Sequence Similarity Analysis Research Articles

A unique view of SARS-CoV-2 through the lens of ORF8 protein

Immune evasion is one of the unique characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attributed to its ORF8 protein. This protein modulates the adaptive host immunity through down-regulation of MHC-1 (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the host's interferon-mediated antiviral response. To understand the host's immune perspective in reference to the ORF8 protein, a comprehensive study of the ORF8 protein and mutations possessed by it have been performed. Chemical and structural properties of ORF8 proteins from different hosts, such as human, bat, and pangolin, suggest that the ORF8 of SARS-CoV-2 is much closer to ORF8 of Bat RaTG13-CoV than to that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 in SARS-CoV-2 can be grouped into four classes based on their predicted effects (Hussain et al., 2021) [1]. Based on the geo-locations and timescale of sample collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were found upon sequence similarity analyses and consideration of the amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of the rapidly evolving SARS-CoV-2 through the ORF8.

Ten Years of Collaborative Progress in the Quest for Orthologs.

Accurate determination of the evolutionary relationships between genes is a foundational challenge in biology. Homology—evolutionary relatedness—is in many cases readily determined based on sequence similarity analysis. By contrast, whether or not two genes directly descended from a common ancestor by a speciation event (orthologs) or duplication event (paralogs) is more challenging, yet provides critical information on the history of a gene. Since 2009, this task has been the focus of the Quest for Orthologs (QFO) Consortium. The sixth QFO meeting took place in Okazaki, Japan in conjunction with the 67th National Institute for Basic Biology conference. Here, we report recent advances, applications, and oncoming challenges that were discussed during the conference. Steady progress has been made toward standardization and scalability of new and existing tools. A feature of the conference was the presentation of a panel of accessible tools for phylogenetic profiling and several developments to bring orthology beyond the gene unit—from domains to networks. This meeting brought into light several challenges to come: leveraging orthology computations to get the most of the incoming avalanche of genomic data, integrating orthology from domain to biological network levels, building better gene models, and adapting orthology approaches to the broad evolutionary and genomic diversity recognized in different forms of life and viruses.

Records of three mammal tick species parasitizing an atypical host, the multi-ocellated racerunner lizard, in arid regions of Xinjiang, China

BackgroundTicks are ectoparasites that feed on blood of a broad taxonomical range of terrestrial and flying vertebrates and are distributed across a wide range of environmental settings. To date, the species identity, diversity, and relationships among the ticks on lizards in China have been poorly understood.MethodsIn this study, 30 ticks, collected from the multi-ocellated racerunner (Eremias multiocellata) lizard in the Tarim Basin and adjacent Yanqi Basin of the Xinjiang Uygur Autonomous Region in China, were identified by morphological observation and confirmed by DNA-based techniques. The mitochondrially encoded 12S rRNA, 16S rRNA, and COI gene fragments of ticks were amplified and sequenced. To understand the genetic polymorphisms, 47 ticks collected from hedgehogs and 1 from brushwood in the Tarim Basin were also included. Species identification was based on both morphological and molecular characters. The median-joining network approach was used to evaluate the intraspecific genealogies of the ticks and their relatedness with the geographical origin or hosts.ResultsThe sequence similarity analysis confirmed that the 30 ticks belong to three genera and three species including 11 individuals of Hyalomma asiaticum, 3 of Rhipicephalus turanicus, and 16 of Haemaphysalis sulcata. A network approach revealed paraphyletic populations of R. turanicus and Hy. asiaticum at the intraspecies level regarding geographical origin and low host specificity. For R. turanicus and Hy. asiaticum, common ancestry was observed between COI sequences from lizards and other sequence types from different hosts and countries.ConclusionsTo our knowledge, our study is the first to conduct a molecular survey of ticks from lizards in the arid regions of Xinjiang, China. Eremias multiocellata is an atypical host of the three tick species. Notably, two species of ticks, Hy. asiaticum and R. turanicus, have been collected and identified from lizards in China for the first time. Star-like networks suggest both of them might have experienced recent population expansion. The discoveries are closely related to the geographical environments in Xinjiang and will provide information for the control of ticks and tick-borne pathogens in Northwest China.Graphical

Efficient Multiple Sequences Alignment Algorithm Generation via Components Assembly Under PAR Framework.

As a key algorithm in bioinformatics, sequence alignment algorithm is widely used in sequence similarity analysis and genome sequence database search. Existing research focuses mainly on the specific steps of the algorithm or is for specific problems, lack of high-level abstract domain algorithm framework. Multiple sequence alignment algorithms are more complex, redundant, and difficult to understand, and it is not easy for users to select the appropriate algorithm; some computing errors may occur. Based on our constructed pairwise sequence alignment algorithm component library and the convenient software platform PAR, a few expansion domain components are developed for multiple sequence alignment application domain, and specific multiple sequence alignment algorithm can be designed, and its corresponding program, i.e., C++/Java/Python program, can be generated efficiently and thus enables the improvement of the development efficiency of complex algorithms, as well as accuracy of sequence alignment calculation. A star alignment algorithm is designed and generated to demonstrate the development process.

Fluorescent thermal shift-based method for detection of NF-\u03baB binding to double-stranded DNA

The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

Paenibacillus silvestris sp. nov., Isolated from Forest Soil.

A bacterial strain, designated 5J-6T, was isolated from soil in Cheongnyeongpo, Republic of Korea. Cells were Gram-stain-positive, strictly aerobic, and motile rods and their catalase and oxidase activities were positive. Strain 5J-6T grew at 10-30°C, pH 6.0-9.0, and 0-0.8% (w/v) NaCl concentration, with optimum growth at 25°C, pH 6.5, and 0.4% NaCl concentration. Anteiso-C15:0 and iso-C16:0 were detected as the predominant fatty acids and menaquinone-7 was the sole isoprenoid quinone detected. Strain 5J-6T contained phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid as major polar lipids. The peptidoglycan belonged to the type A1γ meso-diaminopimelic acid. The G+C content of the genomic DNA calculated from the whole genomic sequence was 46.1mol%. Phylogenetic analysis of strain 5J-6T based on 16S rRNA gene sequences placed the isolate into a member of the genus Paenibacillus. Sequence similarity analysis of 16S rRNA gene sequences revealed that strain 5J-6T was most closely related to Paenibacillus aceris KUDC4121T and Paenibacillus chondroitinus DSM 5051T with 98.76% and 98.42% similarities, respectively. Average nucleotide identity and in silico DNA-DNA hybridization values between strain 5J-6T and the type strain of P. aceris were 83.97% and 28.60%, respectively. Based on the phylogenetic and phenotypic characteristics and genomic data, strain 5J-6T could be considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus silvestris sp. nov. is proposed. The type strain is 5J-6T (= KACC 21430T = JCM 33812T).

Full-length genome characterization of a new fusagravirus hosted by the spring orange peel fungus Caloscypha fulgens.

Mycoviruses widely exist in diverse lineages of fungi, yet there are only a few studies on mycovirus infection in uncultivated fungi. We here report the presence of a dsRNA mycovirus in saprotrophic spring orange peel fungus Caloscypha fulgens. A novel dsRNA virus, named "Caloscypha fulgens fusagravirus 1" (CfFV1), was isolated from a single ascocarp of C. fulgens, and its molecular features were revealed. The full-length cDNA of CfFV1 comprises 9,548 nucleotides with a calculated GC content of 47.9% and two discontinuous open reading frames (ORF 1 and 2). A-1 ribosomal frameshift region with two distinctive elements, including a canonical slippery heptanucleotide (AAAAAAC) and a pseudoknot structure, predicted as a Recoding Stimulatory Element, was detected in the junction region of ORF1 and ORF2. The deduced amino acid sequence of ORF1 and ORF2 showed the highest similarity to the putative structural protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 4 (RnFGV4). Genome organization, sequence similarity, and phylogenetic analysis indicate that this virus belongs to a new member of the proposed family Fusagraviridae. This is the first report of the presence of a mycovirus in the spring orange peel fungus C. fulgens. Keywords: mycovirus; dsRNA; proposed Fusagraviridae; uncultivated fungi; Caloscypha fulgens.

Investigation of data mining technique and artificial intelligence algorithm in microflora bioinformatics

Bioinformatics has gradually received widespread attention and has shown the characteristics of a large amount of calculation and high complexity. Therefore, it is required to adopt computer algorithms in bioinformatics to improve the efficiency of bioinformatics processing problems. Big data and artificial intelligence technologies have the characteristics of supporting bioinformatics and have achieved certain results in the field of bioinformatics. Introduced the application basis of big data and artificial intelligence in bioinformatics, analyzed data collection, preprocessing, data storage and management, data analysis, and mining technology. Furthermore, typical applications in bioinformatics are discussed in terms of gene expression data analysis, genome sequence information analysis, biological sequence difference and similarity analysis, genetic data analysis, and protein structure and function prediction. Finally, the bottlenecks and challenges in the application of big data and artificial intelligence in bioinformatics are discussed, and the application prospects of related technologies in bioinformatics have prospected.

Identification, sequence characteristics and expression analyses of four spore wall protein genes of <italic>Enterocytozoon hepatopenaei</italic> (EHP) in <italic>Litopenaeus vannamei</italic>

<italic>Enterocytozoon hepatopenaei</italic> (EHP) is a newly emerged Microsporidian parasite that causes retarded growth of shrimp, which severely affects the normal development of shrimp industry. By now the mechanism that how this pathogen infects the shrimp has remained unclear, which severely impairs the effectiveness of the prevention and control of EHP. Some spore wall proteins (SWPs) of Microsporidia, as the major structure components, appear to be involved in the invasions in certain hosts. Therefore, identification and characterization of EHP SWPs are of great significance to the study of pathogenic biology and infection mechanism of EHP mediated by SWPs, which would facilitate the control of this pathogen. However, the reports on the EHP SWPs are few, and the potential functions of these molecules are largely unknown. In order to understand the biological characteristics and infection mechanism of local EHP in Shanghai, farmed shrimp (<italic>Litopenaeus vannamei</italic>) in the suburban farm of Shanghai confirmed to be infected by EHP were sampled to the following assays. The data from transcriptome sequencing with the hepatopancreas of EHP-infected shrimp revealed that four putative SWP-like protein genes were found and they were designated as <italic>EhEnP</italic>1, <italic>EhSWP</italic>7, <italic>EhSWP</italic>12, and <italic>EhSWP</italic>26. Sequence similarity analyses revealed that the amino acid sequences of these four molecules were identical with the ones reported in Thailand, which were obtained from the data of the genome sequence of EHP. Then the corresponding DNA sequences of these four SWP genes were cloned and sequenced. The results showed that the cDNA sequences of four SWP genes contained non-coding sequences which were the same as their respective DNA sequences. These results revealed that these four SWP genes had no introns. We also found that only <italic>Eh</italic>SWP7 of these four proteins had a putative signal peptide and a functional domain MICSWaP was found in <italic>Eh</italic>SWP26. Quantitative real-time PCR (qRT-PCR) was performed to analyze the expression levels. All four SWP genes expressed the highest in hepatopancreas, moderately in intestine and stomach but very low in gills. We further compared the four gene expression levels in hepatopancreas and found that the transcripts of <italic>EhSWP</italic>12 was significantly higher than other three genes, implying that <italic>Eh</italic>SWP12 might be a major structure protein in the spore wall. This study provides new data on the study of EHP pathogenic biology and its invading mechanisms.

Molecular Detection and Characterization of Carbapenemases Among Carbapenem-Resistant Escherichia Coli And Klebsiella Pneumoniae Isolated from Urine

Objective: Carbapenem resistance mediated by carbapenemases poses a significant public health threat due to its transferable nature, unlike other carbapenem resistance mechanisms. This study aimed to molecularly detect and characterize carbapenemases among carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolated from urine samples. Methods: A total of 123 non-duplicate isolates (70 E. coli and 53 K. pneumoniae) were screened for carbapenem resistance using Clinical Laboratory Standard Institute guidelines. Carbapenem-resistant isolates were further tested for the presence of carbapenemase genes (blaKPC, blaOXA, blaNDM) using PCR. Positive PCR products were sequenced, and sequence similarity was analyzed using nucleotide BLAST. Multiple sequence alignment was performed using ClustalW and BioEdit. Results: Among the 123 isolates screened, 6 (4.88%) were carbapenem-resistant, including 2 (2.86%) E. coli and 4 (7.55%) K. pneumoniae isolates. Carbapenemase genes were detected in five out of the six carbapenem-resistant isolates. The most frequently detected carbapenemase gene was blaOXA (57.14%), followed by blaNDM (42.86%). No blaKPC gene was detected. Co-harboring of blaNDM and blaOXA genes was observed in two isolates. Sequence similarity analysis showed 98–100% identity with carbapenemase genes from GenBank. Nucleotide substitutions were absent in blaNDM gene sequences, while nucleotide substitutions leading to corresponding amino acid changes were observed in blaOXA gene sequences at various positions. Conclusion: Carbapenem resistance in the studied isolates was predominantly mediated by OXA and NDM carbapenemases. These findings underscore the importance of monitoring carbapenemase spread among Gram-negative bacteria to mitigate the emergence and dissemination of carbapenem-resistant strains, which jeopardize the efficacy of carbapenems as last-resort antibiotics.

Application of Graph Theory in DNA similarity analysis of Evolutionary Closed Species.

DNA is a complex molecule that consists of biological information that is passed down from generation to generation. With the evolution over time, there are different kinds of species that evolved from a common ancestor because of the occurrence of DNA sequence rearrangements. DNA sequence similarity analysis is a major challenge since the number of sequences is rapidly increasing in the DNA database. In this research, we based a mathematical method to analyze the similarity of two DNA sequences using Graph Theory. This mathematical method started by modeling a weighted directed graph for each DNA sequence, constructing its adjacency matrix, and converting it to the representative vector for each graph. From these vectors, the similarity was determined by distance measurements such as Euclidean, Cosine, and Correlation. By keeping this method as the based method, we will check whether it is applicable for any DNA fragments in considered genomes and molecular similarity coefficients can be used as distance measurements. We will obtain similarities using the graph spectrum instead of the representative vector. Then we will compare the results from the representative vector and that of the graph spectrum. The modified method is tested by using the mitochondrial DNA of Human, Gorilla, and Orangutan. It gives the same result when the number of nucleotides in DNA fragments is increased.

Molecular Cloning and Expression Analysis of the Endogenous Cellulase Gene MaCel1 in Monochamus alternatus

The purpose of this study was to characterize the endogenous cellulase gene MaCel1 of Monochamus alternatus, which is an important vector of Bursaphelenchus xylophilus, a pine wood nematode, which causes pine wilt disease (PWD). In this study, MaCel1 was cloned by rapid amplification of cDNA end (RACE), and its expression analyzed by RT-qPCR (real-time quantitative PCR detecting). A total of 1778 bp of cDNA was obtained. The encoding region of this gene was 1509 bp in length, encoding a protein containing 502 amino acids with a molecular weight of 58.66 kDa, and the isoelectric point of 5.46. Sequence similarity analysis showed that the amino acids sequence of MaCel1 had high similarity with the β-Glucosinolate of Anoplophoraglabripennis and slightly lower similarity with other insect cellulase genes (GH1). The β-D-Glucosidase activity of MaCel1 was 256.02 ± 43.14 U/L with no β-Glucosinolate activity. MaCel1 gene was widely expressed in the intestine of M. alternatus. The expression level of MaCel1 gene in male (3.46) and female (3.51) adults was significantly higher than that in other developmental stages, and the lowest was in pupal stage (0.15). The results will help reveal the digestive mechanism of M. alternatus and lay the foundation for controlling PWD by controlling M. alternatus.

Antimicrobial activity of Streptomyces spp. isolated from Apis dorsata combs against some phytopathogenic bacteria.

The aim of this study was to investigate the antimicrobial potential of actinomycetes isolated from combs of the giant honey bee, Apis dorsata. In total, 25 isolates were obtained from three different media and were screened for antimicrobial activity against four plant pathogenic bacteria (Ralstonia solanacearum, Xanthomonas campestris pv. campestris, Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum). Following screening using a cross-streaking method, three isolates showed the potential to inhibit the growth of plant pathogenic bacteria. Based on a 96-well microtiter assay, the crude extract of DSC3-6 had minimum inhibitory concentration (MIC) values against X. oryzae pv. oryzae, X. campestris pv. campestris, R. solanacearum and P. carotovorum of 16, 32, 32 and 64 mg L−1, respectively. The crude extract of DGA3-20 had MIC values against X. oryzae pv. oryzae, X. campestris pv. campestris, R. solanacearum and P. carotovorum of 32, 32, 32 and 64 mg L−1, respectively. The crude extract of DGA8-3 at 32 mgL−1 inhibited the growth of X. oryzae pv. oryzae, X. campestris pv. campestris, R. solanacearum and P. carotovorum. Based on their 16S rRNA gene sequences, all isolates were identified as members of the genus Streptomyces. The analysis of 16S rRNA gene sequence similarity and of the phylogenetic tree based on the maximum likelihood algorithm showed that isolates DSC3-6, DGA3-20 and DGA8-3 were closely related to Streptomyces ramulosus (99.42%), Streptomyces axinellae (99.70%) and Streptomyces drozdowiczii (99.71%), respectively. This was the first report on antibacterial activity against phytopathogenic bacteria from actinomycetes isolated from the giant honey bee.

Genetic Similarity Analysis Based on Positive and Negative Sequence Patterns of DNA

Similarity analysis of DNA sequences can clarify the homology between sequences and predict the structure of, and relationship between, them. At the same time, the frequent patterns of biological sequences explain not only the genetic characteristics of the organism, but they also serve as relevant markers for certain events of biological sequences. However, most of the aforementioned biological sequence similarity analysis methods are targeted at the entire sequential pattern, which ignores the missing gene fragment that may induce potential disease. The similarity analysis of such sequences containing a missing gene item is a blank. Consequently, some sequences with missing bases are ignored or not effectively analyzed. Thus, this paper presents a new method for DNA sequence similarity analysis. Using this method, we first mined not only positive sequential patterns, but also sequential patterns that were missing some of the base terms (collectively referred to as negative sequential patterns). Subsequently, we used these frequent patterns for similarity analysis on a two-dimensional plane. Several experiments were conducted in order to verify the effectiveness of this algorithm. The experimental results demonstrated that the algorithm can obtain various results through the selection of frequent sequential patterns and that accuracy and time efficiency was improved.

The complete mitochondrial genome of the hybrid grouper Epinephelus moara (♀)×Epinephelus tukula (♂), and phylogenetic analysis in subfamily Epinephelinae

The mitochondrial genome (mitogenome) of hybrid grouper Epinephelus moara (♀)×Epinephelus tukula (♂), a new hybrid progeny, can provide valuable information for analyzing phylogeny and molecular evolution. In this study, the mitogenome was analyzed using PCR amplification and sequenced, then the phylogenetic relationship of E. moara (♀)×E. tukula (♂) and 35 other species were constructed using Maximum Likelihood and Neighbor-Joining methods with the nucleotide sequences of 13 conserved protein-coding genes (PCGs). The complete mitogenome of E. moara (♀)×E. tukula (♂) was 16 695 bp in length, which contained 13 PCGs, 2 rRNA genes, 22 tRNA genes, a replication origin and a control region. The composition and order of these genes were consistent with most other vertebrates. Of the 13 PCGs, 12 PCGs were encoded on the heavy strand, and ND6 was encoded on the light strand. The mitogenome of the E. moara (♀)×E. tukula (♂) had a higher AT nucleotide content, a positive AT-skew and a negative GC-skew. All protein initiation codons were ATG, except for COX and ND4 (GTG), ATP6 (CTG), and ND3 (ATA). ND2, COXII, ND3, ND4 and Cytb had T as the terminating codon, COXIII’s termination codon was TA, and the remaining PCGs of that were TAA. All tRNA genes, except for the lacking DHU-arm of tRNASer(AGN), were predicted to form a typical cloverleaf secondary structure. In addition, sequence similarity analysis (99% identity) and phylogenetic analysis (100% bootstrap value) indicated that the mitochondrial genome was maternally inherited. This study provides mitogenome data for studying genetic, phylogenetic relationships and breeding of grouper.

Japanese eel retinol dehydrogenases 11/12-like are 17-ketosteroid reductases involved in sex steroid synthesis

The synthesis of 11-ketotestosterone (11KT) and estradiol-17β (E2), which play important roles in the regulation of gametogenesis in teleost fishes, is catalyzed by several steroidogenic enzymes. In particular, 17β-hydroxysteroid dehydrogenases (Hsd17bs) with 17-ketosteroid reducing activity (17KSR activity) are essential enzymes in the formation of these sex steroid hormones in the gonads and other tissues. Retinol dehydrogenase 11 (RDH11) has been suggested to be a novel tentative HSD17B (HSD17B15) in humans for a decade, however no definitive proof has been provided yet. In this study, three cDNAs related to human RDH11 were isolated from Japanese eel testis and characterized. Sequence similarity and phylogenetic analyses revealed their close relationship to human rdh11 and rdh12 gene products and they were designated as rdh11/12-like 1, rdh11/12-like 2, and rdh11/12-like 3. Three recombinant Rdh11/12-like proteins expressed in HEK293T cells catalyzed the transformation of estrone into E2 and androstenedione into testosterone. Only Rdh11/12-like 1 catalyzed the conversion of 11-ketoandrostenedione into 11KT. Tissue-distribution analysis by quantitative real-time polymerase chain reaction revealed, in immature male Japanese eel, that rdh11/12-like 1 and rdh11/12-like 2 are predominantly expressed in testis and brain, while rdh11/12-like 3 is expressed ubiquitously. Moreover, we analyzed the effects of gonadotropins and 11KT on the expression of the three rdh11/12-like mRNAs in the immature testis. In vitro incubation of immature testes with various doses of recombinant Japanese eel follicle stimulating hormone, luteinizing hormone, and 11KT indicated that the expression of rdh11/12-like 1 mRNA, rdh11/12-like 2, and rdh11/12-like 3 did not change. These findings suggest that the three Rdh11/12-like proteins metabolize sex steroids. Rdh11/12-like 1 may be one of the enzymes with 17KSR activity involved in the production of 11KT in the testis.

A Novel Major Pilin Subunit Protein FimM Is Involved in Adhesion of Bifidobacterium longum BBMN68 to Intestinal Epithelial Cells.

Adhesion to the gastrointestinal tract is considered to be important for bifidobacteria to colonize the human gut and exert their probiotic effects. Some cell surface proteins of bifidobacteria, known as adhesins, play critical roles in the binding to host cells or the extracellular matrix (ECM). To elucidate the mechanisms associated with the adhesion of Bifidobacterium longum BBMN68, a centenarian originated potential probiotic, PSORTdb was employed to identify putative extracellular localized proteins in the B. longum BBMN68. Of the 560 predicted extracellular proteins, 21 were further identified as putative adhesion proteins using the conserved domain database of NCBI, and four were successfully overexpressed in the heterologous host, Lactococcus lactis NZ9000. Notably, a recombinant strain expressing FimM showed a significantly increased adhesive affinity for both HT-29 and mucus-secreting LS174T goblet cells (2.2- and 5.4-fold higher than that of the control strain, respectively). Amino acid sequence alignment showed that FimM is a major pilin subunit protein containing a Cna-B type domain and a C-terminal LPKTG sequence. However, in silico analysis of the fimM-coding cluster revealed that BBMN68_RS10200, encoding a pilus-specific class C sortase, was a pseudogene, indicating that FimM may function as a surface adhesin that cannot polymerize into a pili-like structure. Immunogold electron microscopy results further confirmed that FimM localized to the surface of L. lactis NZfimM and B. longum BBMN68 but did not assemble into pilus filaments. Moreover, the adhesive affinity of L. lactis NZfimM to fibronectin, fibrinogen, and mucin were 3.8-, 2.1-, and 3.1-fold higher than that of the control. The affinity of FimM for its attachment receptors was further verified through an inhibition assay using anti-FimM antibodies. In addition, homologs of FimM were found in Bifidobacterium bifidum 85B, Bifidobacterium gallinarum CACC 514, and 23 other B. longum strains by sequence similarity analysis using BLASTP. Our results suggested that FimM is a novel surface adhesin that is mainly present in B. longum strains.

Fungus-originated genes in the genomes of cereal and pasture grasses acquired through ancient lateral transfer

Evidence for ancestral gene transfer between Epichloë fungal endophyte ancestors and their host grass species is described. From genomes of cool-season grasses (the Poeae tribe), two Epichloë-originated genes were identified through DNA sequence similarity analysis. The two genes showed 96% and 85% DNA sequence identities between the corresponding Epichloë genes. One of the genes was specific to the Loliinae sub-tribe. The other gene was more widely conserved in the Poeae and Triticeae tribes, including wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). The genes were independently transferred during the last 39 million years. The transferred genes were expressed in plant tissues, presumably retaining molecular functions. Multiple gene transfer events between the specific plant and fungal lineages are unique. A range of cereal crops is included in the Poeae and Triticeae tribes, and the Loliinae sub-tribe is consisted of economically important pasture and forage crops. Identification and characterisation of the 'natural' adaptation transgenes in the genomes of cereals, and pasture and forage grasses, that worldwide underpin the production of major foods, such as bread, meat, and milk, may change the ‘unnatural’ perception status of transgenic and gene-edited plants.

Co-infection of a novel fusagravirus and a partitivirus in a Korean isolate of Rosellinia necatrix KACC40168.

We report here the presence of dsRNA mycoviruses in a Korean isolate of Rosellinia necatrix. A multiple band pattern of double-stranded RNA (dsRNA) from R. necatrix suggested mixed mycovirus infection. Next-generation sequencing analysis of purified dsRNAs indicated the presence of two dsRNA mycoviruses related to the members of families "Fusagraviridae" (proposed) and Partitiviridae. The first dsRNA virus revealed that the complete genome sequence was 8868bp in size and contained two large open reading frames (ORFs 1 and 2), overlapped by 22bp containing a canonical (- 1) slippery heptanucelotide sequence of UUUAAAC. The deduced amino acid sequence of ORF1 and ORF2 showed highest similarity to the hypothetical protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 3 (RnFGV3). Phylogenetic analysis showed that this dsRNA virus clustered with RnFGV3 and other fusagraviruses. Gene organization, sequence similarity, and phylogenetic analysis indicate that this virus seems to belong to a novel species of "Fusagraviridae", which we have named Rosellinia necatrix fusagravirus 4. The second virus has two dsRNA segments with sizes of 1907bp and 1918bp, each of which encoded a single ORF showing highest similarity to the RdRp and capsid protein of known members of Partitiviridae. Evaluation of genome structure, sequence similarity, and phylogeny indicate this to be a new member of the genus Alphapartitivirus in the family Partitiviridae, hereafter designated as Rosellinia necatrix partitivirus 26. This is the first report of the presence of a fusagravirus in an Asian R. necatrix isolate and of its mixed infection with a partitivirus.

Characterization of a novel dsRNA mycovirus of Trichoderma atroviride NFCF377 reveals a member of "Fusagraviridae" with changes in antifungal activity of the host fungus.

Trichoderma atroviride is a common fungus found in various ecosystems that shows mycoparasitic ability on other fungi. A novel dsRNA virus was isolated from T. atroviride NFCF377 strain and its molecular features were analyzed. The viral genome consists of a single segmented double-stranded RNA and is 9,584 bp in length, with two discontinuous open reading frames (ORF1 and ORF2). A mycoviral structural protein and an RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively, between which is found a canonical shifty heptameric signal motif (AAAAAAC) followed by an RNA pseudoknot. Analysis of sequence similarity and phylogeny showed that it is closely related to members of the proposed family "Fusagraviridae", with a highest similarity to the Trichoderma atroviride mycovirus 1 (TaMV1). Although the sequence similarity of deduced amino acid to TaMV1 was evident, sequence deviations were distinctive at untranslated regions (UTRs) due to the extended size. Thus, we inferred this dsRNA to be a different strain of Trichoderma atroviride mycovirus 1 (TaMV1-NFCF377). Electron microscopy image exhibited an icosahedral viral particle of 40 nm diameter. Virus-cured isogenic isolates were generated and no differences in growth rate, colony morphology, or conidia production were observed between virus-infected and virus-cured strains. However, culture filtrates of TaMV1-NFCF377-infected strain showed enhanced antifungal activity against the plant pathogen Rhizoctonia solani but not to edible mushroom Pleurotus ostreatus. These results suggested that TaMV1-NFCF377 affected the metabolism of the fungal host to potentiate antifungal compounds against a plant pahogen, but this enhanced antifungal activity appeared to be species-specific.