Abstract:The rumen is inhabited by a multitude of microorganisms that play an important role in the production of ammonia. Microbe-mediated amino acid deamination and non-protein nitrogen hydrolysis are the main pathways for ruminal ammonia production. Ammonia produced by rumen microorganisms acts in a feedback loop to affect the structure of microbial flora and the function of ruminal epithelium, thus affecting the ruminal fermentation and host health. This review summarizes the production of ammonia and the influence of ammonia on digestion and epithelial function in rumen, aiming to provide a reference to the follow-up related research.

Abstract:Protein modification by methylation is one of the main ways of protein post-translational modification. More and more reports have confirmed the existence of protein methylation in archaea. Methyltransferases that are involved in the post-translational modification in archaea have been identified but their mechanism is still unclear. This article summarizes the methyltransferases reported so far and the possible roles of protein methylation. Protein methylation in archaea can improve the protein stability, affect side chain conformational changes, and facilitate interaction with other molecules. The modified proteins include those in DNA damage repair and stress response. Finally, future research directions of archaeal protein methylation are proposed.

Abstract:The genus Kibdelosporangium is a classical group of actinomycetes. Since the establishment of this genus in 1986 by Shearer et al., new strains have been isolated from different habitats by pure culture methods, and 8 species and 2 subspecies have been published. Kibdelosporangium is a rare actinomycete genus and an important resource for obtaining new secondary metabolites. Based on the function and genome analysis of a newly isolated strain and the related literature of the genus Kibdelosporangium, we systematically summarize the research advances of the genus Kibdelosporangium, including the genus establishment, taxonomic characteristics, distribution of the species, the discovery of active secondary metabolites and other functional applications and development prospects. This review provides references for the classification and identification of other new isolates, the discovery of novel secondary metabolites, and the exploitation of functional gene resources from the genus Kibdelosporangium in the future.

Abstract:Viruses attach and invade target-cells by binding to a specific receptor(s) on the surface of target-cells, and hijack the cellular replication machinery to complete the life cycle. The cellular receptor is the gateway for viruses to infect host cells, and the structure and function of cellular receptors and the mechanism of virus entry mediated by cellular receptors have been one of the hotspots of molecular virology. The discovery of cellular receptors is helpful to understand the pathogenesis of viruses and develop prevention and control strategies for viral diseases. In recent years, the progress in screening functional viral receptors using functional genomics has greatly expanded our understanding of the mechanism of virus entry. At present, widely used functional viral receptor screening strategies include RNA interference, random retroviral insertional mutagenesis using haploid cell lines and the recently emerging CRISPR/Cas9 system. In this review, three novel strategies of functional viral receptors screening are systematically introduced and compared, and their applications, advantages and disadvantages are summarized to provide references for relevant researchers.

Abstract:Microcins are antibacterial peptides produced by Enterobacteria with low molecular masses below 10 kDa. They are encoded by cluster genes on the plasmid or on the genome. Microcins have narrow antibacterial spectrum. They have potent antibacterial activity against closely related bacteria in Enterobacteria. Typical gene clusters in microcins include microcin precursor gene, the self-immunity gene, the secretion gene and frequently the post-translational modification gene. Microcins are ribosomally synthesized as precursors, in contrast to most microbial antibiotics that are non-ribosomally synthesized. Fifteen microcins have been discovered so far. They show a variety of structures and antibacterial mechanisms.

Abstract:[Objective] In this study, we demonstrated the mechanism of ToxR activating virulence gene expression by modifying its protein function in Vibrio cholerae. [Methods] ToxR redox status assay with or without DsbA was analyzed by thiol-trapping assay. ToxR cysteine mutant ToxR_C263/293S was obtained by site-directed mutagenesis. Escherichia coli strains containing ctxAB promoter luxCDABE transcriptional fusion and a plasmid-coded ToxR expressed under the control of arabinose were used to analyze the transcriptional level. ToxR and ToxS proteins interaction was detected by the bacterial two-hybrid system. [Results] The two cysteines in the periplasmic domain of ToxR could be oxidized by DsbA. When ToxR and ToxS were co-transcribed under the control of the same promoter, ToxR activated ctxAB expression to a higher level in E. coli dsbA knock out mutant. However, when ToxR was expressed alone without ToxS, the redox status of ToxR had no effect on its activation of ctxAB expression in E. coli. Bacterial two-hybrid system assay showed that ToxS greatly enhanced ToxR homodimerization regardless of the redox status of ToxR, and DsbA strongly repressed ToxS-ToxS interaction. [Conclusion] The redox status of ToxR has no effect on its activating virulence gene expression and ToxS enhances ToxR activity by promoting ToxR homodimerization. DsbA indirectly affects ToxR activating virulence genes expression by repressing ToxS homodimerization.

Abstract:[Objective] In this study, an unknown bacterium was isolated from Dermacentor nuttalli-a dominant tick species in the area of Hulunbuir. Then we identified the bacterium by molecular biology. We also explored its pathogenicity. [Methods] First, through conventional morphological observation, physiological and biochemical analysis, drug sensitivity test, 16S rRNA gene sequence analysis and constructing phylogenetic tree, we confirmed the taxonomic status of the bacterium. Then we did pathogenicity experiment on mice. [Results] This bacterium was Gram-positive and grew well in high-salt medium (5% NaCl) and alkaline medium (pH 9), but its ability was limited to ferment sugar, instead it only used some sugar (Simon's citrate test showed positive results) to make aerogenesis but not acid. It is sensitive for individual chemicals like ciprofloxacin, and most antibiotics like neomycin, gentamicin, erythromycin and cefotaxime, whereas it was resistance to kanamycin and amoxicillin. From the analysis of the 16S rRNA gene sequence and the phylogenetic trees, we found that the homology of this bacterial strain and Oceanobacillus oncorhynchi from the GenBank database (GenBank:GQ121034.1) is over 99%. Besides, this bacterial strain shows incomplete hemolysis and no obvious pathogenicity was observed in Kunming mice 7 days after infection. [Conclusion] A tick-borne O. ncorhynchi strain was isolated, identified and named O. oncorhynchi IMH and registered in Genbank (No. LC213016.1), which enriched the relevant data in GenBank.

Abstract:[Objective] DNA phosphorothioate (PT) modification, in which the sulfur replaces a nonbridging oxygen, occurs naturally in diverse bacteria, archaea and human pathogens as a new kind of epigenetic modification. However, the regulatory mechanism of PT modification has not been fully characterized. In this study, the regulatory mechanism of spfB on DNA phosphorothioate (PT) modification in Pseudomonas fluorescens Pf0-1 was demonstrated. [Methods] Firstly, the spfB genetic interruption and complementary strains were constructed by homologous recombination and then tested the modification frequency in these strains by iodine cleavage. The operons within spf gene cluster were grouped by RT-PCR and the transcriptional level was analyzed in the ΔspfB mutant by quantitative real-time PCR. Finally, the possible regulatory region of SpfB on the spf operon was characterized by EMSA and DNase I footprinting assay.[Results] The inactivation of spfB led to more dispersed small fragments in genomic DNA of ΔspfB mutant and its complementation obviously restored the phenotype of wild type strain. Genes in spf gene cluster were assigned into one co-transcription unit, and the disruption of spfB directly up-regulated the transcription of the operon. In vitro SpfB directly protected two separate sequences within the spf promoter region from DNase I cleavage, and each protected sequence contained a direct repeat (5'-TGTTTGT-3'). [Conclusion] SpfB in Pseudomonas fluorescens Pf0-1 was a negative regulator in DNA phosphorothioate modification.

Abstract:[Objective] In this study, we studied the role of HOG1 MAPK in sodium arsenite-induced apoptosis in yeast cells. [Methods] Yeast wild-type (BY4741) and HOG1 mutant (ΔHOG1) strains were used to study the effects of sodium arsenite on the growth and relative survival rate and oxidative damages of yeast cells. Further, the apoptotic rate, intracellular reactive oxygen species (ROS) level, and mitochondrial membrane potential of the yeast cells under sodium arsenite-induced stress were determined by flow cytometry. [Results] Sodium arsenite inhibited the growth of yeast cells and induced their apoptosis. Compared to the wild-type strain in the same treatment group, ΔHOG1 strain showed higher sensitivity to sodium arsenite with a lower cell survival rate and higher apoptotic rate. Under sodium arsenite-induced stress, ΔHOG1 strain showed significantly higher intracellular ROS and malondialdehyde (MDA) levels than the wild-type BY4741 strain. On the contrary, the mitochondrial membrane potential of ΔHOG1 strain was significantly lower than that of the wild-type BY4741 strain. [Conclusion] These results indicated that HOG1 MAPK gene was involved in the regulation of sodium arsenite-induced apoptosis by affecting intracellular ROS level and changing △ψ_M in yeast cells.

Abstract:[Objective] For the excavation of plant growth promoting rhizobacteria (PGPR) strains, the functional genes were analyzed and mapped in order to find out their scientific value and industrialization development and serve agricultural production. [Methods] Bacillus mycoides Gnyt1 strain was used as the material, and the second and third generation sequencing technology were combined to carry out the whole genome sequencing of the strain. The possible functional genes of the nuclear genome of the strain were analyzed, and genes secreting iron carrier-related strain were analyzed. [Results] The total length of the genomic sequence after splicing was 5597907 bp. The percentage of GC was 35.57%. There were 9 genes involved in the secretion of siderophore in this strain, of which 2 genes mainly existed in the Porphyrin metabolism pathway and were associated with intracellular iron transport metabolism related. [Conclusion] Through whole genome sequencing, the functional genes related to the secretion of siderophores were identified as GYT1 and GYT2, which makes it possible for verifying the gene functions.

Abstract:[Objective] Ectropis obliqua Prout (Lepidoptera:Geometridae) is the most devastating insect pest of tea plants in China. Studying on the relationship among feed, gut microbial flora and fitness of Ectropis obliqua is of relatively high theoretical value for the management of E. obliqua. [Methods] We analyzed the effect of artificial diet (without tea ingredients) and tea leaves on survival of Ectropis obliqua larvae. We also analyzed the similarities and differences of gut microbial flora in Ectropis obliqua larvae fed with different feeds based on high-throughput sequencing technology. [Results] The mortality of larvae were significantly different. Larvae fed with artificial diet had high mortality. Larvae fed with artificial diet had higher bacterial diversity and evenness than larvae fed with tea leaves. Many bacteria were found in the gut of Ectropis obliqua larvae. [Conclusion] Feed affects the survival and the gut microbial flora of Ectropis obliqua larvae.

Abstract:Fusarium oxysporum f. sp. cubense race 4 (Foc4), the strong virulent pathogen of Banana Fusarium Wilt, must face to oxidative burst produced by the host in the early stage of infecting banana plants. [Objective] To study the molecular mechanism how Foc4 responses exogenous oxidative stress, [Methods] RNA-Seq was done between the wild type B2 strain of Foc4 treated by H₂O₂ and the control using Illumina 2500 sequencing platform, and differentially expression genes (DEGs) was analyzed. [Results] The growth of Foc4 was inhibited under exogenous oxidative stress, and more than 20 million clean reads were obtained after RNA-Seq. Compared with the control, a total of 496 genes were up-regulated and 298 genes were down-regulated, respectively, with FDA value ≤ 0.001 and Fold Change (FC) ≥ 2 used as the selection criteria. Gene ontology (GO) functional enrichment analysis showed that 429 genes were annotated in the GO functional analysis database. Many of them were related to metabolic processes, biological regulation, cellular processes and response to stimuli. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis showed that 141 DEGs were annotated in 50 metabolic pathways in KEGG. These metabolic pathways mainly include the metabolic pathway of various amino acids and fatty acid. It also includes metabolic pathways that are directly related to antioxidant stress, including damage repair of DNA, biosynthesis of carotenoids, peroxisome and glutathione metabolism. [Conclusion] Material metabolism, energy metabolism and signal regulation pathways directly dealing with oxidative stress must be changed in Foc4 cells to cope with environmental stress and to survive in strong oxidative stress environment.

Abstract:[Objective] To structure a type A foot-and-mouth disease (FMD) marker virus with deletion of 104-115 amino acids of the nonstructural protein 3A, then to analyze its biological characteristics and the potential of developing marker vaccine.[Methods] Using overlap extension PCR method, we introduced the deletion of 104-115 amino acids of the nonstructural protein 3A into FMDV A/Sea-97/CHA/2014 full-length infectious clone pQAHN to generate a recombinant full-length plasmid. The marker virus was rescued after transfecion linearized recombinant plasmid into BSR/T7 cells expressing T7 RNA polymerase and identified by RT-PCR, sequencing analysis, indirect immunofluorescence and Western blotting. The plaque and one-step growth curves were used to analyze the biological characteristics of the marker virus. A developed block ELISA method targeting to the deleted epitope of 3A was used to analyze the potential of differentiating animals infected with the marker virus and the wild type virus.[Results] We rescued a type A FMD marker virus containing deletion of 104-115 amino acids of 3A protein. The deletion did not affect the plaque phenotype and one-step growth curve of the marker virus. A developed block ELISA method targeted to the deleted epitope could clearly differentiate animals infected with the marker virus and the wild type virus.[Conclusion] The marker virus containing deletion of the dominant epitope of 3A is suitable as a vaccine candidate strain of differentiating infected from vaccinated animals to effectively prevent and control type A FMD in future.

Abstract:[Objective] To compare the differences of degradation properties of coexisting phenanthrene and anthracene, we studied factors affecting the degradation by Irpex lacteus F17. We also discussed the degradation pathway preliminarily combining with the analysis of intermediate products. [Methods] We determined the concentration of phenanthrene and anthracene by GC-MS, and analyzed the intermediate products of biodegradation with mass spectrogram. [Results] When the initial concentration of phenanthrene and anthracene was 5 mg/L, 93% of the former and 85% of the latter was degraded. Phenanthrene could be degraded preferably between pH 3.0 and 8.0, and coexisting anthracene could be degraded well between pH 4.0 and 8.0 by Irpex lacteus F17. Phenanthrene was degraded better than anthracene at low temperature, and the optimal temperature for coexisting system was 30℃. Anthracene was transformed into phthalic acid, whereas phenanthrene was converted into phthalic acid or catechol by enzymes. [Conclusion] The results indicated that phenanthrene was degraded better and faster than coexisting anthracene by Irpex lacteus F17 under different conditions. There were some differences between coexisting phenanthrene and anthracene on degradation properties and degradation pathways due to the different positions of the three benzene rings.

Abstract:[Objective] The aim of this study was to screen a probiotic strain that antagonizes Streptococcus agalactiae isolated from the intestine of healthy Nile tilapia (Oreochromis niloticus). [Methods] The gut of healthy tilapia was collected, homogenized, and diluted by a 10-fold series. Then 0.3mL suspension was streaked on brain heart infusion plates and incubated for 1-2 d at 30℃, and then single colonies were selected for purification. Strains had antagonistic function against S. agalactiae were screened by the dot-inoculating method, and a strain named LF01 with a better antagonistic effect was selected. In this study, the strain LF01 was identified by physiological, biochemical characteristics and molecular biological analysis. In addition, some characteristics and biosafety of the strain LF01 were determined and analyzed. [Results] A total of 64 strains were isolated from the intestine of healthy tilapia and 6 strains with obvious antagonistic effects were obtained, with the antagonism of LF01 strain considered the best. Strain LF01 was identified as Bacillus velezensis. The best growth temperature, pH, and salinities of strain LF01 were 30℃, 7, and 5‰, respectively. Furthermore, the strain LF01 exhibited the ability to hydrolyze starch and casein. Strain LF01 was sensitive to most antibiotics and resistant only to bacitracin, and showed antimicrobial activity against a broad range of fish pathogens including Streptococcus agalactiae, Streptococcus iniae, Edwardsiella tarda, Edwardsiella ictaluri, Aeromonas hydrophila, Aeromonas schubertii, Aeromonas veronii, Aeromonas jandaei and Nocardia seriolae. Biosafety tests suggested that the strain LF01 was not pathogenic to Nile tilapia, Danio rerio, and Channa argus. [Conclusion] In this study, it was found that B. velezensis LF01 strain isolated from the intestine of healthy tilapia has good biosafety and could antagonize the common aquatic pathogens. It has the potential to prevent and control a variety of aquatic animal diseases, and has broad application prospects.

Abstract:[Objective] For yeast gene functional studies, we constructed a plasmid set combining the advantages of pUG and pFA6a plasmid series, and allowing convenient insertion of tandem epitope tags using isocaudomers. [Methods] We cloned the loxP locus of pUG plasmids, the multiple restriction site of pFA6a plasmids and ADH1 terminator cassette by PCR. Through homologous recombination of the DNA fragments, we obtained plasmid pCLHN-TRP and pCLHN-URA. Using introduced isocaudomer sites, we then constructed additional tagging plasmids containing either single or multiple tandem copies of various epitope tags. Finally, we tested the performance of our plasmids using ATG1, COX4 and NHX1 as target genes. [Results] We constructed two plasmids for gene deletion purposes, and seventeen plasmids for epitope tagging purposes (covering 1-8 FLAG, 1-12 V5, 3-9 HA, 2-8 MYC, GFP and mCherry). Testing on selected target genes demonstrated the usability of these plasmids. In particular, by combining different copy numbers of tandem epitopes, it was feasible to properly detect proteins with drastically different expression levels on the same blot without saturating the signal of high expression targets. [Conclusion] The pCLHN plasmids constitute a beneficial complement to existing yeast plasmid tools.

Abstract:[Objective] Researches were carried out for biocontrol of green mold decay in citrus by Pseudomonas fluorescens ZX. [Methods] The biocontrol effectiveness in fruits and effects on Penicillium digitatum growth were evaluated along with the antifungal activity of volatile organic compounds (VOCs). Meanwhile, assays of competitive grow between Pseudomonas fluorescens ZX and Penicillium digitatum were conducted, as well as the growth curve and biofilm formation ability of Pseudomonas fluorescens ZX. [Results] Pseudomonas fluorescens ZX suspension showed potent ability for the depression of Penicillium digitatum, so did the VOCs. In addition, Pseudomonas fluorescens ZX made better use of nutrients than Penicillium digitatum, and it entered the logarithmic growth stage quickly (4 h) and formed an effective biofilm after 24 h. [Conclusion] The application of Pseudomonas fluorescens ZX was effective to control green mold, and the potential mechanisms included inhibition of spore germination, competition for nutrients and space, and formation of biofilm.

Abstract:[Objective] The fungal conidial pigments are essential components for the formation of the fungal cell wall. The pigments play very important roles in fungal growth and resistance to various environmental stress responses. Here, the biosynthetic gene of conidial pigment was identified and characterized in Aspergillus flavus. [Methods] By the homologous search of the known pigment biosynthetic gene protein, the key gene involved in the pigment production was identified in A. flavus. The target gene was disrupted using homologous recombination strategy, and the effects of the gene deletion on fungal phenotype, sporulation, sclerotia formation, aflatoxin production, anti-ultraviolet irradiation and invasiveness were investigated. [Results] Compared with the wild-type strain, the Δpks1 mutant lost the conidial color instead of producing the white conidia. No obvious difference was detected in the growth rate, spore yield, formation of stress-resistant structure sclerotia and production of aflatoxin B₁ between WT and Δpks1 mutant strains. However, the deletion of pks1 resulted in a decrease in resistance to UV radiation and the ability to infect corn and peanut seeds. [Conclusion] The pks1 (AFLA_006170) is a key gene for conidia pigment production in A. flavus. The conidial pigment biosynthetic pathway is related to fungal resistance against UV radiation and the infection to cereal crops.