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).

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.

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.

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/.

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.

World data centre for microorganisms: an information infrastructure to explore and utilize preserved microbial strains worldwide.

The World Data Centre for Microorganisms (WDCM) was established 50 years ago as the data center of the World Federation for Culture Collections (WFCC)-Microbial Resource Center (MIRCEN). WDCM aims to provide integrated information services using big data technology for microbial resource centers and microbiologists all over the world. Here, we provide an overview of WDCM including all of its integrated services. Culture Collections Information Worldwide (CCINFO) provides metadata information on 708 culture collections from 72 countries and regions. Global Catalogue of Microorganism (GCM) gathers strain catalogue information and provides a data retrieval, analysis, and visualization system of microbial resources. Currently, GCM includes >368 000 strains from 103 culture collections in 43 countries and regions. Analyzer of Bioresource Citation (ABC) is a data mining tool extracting strain related publications, patents, nucleotide sequences and genome information from public data sources to form a knowledge base. Reference Strain Catalogue (RSC) maintains a database of strains listed in International Standards Organization (ISO) and other international or regional standards. RSC allocates a unique identifier to strains recommended for use in diagnosis and quality control, and hence serves as a valuable cross-platform reference. WDCM provides free access to all these services at www.wdcm.org.

Insights from the genome of Ophiocordyceps polyrhachis-furcata to pathogenicity and host specificity in insect fungi.

BACKGROUND: Ophiocordyceps unilateralis is an outstanding insect fungus for its biology to manipulate host ants' behavior and for its extreme host-specificity. Through the sequencing and annotation of Ophiocordyceps polyrhachis-furcata, a species in the O. unilateralis species complex specific to the ant Polyrhachis furcata, comparative analyses on genes involved in pathogenicity and virulence between this fungus and other fungi were undertaken in order to gain insights into its biology and the emergence of host specificity. RESULTS: O. polyrhachis-furcata possesses various genes implicated in pathogenicity and virulence common with other fungi. Overall, this fungus possesses protein-coding genes similar to those found on other insect fungi with available genomic resources (Beauveria bassiana, Metarhizium robertsii (formerly classified as M. anisopliae s.l.), Metarhizium acridum, Cordyceps militaris, Ophiocordyceps sinensis). Comparative analyses in regard of the host ranges of insect fungi showed a tendency toward contractions of various gene families for narrow host-range species, including cuticle-degrading genes (proteases, carbohydrate esterases) and some families of pathogen-host interaction (PHI) genes. For many families of genes, O. polyrhachis-furcata had the least number of genes found; some genes commonly found in other insect fungi are even absent (e.g. Class 1 hydrophobin). However, there are expansions of genes involved in 1) the production of bacterial-like toxins in O. polyrhachis-furcata, compared with other entomopathogenic fungi, and 2) retrotransposable elements. CONCLUSIONS: The gain and loss of gene families helps us understand how fungal pathogenicity in insect hosts evolved. The loss of various genes involved throughout the pathogenesis for O. unilateralis would result in a reduced capacity to exploit larger ranges of hosts and therefore in the different level of host specificity, while the expansions of other gene families suggest an adaptation to particular environments with unexpected strategies like oral toxicity, through the production of bacterial-like toxins, or sophisticated mechanisms underlying pathogenicity through retrotransposons.

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.

Global catalogue of microorganisms (gcm): a comprehensive database and information retrieval, analysis, and visualization system for microbial resources.

BACKGROUND: Throughout the long history of industrial and academic research, many microbes have been isolated, characterized and preserved (whenever possible) in culture collections. With the steady accumulation in observational data of biodiversity as well as microbial sequencing data, bio-resource centers have to function as data and information repositories to serve academia, industry, and regulators on behalf of and for the general public. Hence, the World Data Centre for Microorganisms (WDCM) started to take its responsibility for constructing an effective information environment that would promote and sustain microbial research data activities, and bridge the gaps currently present within and outside the microbiology communities. DESCRIPTION: Strain catalogue information was collected from collections by online submission. We developed tools for automatic extraction of strain numbers and species names from various sources, including Genbank, Pubmed, and SwissProt. These new tools connect strain catalogue information with the corresponding nucleotide and protein sequences, as well as to genome sequence and references citing a particular strain. All information has been processed and compiled in order to create a comprehensive database of microbial resources, and was named Global Catalogue of Microorganisms (GCM). The current version of GCM contains information of over 273,933 strains, which includes 43,436 bacterial, fungal and archaea species from 52 collections in 25 countries and regions.A number of online analysis and statistical tools have been integrated, together with advanced search functions, which should greatly facilitate the exploration of the content of GCM. CONCLUSION: A comprehensive dynamic database of microbial resources has been created, which unveils the resources preserved in culture collections especially for those whose informatics infrastructures are still under development, which should foster cumulative research, facilitating the activities of microbiologists world-wide, who work in both public and industrial research centres. This database is available from http://gcm.wfcc.info.

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.

ChemEx: information extraction system for chemical data curation.

BACKGROUND: Manual chemical data curation from publications is error-prone, time consuming, and hard to maintain up-to-date data sets. Automatic information extraction can be used as a tool to reduce these problems. Since chemical structures usually described in images, information extraction needs to combine structure image recognition and text mining together. RESULTS: We have developed ChemEx, a chemical information extraction system. ChemEx processes both text and images in publications. Text annotator is able to extract compound, organism, and assay entities from text content while structure image recognition enables translation of chemical raster images to machine readable format. A user can view annotated text along with summarized information of compounds, organism that produces those compounds, and assay tests. CONCLUSIONS: ChemEx facilitates and speeds up chemical data curation by extracting compounds, organisms, and assays from a large collection of publications. The software and corpus can be downloaded from http://www.biotec.or.th/isl/ChemEx.

A computational tool for the design of live attenuated virus vaccine based on microRNA-mediated gene silencing.

BACKGROUND: The microRNA-based gene-silencing machinery has been recognized as a promising approach to control viral replication and used for improving safety for the live attenuated virus vaccines. The effective host microRNA response elements (MREs) have been incorporated into a virus sequence mainly based on the experimental trials for identifying both microRNA binding sites and effective mutations. The design of MREs for viral genomes or with multiple host microRNAs of interest, then, will be time and cost consuming. RESULTS: In this paper, we introduced a computational flow that could be used to design MREs of human microRNAs within Influenza A H1N1 virus gene segments. The main steps of the flow includes locating possible binding sites; MREs, of human microRNAs within the viral sequences using a miRNA target prediction tool (miranda), performing various mutations among mismatched binding positions, calculating the binding energy, score, identity, and the effects of changed physical properties of amino acids according to the changed bases in RNA level, and prioritizing the mutated binding sites. The top ranked MREs of human microRNA hsa-miR-93 is consistent with previous literature while other results waited to be experimentally verified. To make the computational flow easily accessible by virologists, we also developed MicroLive, a web server version of the MRE design flow together with the database of miranda-predicted MREs within gene sequences of seven RNA viruses including Influenza A, dengue, hepatitis C, measles, mumps, poliovirus, and rabies. Users may design MREs of specific human microRNAs for their input viral sequences using MRE design tool or optimize the miranda-predicted MREs of seven viruses available on the system. Also, users could design varied number of MREs for multiple human microRNAs to modulate the degree of live vaccine attenuation and reduce the likelihood of escape mutants. CONCLUSIONS: The computational design of MREs helps reduce time and cost for experimental trials. While the flow was demonstrated using human microRNAs and Influenza A H1N1 virus, it could be flexibly applied to other hosts (e.g., animals) and viruses of interest for constructing host-specific live attenuated vaccines. Also, it could be deployed for engineering tissue-specific oncolytic viruses in cancer virotherapeutics. The MicroLive web server is freely accessible at http://www.biotec.or.th/isl/microlive.

C-mii: a tool for plant miRNA and target identification.

BACKGROUND: MicroRNAs (miRNAs) have been known to play an important role in several biological processes in both animals and plants. Although several tools for miRNA and target identification are available, the number of tools tailored towards plants is limited, and those that are available have specific functionality, lack graphical user interfaces, and restrict the number of input sequences. Large-scale computational identifications of miRNAs and/or targets of several plants have been also reported. Their methods, however, are only described as flow diagrams, which require programming skills and the understanding of input and output of the connected programs to reproduce. RESULTS: To overcome these limitations and programming complexities, we proposed C-mii as a ready-made software package for both plant miRNA and target identification. C-mii was designed and implemented based on established computational steps and criteria derived from previous literature with the following distinguishing features. First, software is easy to install with all-in-one programs and packaged databases. Second, it comes with graphical user interfaces (GUIs) for ease of use. Users can identify plant miRNAs and targets via step-by-step execution, explore the detailed results from each step, filter the results according to proposed constraints in plant miRNA and target biogenesis, and export sequences and structures of interest. Third, it supplies bird's eye views of the identification results with infographics and grouping information. Fourth, in terms of functionality, it extends the standard computational steps of miRNA target identification with miRNA-target folding and GO annotation. Fifth, it provides helper functions for the update of pre-installed databases and automatic recovery. Finally, it supports multi-project and multi-thread management. CONCLUSIONS: C-mii constitutes the first complete software package with graphical user interfaces enabling computational identification of both plant miRNA genes and miRNA targets. With the provided functionalities, it can help accelerate the study of plant miRNAs and targets, especially for small and medium plant molecular labs without bioinformaticians. C-mii is freely available at http://www.biotec.or.th/isl/c-mii for both Windows and Ubuntu Linux platforms.

LinkinPath: from sequence to interconnected pathway.

SUMMARY: LinkinPath is a pathway mapping and analysis tool that enables users to explore and visualize the list of gene/protein sequences through various Flash-driven interactive web interfaces including KEGG pathway maps, functional composition maps (TreeMaps), molecular interaction/reaction networks and pathway-to-pathway networks. Users can submit single or multiple datasets of gene/protein sequences to LinkinPath to (i) determine the co-occurrence and co-absence of genes/proteins on animated KEGG pathway maps; (ii) compare functional compositions within and among the datasets using TreeMaps; (iii) analyze the statistically enriched pathways across the datasets; (iv) build the pathway-to-pathway networks for each dataset; (v) explore potential interaction/reaction paths between pathways; and (vi) identify common pathway-to-pathway networks across the datasets. AVAILABILITY: LinkinPath is freely available to all interested users at http://www.biotec.or.th/isl/linkinpath/.

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.

d-Omix: a mixer of generic protein domain analysis tools.

Domain combination provides important clues to the roles of protein domains in protein function, interaction and evolution. We have developed a web server d-Omix (a Mixer of Protein Domain Analysis Tools) aiming as a unified platform to analyze, compare and visualize protein data sets in various aspects of protein domain combinations. With InterProScan files for protein sets of interest provided by users, the server incorporates four services for domain analyses. First, it constructs protein phylogenetic tree based on a distance matrix calculated from protein domain architectures (DAs), allowing the comparison with a sequence-based tree. Second, it calculates and visualizes the versatility, abundance and co-presence of protein domains via a domain graph. Third, it compares the similarity of proteins based on DA alignment. Fourth, it builds a putative protein network derived from domain-domain interactions from DOMINE. Users may select a variety of input data files and flexibly choose domain search tools (e.g. hmmpfam, superfamily) for a specific analysis. Results from the d-Omix could be interactively explored and exported into various formats such as SVG, JPG, BMP and CSV. Users with only protein sequences could prepare an InterProScan file using a service provided by the server as well. The d-Omix web server is freely available at http://www.biotec.or.th/isl/Domix.