiae natural strain • Deletion of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin-sensitivity • Replacement of VMS1 and MRH1 with natural strain alleles increased ethanol production titers in the presence of hydrolysate toxins.

AirDNA sampler: An efficient and simple device enabling high-yield, high-quality airborne environment DNA for metagenomic applications.

Analyzing temporal and spatial distributions of airborne particles of biological origins is vital for the assessment and monitoring of air quality, especially with regard to public health, environmental ecology, and atmospheric chemistry. However, the analysis is frequently impeded by the low levels of biomass in the air, especially with metagenomic DNA analysis to explore diversity and composition of living organisms and their components in the air. To obtain sufficient amounts of metagenomic DNA from bioaerosols, researchers usually need a long sampling time with an expensive high-volume air sampler. This work shows the utilization of an air sampling device containing an economical, high-volume portable ventilation fan in combination with customized multi-sheet filter holders to effectively obtain high yields of genomic DNA in a relatively short time. The device, named 'AirDNA' sampler, performed better than other commercial air samplers, including MD8 Airport and Coriolis compact air samplers. Using the AirDNA sampler, an average DNA yield of 40.49 ng (12.47-23.24 ng at 95% CI) was obtained in only 1 hour of air sampling with a 0.85 probability of obtaining ≥10 ng of genomic DNA. The genomic DNA obtained by the AirDNA system is of suitable quantity and quality to be further used for amplicon metabarcoding sequencing of 16S, 18S, and cytochrome c oxidase I (COI) regions, indicating that it can be used to detect various prokaryotes and eukaryotes. Our results showed the effectiveness of our AirDNA sampling apparatus with a simple setup and affordable devices to obtain metagenomic DNA for short-term or long-term spatiotemporal analysis. The technique is well suited for monitoring air in built environments, especially monitoring bioaerosols for health purposes and for fine-scale spatiotemporal environmental studies.

Incidence, genetic diversity, and antimicrobial resistance profiles of Vibrio parahaemolyticus in seafood in Bangkok and eastern Thailand.

Background: Emergence of Vibrio parahaemolyticus pandemic strain O3:K6 was first documented in 1996. Since then it has been accounted for large outbreaks of diarrhea globally. In Thailand, prior studies on pandemic and non-pandemic V. parahaemolyticus had mostly been done in the south. The incidence and molecular characterization of pandemic and non-pandemic strains in other parts of Thailand have not been fully characterized. This study examined the incidence of V. parahaemolyticus in seafood samples purchased in Bangkok and collected in eastern Thailand and characterized V. parahaemolyticus isolates. Potential virulence genes, VPaI-7, T3SS2, and biofilm were examined. Antimicrobial resistance (AMR) profiles and AMR genes (ARGs) were determined. Methods: V. parahaemolyticus was isolated from 190 marketed and farmed seafood samples by a culture method and confirmed by polymerase chain reaction (PCR). The incidence of pandemic and non-pandemic V. parahaemolyticus and VPaI-7, T3SS2, and biofilm genes was examined by PCR. AMR profiles were verified by a broth microdilution technique. The presence of ARGs was verified by genome analysis. V. parahaemolyticus characterization was done by multilocus sequence typing (MLST). A phylogenomic tree was built from nucleotide sequences by UBCG2.0 and RAxML softwares. Results: All 50 V. parahaemolyticus isolates including 21 pathogenic and 29 non-pathogenic strains from 190 samples had the toxRS/old sequence, indicating non-pandemic strains. All isolates had biofilm genes (VP0950, VP0952, and VP0962). None carried T3SS2 genes (VP1346 and VP1367), while VPaI-7 gene (VP1321) was seen in two isolates. Antimicrobial susceptibility profiles obtained from 36 V. parahaemolyticus isolates revealed high frequency of resistance to colistin (100%, 36/36) and ampicillin (83%, 30/36), but susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (100%, 36/36). Multidrug resistance (MDR) was seen in 11 isolates (31%, 11/36). Genome analysis revealed ARGs including blaCARB (100%, 36/36), tet(34) (83%, 30/36), tet(35) (42%, 15/36), qnrC (6%, 2/36), dfrA6 (3%, 1/36), and blaCTX-M-55 (3%, 1/36). Phylogenomic and MLST analyses classified 36 V. parahaemolyticus isolates into 5 clades, with 12 known and 13 novel sequence types (STs), suggesting high genetic variation among the isolates. Conclusions: Although none V. parahaemolyticus strains isolated from seafood samples purchased in Bangkok and collected in eastern Thailand were pandemic strains, around one third of isolates were MDR V. parahaemolyticus strains. The presence of resistance genes of the first-line antibiotics for V. parahaemolyticus infection raises a major concern for clinical treatment outcome since these resistance genes could be highly expressed under suitable circumstances.

Temporal, compositional, and functional differences in the microbiome of Bangkok subway air environment.

With the growing numbers of the urban population, an increasing number of commuters have relied on subway systems for rapid transportation in daily life. Analyzing the temporal distribution of air microbiomes in subway environments is crucial for the assessment and monitoring of air quality in the subway system, especially with regard to public health. This study employed culture-independent metabarcode sequencing to analyze bacterial diversity and variations in bacterial compositions associated with bioaerosols collected from a subway station in Bangkok over a four-month period. The bacteria obtained were found to consist primarily of Proteobacteria, Firmicutes, and Actinobacteria, with variations at the family, genus, and species levels among samples obtained in different months. The vast majority of these bacteria are most likely derived from outside environments and human body sources. Many of the bacteria found in Bangkok subway station were also identified as "core microorganisms" of subway environments around the world, as suggested by the MetaSUB Consortium. The diversity of bacterial communities was shown to be influenced by several air quality variables, especially ambient temperature and the quantity of particulate matters, which showed positive correlations with several bacterial species such as Acinetobacter lwoffii, Staphylococcus spp., and Moraxella osloensis. In addition, metabolic profiles inferred from metabarcode-derived bacterial diversity showed significant variations across different sampling times and sites and can be used as a starting point to further explore the functional roles of specific groups of bacteria in the subway environment. This study thus introduced the information required for surveillance of microbiological impacts and their contributions to the well-being of subway commuters in Bangkok.

Beneficial bacterial-Auricularia cornea interactions fostering growth enhancement identified from microbiota present in spent mushroom substrate.

Complex dynamic bacterial-fungal interactions play key roles during mushroom growth, ranging from mutualism to antagonism. These interactions convey a large influence on mushroom's mycelial and fruiting body formation during mushroom cultivation. In this study, high-throughput amplicon sequencing was conducted to investigate the structure of bacterial communities in spent mushroom substrates obtained from cultivation of two different groups of Auricularia cornea with (A) high yield and (B) low yield of fruiting body production. It was found that species richness and diversity of microbiota in group (A) samples were significantly higher than in group (B) samples. Among the identified 765 bacterial OTUs, 5 bacterial species found to exhibit high differential abundance between group (A) and group (B) were Pseudonocardia mangrovi, Luteimonas composti, Paracoccus pantotrophus, Sphingobium jiangsuense, and Microvirga massiliensis. The co-cultivation with selected bacterial strains showed that A. cornea TBRC 12900 co-cultivated with P. mangrovi TBRC-BCC 42794 promoted a high level of mycelial growth. Proteomics analysis was performed to elucidate the biological activities involved in the mutualistic association between A. cornea TBRC 12900 and P. mangrovi TBRC-BCC 42794. After co-cultivation of A. cornea TBRC 12900 and P. mangrovi TBRC-BCC 42794, 1,616 proteins were detected including 578 proteins of A. cornea origin and 1,038 proteins of P. mangrovi origin. Functional analysis and PPI network construction revealed that the high level of mycelial growth in the co-culture condition most likely resulted from concerted actions of (a) carbohydrate-active enzymes including hydrolases, glycosyltransferases, and carbohydrate esterases important for carbohydrate metabolism and cell wall generation/remodeling, (b) peptidases including cysteine-, metallo-, and serine-peptidases, (c) transporters including the ABC-type transporter superfamily, the FAT transporter family, and the VGP family, and (d) proteins with proposed roles in formation of metabolites that can act as growth-promoting molecules or those normally contain antimicrobial activity (e.g., indoles, terpenes, ß-lactones, lanthipeptides, iturins, and ectoines). The findings will provide novel insights into bacterial-fungal interactions during mycelial growth and fruiting body formation. Our results can be utilized for the selection of growth-promoting bacteria to improve the cultivation process of A. cornea with a high production yield, thus conveying potentially high socio-economic impact to mushroom agriculture.

Iron homeostasis in the absence of ferricrocin and its consequences in fungal development and insect virulence in Beauveria bassiana.

The putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.

Identification of proteins responsive to heterologous protein production in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label-free comparative proteomic approach. Four hundred sixty-four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%-20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%-25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.

Uncovering multi-faceted taxonomic and functional diversity of soil bacteriomes in tropical Southeast Asian countries.

Environmental microbiomes encompass massive biodiversity and genetic information with a wide-ranging potential for industrial and agricultural applications. Knowledge of the relationship between microbiomes and environmental factors is crucial for translating that information into practical uses. In this study, the integrated data of Southeast Asian soil bacteriomes were used as models to assess the variation in taxonomic and functional diversity of bacterial communities. Our results demonstrated that there were differences in soil bacteriomes across different geographic locality with different soil characteristics: soil class and pH level. Such differences were observed in taxonomic diversity, interspecific association patterns, and functional diversity of soil bacteriomes. The bacterial-mediated biogeochemical cycles of nitrogen, sulfur, carbon, and phosphorus illustrated the functional relationship of soil bacteriome and soil characteristics, as well as an influence from bacterial interspecific interaction. The insights from this study reveal the importance of microbiome data integration for future microbiome research.

The Fungus Metarhizium sp. BCC 4849 Is an Effective and Safe Mycoinsecticide for the Management of Spider Mites and Other Insect Pests.

Five isolates of Metarhizium sp. were evaluated for their pathogenicity against the spider mite (Tetranychus truncatus Ehara) (Acari: Tetranychidae) and Metarhizium sp. BCC 4849 resulted in the highest mortality (82%) on the 5th day post-inoculation (DPI). Subsequent insect bioassay data indicated similar high virulence against five other insects: African red mites (Eutetranychus africanus Tucker) (Acari: Tetranychidae), bean aphid (Aphis craccivora Koch) (Hemiptera: Aphididae), cassava mealybug (Phenacoccus manihoti Matile-Ferrero) (Hemiptera: Pseudococcidae), sweet potato weevil (Cylas formicarius Fabricius) (Coleoptera: Brentidae), and oriental fruit fly (Bactrocera dorsalis Hendel) (Diptera: Tephritidae), at mortalities of 92-99%, on 3rd-6th DPI, and in laboratory conditions. The pathogenicity assay against E. africanus in hemp plants under greenhouse conditions indicated 85-100% insect mortality on 10th DPI using the fungus alone or in combination with synthetic acaricide. Genome sequencing of Metarhizium sp. BCC 4849 revealed the high abundance of proteins associated with zinc-, heme-, and iron-binding; oxidation-reduction; and transmembrane transport, implicating its versatile mode of interaction with the environment and adaptation to various ion homeostasis. The light and scanning electron microscopy indicated that at 24 h post inoculation (PI), adhesion and appressorial formation occurred, notably near the setae. Most infected mites had stopped moving and started dying by 48-72 h PI. Elongated hyphal bodies and oval blastospores were detected in the legs. At 96-120 h PI or longer, dense mycelia and conidial mass had colonized the interior and exterior of dead mites, primarily at the bottom than the upper part. The shelf-life study also indicated that conidial formulation combined with an oxygen-moisture absorber markedly enhanced the viability and germination after storage at 35 °C for four months. The fungus was tested as safe for humans and animals, according to our toxicological assays.

gcType: a high-quality type strain genome database for microbial phylogenetic and functional research.

Taxonomic and functional research of microorganisms has increasingly relied upon genome-based data and methods. As the depository of the Global Catalogue of Microorganisms (GCM) 10K prokaryotic type strain sequencing project, Global Catalogue of Type Strain (gcType) has published 1049 type strain genomes sequenced by the GCM 10K project which are preserved in global culture collections with a valid published status. Additionally, the information provided through gcType includes >12 000 publicly available type strain genome sequences from GenBank incorporated using quality control criteria and standard data annotation pipelines to form a high-quality reference database. This database integrates type strain sequences with their phenotypic information to facilitate phenotypic and genotypic analyses. Multiple formats of cross-genome searches and interactive interfaces have allowed extensive exploration of the database's resources. In this study, we describe web-based data analysis pipelines for genomic analyses and genome-based taxonomy, which could serve as a one-stop platform for the identification of prokaryotic species. The number of type strain genomes that are published will continue to increase as the GCM 10K project increases its collaboration with culture collections worldwide. Data of this project is shared with the International Nucleotide Sequence Database Collaboration. Access to gcType is free at http://gctype.wdcm.org/.

Mating-type switching and mating-type gene array expression in the methylotrophic yeast Ogataea thermomethanolica TBRC656.

The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.

A novel sucrose-based expression system for heterologous proteins expression in thermotolerant methylotrophic yeast Ogataea thermomethanolica.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein expression. In this study, a novel expression system was developed for O. thermomethanolica based on the maltase (mal) gene promoter from this organism. The OtMal promoter function was tested for expression of fungal enzymes as reporter genes. Measurement of xylanase reporter enzyme activity showed that the OtMal promoter was repressed during growth on glucose and was activated by sucrose. When sucrose was used as a carbon source, the OtMal promoter was approximately twice as strong as the constitutive OtGAP promoter. Comparison of the OtMal promoter with the methanol-inducible OtAOX promoter showed that OtMal promoter drove 1.2 and 1.7-fold higher expression of xylanase and phytase reporter, respectively, than OtAOX promoter under inducing conditions at 24 h. Our results indicated that this novel expression system could be useful for the production of heterologous proteins from sucrose in yeast O. thermomethanolica.

The global catalogue of microorganisms 10K type strain sequencing project: closing the genomic gaps for the validly published prokaryotic and fungi species.

Genomic information is essential for taxonomic, phylogenetic, and functional studies to comprehensively decipher the characteristics of microorganisms, to explore microbiomes through metagenomics, and to answer fundamental questions of nature and human life. However, large gaps remain in the available genomic sequencing information published for bacterial and archaeal species, and the gaps are even larger for fungal type strains. The Global Catalogue of Microorganisms (GCM) leads an internationally coordinated effort to sequence type strains and close gaps in the genomic maps of microorganisms. Hence, the GCM aims to promote research by deep-mining genomic data.

CRISPR-Cas9 enabled targeted mutagenesis in the thermotolerant methylotrophic yeast Ogataea thermomethanolica.

Ogataea thermomethanolica TBRC656 is a thermotolerant methylotrophic yeast suitable for heterologous protein expression at various temperatures. However, the lack of efficient methods for targeted gene mutagenesis limits strain engineering in this yeast. In this study, we applied a CRISPR-Cas9-based tool for targeted gene mutagenesis in O. thermomethanolica. The putative unfolded protein response regulator OtHAC1, and the OtMAL1 (maltase) and OtMAL2 (maltose permease) genes involved with sucrose and maltose utilization were targeted for CRISPR-Cas9 mutagenesis. Plasmids were constructed for integrative and episomal expression of CRISPR-Cas9 elements in O. thermomethanolica in which Cas9 and gRNA are transcribed from the alcohol oxidase (AOX) promoter. The expression of these genome-editing elements is controlled by derepression with glycerol and gRNA are flanked by self-cleaving ribozymes. For integrative system, OtHAC1, OtMAL1 and OtMAL2 were disrupted at 63%, 97% and 93%, respectively. In addition, OtMAL1 was also disrupted with episomal system at 92%. These findings indicate that the CRISPR-Cas9 system described herein is thus applicable for studying gene function and strain engineering in yeast O. thermomethanolica.

Transcriptome-based discovery of pathways and genes related to reproduction of the black tiger shrimp (Penaeus monodon).

The black tiger shrimp (Penaeus monodon) is an aquatic animal with considerable economic importance. Poor reproductive maturation in captivity impedes sustainable aquaculture production of this species. This study aims to provide transcriptomic information on reproductive organs using 454 pyrosequencing technology. The transcriptome analysis of ovaries and testes revealed 41,136 transcripts with 20,192 contigs. We found novel sets of transcripts completing several important reproductive pathways such as gonadotropin-releasing hormone (GnRH) signaling and progesterone-mediated oocyte maturation. In addition, we found transcripts encoding for receptors crucial for initiation of the maturation process, such as GnRH receptor (GnRHR), voltage-dependent calcium channel L type alpha-1C (CACNA1C) and epidermal growth factor receptor (EGFR). Moreover, we found a putative novel vigillin encoding for an estrogen-induced polysome-associated protein, which has not been reported in penaeid shrimp. These results suggest that the regulatory mechanism of the pathways important to reproductive maturation might be similar to those in the vertebrate. The obtained data will consequently accelerate the study of reproductive biology of this important species to ensure a sustainable shrimp farming industry.