Abstract:Innate immune response serves as the first line and the most rapid system of host defense against the invasion of microbial pathogens. Pattern recognition receptors in the host innate immune system activate a number of proinflammatory cytokines and cause inflammatory response after recognizing the invasion signal. Viral infections may activate immune responses of host. The strong regulatory network of inflammatory response plays a key role in the antiviral process of host to maintain the homeostasis. This paper reviews the inflammatory response induced by viral infections, focusing on the host regulatory network of inflammatory response, and the mechanisms of DNA and RNA viruses in regulating inflammatory response, aiming to provide some references for the treatment of immune diseases caused by viral infections.

Abstract:Pyrazinamide (PZA) is an indispensable first-line drug for the treatment of tuberculosis. It plays a key role in shortening the course of the treatment from 9–12 months to 6 months. The antibiotics rifampicin (R), isoniazid (H), ethambutol (E), and PZA (Z) form the core control regimen for the drug-sensitive Mycobacterium tuberculosis. However, PZA resistance has led to treatment failure in many patients with the emergence of MDR-TB in recent years. Therefore, it is particularly important for reducing PZA resistance to carry out the susceptibility test. Nevertheless, the test is challenging and often unreliable, as the drug is active only at pH 5.5 which affects the in vitro growth of M. tuberculosis, and thus causes both false-susceptible and false-resistant results. In this review, we summarized the research on susceptibility testing of PZA, hoping to provide a reference for the effective diagnosis and treatment of tuberculosis.

Abstract:The genus Saccharothrix was established in 1984 by Labeda, a famous American taxonomist of actinobacteria. This genus is an important group of rare actinomycetes with filamentous hyphae, and most species have zigzag aerial hyphae during sporulation. They contained meso-diaminopimelic acid in cell wall, abundant phosphatidylethanolamine in phospholipids, MK-9(H₄) and MK-10(H₄) as the principal menaquinones, and the genus-specific motifs CAC (607–609) and GTG (617–619) in the 16S rRNA gene. Recent studies of genome mining have confirmed the existence of non-ribosomal peptide synthetase genes with high frequency in the genomes of Saccharothrix spp., which have the potential to produce structurally-novel secondary metabolites and enzyme products with diverse activities. Further, Saccharothrix can produce derivatives of known antibiotics or antibiotics with new structures, such as dithiopyrrolones, lactams, anthracyclines, and chloramphenicol, which have a great value in antiviral, antibacterial and antitumor treatment. Moreover, Saccharothrix serves as a new microbial resource for the production of industrial enzyme preparations, which has strong development potential in the application of enzyme preparations. New active enzymes such as chitinase and cellulase generated by the metabolism of Saccharothrix are widely used in modern agriculture, light industry and other fields. Owing to the unique genetic and metabolic diversity, Saccharothrix plays a key role in the degradation of organic pollutants and remediation of heavy metal pollution in soil. On the basis a novel species of Saccharothrix discovered by our laboratory and relevant literature, we reviewed the typical taxonomic characteristics, genomics, secondary metabolites, and enzyme development of Saccharothrix, aiming to provide a scientific basis for further mining the Saccharothrix strains with application potential.

Abstract:Extracellular vesicles (EVs), the products of cell life activities, are the nanoscale phospholipid bilayers encapsulating nucleic acids, proteins, lipids and other molecules. A growing number of studies have demonstrated that EVs can be secreted by bacteria as “bait” for antibiotics and phages to perform defense functions. In addition, EVs play a role in the delivery of virulence factors, cellular communication, horizontal gene transfer, nutrient and electron transfer, and biofilm formation. Therefore, EVs are essential for organism individuals and communities. Here, we review the formation mechanism, extraction and identification methods, and the factors that influence the secretion of EVs. We focus on their biological functions and the research progress in the environmental field to provide a reference for relevant studies in the future.

Abstract:Cyanobacteria have strong capacities of tolerance and accumulation for different speciations of mercury, make the concentrations of different mercury-speciations convert and influence the biogeochemical cycling of mercury. Meanwhile, the various mecury-speciations accumulated by cyanobacteria which are significant primary producers in the ecosystem, are more easily delivered into the food chain, which affects human health. This paper systematically summarized the mechanisms of mercury tolerance of cyanobacteria, which included: (1) synthesizing a colloidal sheath outside the cell wall to isolate mercury; (2) mercury stabilization by combining with their own compounds; (3) using their own antioxidant mechanisms to repair the damage of mercury to cells; (4) using their own enzymes to transform the form of mercury to reduce toxicity; (5) symbiosis with mercury-resistant bacteria to resist mercury. On the basis of the above, this paper gave prospects of the further research directions of mercury-tolerance mechanisms for cyanobacteria, the promising future of mercury detoxification and pollution remediation utilizing cyanobacteria.

Abstract:Helicobacter pylori induces the incidence and development of many human gastrointestinal diseases via various virulence factors. Due to the lack of commercial vaccine and multi-drug resistance, it is difficult to eradicate this pathogen. Therefore, accurate detection technology is the key to prevent H.pylori infection and an important means of evaluating the treatment effect. The detection methods mainly include urea breath test, rapid urease test, stool antigen test, serological test, endoscopy, histopathologic examination, polymerase chain reaction, and bacteriological culture, each having specific strengths and weaknesses in clinical application. In the absence of an agreed-upon “gold standard” forH.pylori detection, we reviewed H.pylori detection techniques, analyzing their advantages and limitations. Particularly, we summarized the advantages of accurate, rapid, and convenient detection techniques in epidemiological investigation, aiming to provide a scientific reference for clinical application of and research on them.

Abstract:RNA interference (RNAi) is a conserved eukaryotic mechanism that uses small non-coding RNAs to mediate transcriptional/post-transcriptional gene silencing. Despite the deficiency of RNAi mechanism in some fungi, evidence accumulates that fungal RNAi not only maintains genome integrity but also plays a pivotal role in the regulation of fungal growth and development, heterochromatin formation, centromere evolution, and development of fungal drug resistance and pathogenicity. This review summarizes the biological functions of RNAi in fungi, which is expected to lay a basis for further research on the mechanism of fungal RNAi.

Abstract:Methane is not only a greenhouse gas but also a potential energy substance. The balance of methane source and sink is of great significance to geochemical cycle and engineering application. Anaerobic oxidation of methane (AOM) is an important methane sink in natural habitats such as deep sea, wetland, and farmland, which plays a role in mitigating greenhouse gas emissions. The central metabolic mechanism and energy conversion pathways of methane-oxidizing microorganisms are the key to AOM coupled with the reduction of other substances. Therefore, from the perspective of electron acceptor diversity, we analyzed the physiological and biochemical processes and environmental distribution of sulfate-, nitrate/nitrite-, and metal-reducing anaerobic methane-oxidizing microorganisms, and reviewed the new anaerobic methane-oxidizing microorganisms discovered in recent years. Further, we summarized the intracellular and extracellular electron transport pathways of anaerobic methane-oxidizing microorganisms. According to the environmental distribution and reaction characteristics, we prospected the ecological significance and potential application value of anaerobic methane-oxidizing microorganisms in pollution control and energy recovery. Through the review, we aim to deepen the understanding of the microbial processes of AOM and shed light on its potential engineering application.

Abstract:Antibiotic resistance is one of the global conundrums. Multi-drug resistant (MDR) bacteria-associated infections pose a great threat to the health of both human and animals. Collateral sensitivity is a phenomenon that the bacteria developed resistance to one antibiotic display increased susceptibility to a second antibiotic. Collateral sensitivity has been intensively explored to restrict and/or reverse the evolutionary trajectory of resistance bacteria in the past years. This review mainly focuses on the concept, phenotypes, and molecular mechanisms of collateral sensitivity, to shed light on the development of alternative approaches to treating MDR bacteria.

Abstract:More than half of the crop diseases are caused by fungal pathogens, which comprise the majority of crop pathogens. In addition to germplasm improvement via breeding and application of agrochemicals, another efficient strategy for disease prevention and control is to isolate and apply the biocontrol rhizospheric and/or endophytic microorganisms of the crop. [Objective] In this study we screened, isolated, and identified the endophytic bacteria with antagonistic effect against fungal pathogens infecting major crops from sugarcane to provide a theoretical basis for developing a new strategy for the biocontrol of crop fungal diseases. [Methods] Plate confrontation assay was used to isolate bacteria against multiple crop pathogenic fungi from sugarcane leaves. We identified the isolates based on 16S rRNA gene sequences, tested the inhibitory effect of the candidate strain on the sexual mating/filamentous growth and teliospore germination of Sporisorium scitamineum causing sugarcane smut, and then carried out field trial to explore the effect of the strain on sugarcane smut under field conditions. Furthermore, we examined the inhibitory effect of the candidate strain on the appressorium formation of Pyriculariaoryzae causing rice blast, as well as on diseased spot formation of detached leaves and rice seedlings cultivated in pots. [Results] The isolated bacterial strain CGB15 was identified as Bacillus amyloliquefaciens. CGB15 suppressed sexual mating/filamentous growth of S.scitamineum, making the fungal colony appear smooth under confrontation culture conditions. CGB15 suppressed the teliospore germination of S.scitamineum, with an inhibitory rate of (89.01±0.12)%. Under field conditions, CGB15 treatment showed a relative disease control rate of 48.65%. The supernatant of the fermented liquid of CGB15 inhibited the appressorium formation of P.oryzae, with an inhibitory rate of (93.55±1.11)%. Moreover, such supernatant suppressed the formation of diseased spots on detached leaves and rice seedlings cultivated in pots. We thus infer that CGB15 can produce and secret bioactive compounds to suppress the cell differentiation and pathogenicity of P.oryzae. [Conclusion] B.amyloliquefaciens CGB15 is an endophytic bacterial strain isolated from sugarcane leaves and has the potential to be applied in the prevention and control of sugarcane smut and rice blast.

Abstract:Compared with typical strains of Bacillus thuringiensis (Bt), LM1212 strain can differentiate into spore-formers and crystal-producers. In LM1212, the crystal-producing cell regulator (CpcR) not only participates in cell differentiation but also activates the promoter of crystal protein genecry35-like (P₃₅). [Objective] We aimed to screen out the homologous genes of cpcR and verify their biological functions. [Methods] We cloned two cpcR homologous genes, cpcR-c1 from Bacillus cereus and cpcR-t from B.toyonensis. Then, we inserted cpcR and its homologous genes into pHT304-P₃₅-gfp and pHT304-P₃₅-lacZ vectors, respectively. The recombinant plasmids were transferred into Bt HD73⁻ strain without cpcR and the crystal protein gene. We then observed the cell phenotypes of recombinant strains HD⁻(cpcR-c1-P₃₅-gfp) and HD⁻(cpcR-t-P₃₅-gfp) by using a laser confocal microscope and quantified the sporulation efficiency. The β-galactosidase activities of HD⁻(cpcR-c1-P₃₅-lacZ) and HD⁻(cpcR-t-P₃₅-lacZ) strains were determined. [Results] Compared with the control strain, strain HD⁻(cpcR-c1-P₃₅-gfp) and HD⁻(cpcR-t-P₃₅-gfp) showed the number of spores decreasing by 80.79% and 90.14% and the percentage of crystal-producers increasing by 7 and 9 times, respectively. Gene gfp was expressed in these two strains. The β-galactosidase activity assay demonstrated that promoter P₃₅ had high transcriptional activity in strain HD⁻(cpcR-c1-P₃₅-lacZ) and HD⁻(cpcR-t-P₃₅-lacZ). [Conclusion] The homologous genes of cpcR, cpcR-c1 and cpcR-t can regulate cell differentiation and activate the transcription of P₃₅, and cpcR-c1 had better performance in activating P₃₅ transcription than cpcR.

Abstract:[Objective]To investigate the diversity of culturable yeasts and the correlation with environmental factors in Yilong Lake after wetland restoration.[Methods] The DNA of culturable yeasts in Yilong Lake was extracted and sequenced. Based on the D1/D2 domains of 26S rRNA gene, morphological characteristics, and physiological and biochemical properties, the yeasts were identified. The total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), total hardness (TH), and conductivity (Cond) of each water sample were determined. R 4.0.5 and Canoco 5 were used to analyze the diversity of culturable yeasts, and the correlation with environmental factors. [Results] A total of 519 culturable yeast isolates were obtained from Yilong Lake after wetland restoration, which belonged to 1 potential new specie and 42 species in 24 genera. Rhodotorula mucilaginosa (320 strains, 52.29%), Cutaneotrichosporon dermatis (40, 7.71%), and Aureobasidium melanogenum (37, 7.13%) were the dominant species in Yilong Lake. [Conclusion] Culturable yeast resources were abundant in Yilong Lake after wetland restoration, and its community structure had changed greatly after the restoration. The diversity of culturable yeasts was higher in the northwest of the lake than in the southeast. TN showed a negative correlation with the diversity of culturable yeasts and a positive correlation with the dominant groups in Yilong Lake and it is the main environmental factor affecting the distribution of culturable yeasts in Yilong Lake.

Abstract:[Objective]The present work aims to isolate and identify actinobacteria from rhizosphere sediments of 3 mangrove plants and to investigate the target strain for secondary metabolites based on antibacterial activities. [Methods] Five media were used to isolate actinobacteria from rhizosphere sediments of mangrove plants in Gaoqiao, Zhanjiang. The isolates were identified based on 16S rRNA genes and then screened for antibacterial activities against 6 indicator strains. The target strain was selected for upscaled fermentation and identification of bioactive compounds. We then analyzed the biosynthetic gene cluster of the isolated compounds to predict the biosynthetic pathways. [Results]A total of 49 actinobacterial strains were isolated and categorized into 6 genera including Streptomyces (31 strains), Micromonospora (14 strains), Microbispora (1 strain), Streptosporangium (1 strain), Nonomuraea (1 strain), and Saccharomonospora (1 strain). Six α-pyrones, including germicidins A–C, germicidin I, and isogermicidins A–B, were separated from the crude extract of Streptomyces sp. SCSIO 40067 and identified. The crystal structure of germicidin A was reported for the first time. The type Ⅲ polyketide synthase biosynthetic gene cluster of α-pyrones was localized in the SCSIO 40067 genome by bioinformatics analysis. The biosynthetic pathway of α-pyrones was then proposed. [Conclusion]Actinobacteria in the rhizosphere sediment of mangrove plants in Gaoqiao, Zhanjiang had high species diversity, from which new species capable of producing valuable natural products can be mined.Streptomyces sp. SCSIO 40067 can produce α-pyrones via the type Ⅲ polyketide synthase pathway, which can serve as a start strain for the following studies.

Abstract:[Objective]As a type of ribosomally synthesized and post-translationally modified peptides (RiPPs) rich in Actinomycetes, lasso peptides have received increasing attention for their distinct modified structures and diverse biological activities. Therefore, we endeavored to develop a Streptomyces-based cell-free protein transcription/translation platform suitable for cell-free synthesis of lasso peptides or their precursors. [Methods] We first developed the cell-free platform with different Streptomyces strains and then optimized the preparation method, system components, and reaction conditions to improve the platform efficiency according to the expression of enhanced green fluorescent protein (eGFP). The core genes of the targeted lasso peptides were then introduced to the optimized platform for expression. [Results]We increased the titer of eGFP in a cell-free platform based on Streptomyces lividans TK24 to 90 µg/mL under the optimized conditions. With this cell-free platform, we expressed the precursors of lasso peptides and further fused SUMO-tag to the targeted precursors to increase their stability. [Conclusion] In this study, we constructed a Streptomyces-based cell-free platform for the expression of unexplored genes. Although the applicability of the platform for expressing unknown lasso peptides still calls for further improvement, cell-free platforms will facilitate the research on natural products.

Abstract:[Objective] Through computer-aided design, we improved the catalytic efficiency and stability of Brucella melitensis 7α-hydroxysteroid dehydrogenase and realized the efficient and stable synthesis of products. [Methods]Directed-mutagenesis at the key sites was rationally designed via homology modeling, molecular docking, and protein-ligand interaction analysis. Enzymatic property determination, enzymatic reaction kinetic analysis, and circular dichroism characterization were carried out to determine the catalytic function and stability of the enzyme. The root-mean-square deviation, root-mean-square fluctuation, and protein-ligand interactions were analyzed through all atom dynamics simulation to clarify the molecular mechanism of Met196 mutations improving catalytic efficiency and stability. [Results]Compared with the wild-type 7α-hydroxysteroid dehydrogenase, Met196Ile and Met196Val increased the specific activity to 8.33 and 7.41 folds, the k_cat/K_m values to 4.93 and 4.37 folds, and the T_m values by 1.75 ℃ and 1.10 ℃, respectively. Furthermore, Met196Ile and Met196Val shortened the duration of the synthesis from chenodeoxycholic acid to 7-oxolithocholic acid from 8 h to 2 h, and the mutants had the highest yield of about 91%. The Met196 mutation-induced rigidity enhancement of loop B (residues Ala145–Pro157) and α7 helix (residues Val249–Gly265) was beneficial to the protein stability. The enhanced interaction between substrate and binding sites or active sites (Tyr208 and Lys212) was conducive to the catalytic efficiency.[Conclusion] This study employed homology modeling and site-directed mutagenesis to modify 7α-hydroxysteroid dehydrogenase and thus improved its catalytic efficiency and stability. The findings laid a solid foundation for the efficient and stable synthesis of 7-oxolithocholic acid in industry and provided theoretical guidance for the rational design of steroid dehydrogenase.

Abstract:[Objective] To compare the virulence of different Aspergillus flavus strains and the effect of the mycovirus AfPV1 on A.flavus virulence to the established immunosuppressed mouse model. [Methods]Institute of Cancer Research (ICR) mice were intraperitoneally injected with different concentrations of cyclophosphamide, and the degree of immunosuppression was determined based on the number of white blood corpuscles. Different concentrations of A.flavus spores were inoculated through nasal drip and caudal vein, and the optimal inoculation amount ofA.flavus spores was determined based on the mortality rate of mice within 14 days. Fungal load and pathological changes of lung tissue were employed to determine whether the mice were infected by A.flavus. The effect of AfPV1 on the virulence of A.flavus was finally evaluated with the mouse model.[Results]The cyclophosphamide at a dose of 250 mg/kg caused the immunosuppression of ICR mice. Fungal load and histological changes demonstrated that ICR mice were successfully infected byA.flavus. In the nasal inoculation model, the inoculation with 40 μL (1×10⁶ CFU/mL spores) A.flavus was suitable for the evaluation of A.flavus virulence. In the model of caudal vein inoculation, the suitable inoculation dose ofA.flavus was 50 μL (1×10⁶ CFU/mL spores). AfPV1 weakened the virulence of A.flavus while did not affect the load of A.flavus in mice. [Conclusion] The constructed mouse infection model could evaluate the pathogenicity of A.flavus strains, and the mycovirus AfPV1 infection reduced the pathogenicity ofA.flavus.

Abstract:[Objective]To investigate the transcriptional regulation of mshH gene by the master quorum sensing (QS) regulators AphA and OpaR in Vibrio parahaemolyticus. [Methods] Total RNAs were extracted from the wild-type (WT) strain and the regulatory gene mutants (ΔaphA and ΔopaR). Quantitative real-time PCR (qPCR) was employed to compare the transcriptional variation of mshH gene between WT and ΔaphA (or ΔopaR) and to detect the growth phase-dependent transcription of mshH gene. The promoter region of mshH was cloned into the upstream region of the promoterless LacZ reporter gene in pHRP309. The recombinant plasmid was respectively transferred into WT, ΔaphA, and ΔopaR, and the β-galactosidase activities in the extracts of the recombinant strains were determined via a β-galactosidase enzyme assay system (Promega). Thus, the expression levels ofmshH gene between different strains or different growth phases can be compared based on the results of LacZ fusion. The promoter-proximal DNA region ofmshH was amplified by PCR, and the over-expressed His-AphA and His-OpaR were purified simultaneously under native conditions. The electrophoretic mobility shift assay (EMSA) was adopted to detect the DNA-binding activity of His-AphA or His-OpaR, and the DNase I footprinting assay was further employed to detect the DNA-binding sites of the His-recombinant proteins within the target DNA. [Results]mshH gene expression manifested in a growth phase-dependent manner, and the high expression level occurred at high cell density (HCD). At low cell density (LCD), AphA inhibited the transcription of mshH gene, while His-AphA had no binding activity to the promoter-proximal DNA fragment of mshH. At HCD, OpaR activated the transcription of mshH gene. His-OpaR protected two DNA regions located from −160 bp to −80 bp and −58 bp to −19 bp upstream of mshH gene. [Conclusion]AphA indirectly inhibited the transcription of mshH gene at LCD, whereas OpaR activated the transcription ofmshH gene in a direct manner at HCD.

Abstract:[Objective]Rhodobacteraceae, the dominant group of gut microbiota in Litopenaeus vannamei, usually has higher relative abundance in the gut of healthy shrimps, and some members of this family have been identified as the indicators for shrimp health. Therefore, clarifying the method for the directional enrichment and isolation of Rhodobacteraceae from shrimp gut can provide a basis for the development of probiotics for shrimp farming. [Methods]The 16S rRNA gene high-throughput sequencing was applied in the screening of the suitable carbon sources for the enrichment of Rhodobacteraceae. Then, the bacteria were directionally isolated from the enriched samples via the pure culture method. Finally, the taxonomic status and genetic diversity of the isolates were determined.[Results] The addition of short-chain fatty acids (acetic acid, propionic acid, butyric acid, and valeric acid) and sodium bicarbonate facilitated the enrichment of Rhodobacteraceae, which mainly included Cribrihabitans, Tritonibacter, Rhodovulum, Ruegeria, Sagittula, andThalassobius. A total of 303 bacterial strains belonging to 12 families of 2 phyla were isolated from the samples with high relative abundance of Rhodobacteraceae, and 119 out of the 303 strains were identified as members of Rhodobacteraceae, including 90 strains of Tritonibacter, 25 strains ofPhaeobacter, 1 strain of Sulfitobacter, 1 strain of Ruegeria, 1 strain ofRoseovarius, and 1 strain ofAliiroseovarius. In addition, all the isolates of Rhodobacteraceae had the relative abundance similar to the results of high-throughput sequencing. [Conclusion] We explored the effect of different carbon sources on the enrichment and directional isolation of Rhodobacteraceae by using high-throughput sequencing and screened out five carbon sources for isolating 119 strains of Rhodobacteraceae.

Abstract:[Objective] To investigate the antimicrobial activity and secondary metabolites from the coral-derived fungus Aspergillus sp. SCSIO 40435. [Methods] The coral-associated fungi were isolated by dilution-plating method. One strain-many compounds (OSMAC) approach was used for the analysis of metabolite diversity, and the antibacterial activities of fungal metabolites were tested via the standard disk diffusion method. The bioactive strain SCSIO 40435 was identified by rDNA ITS sequence analysis. The active metabolites of Aspergillus sp. SCSIO 40435 were isolated and purified from the crude extract by chromatographic methods, and their chemical structures were characterized by HRESIMS, 1D and 2D NMR, and single crystal X-ray diffraction analysis. The antibacterial activities of the isolated compounds were measured by the broth microdilution method. [Results] A total of 19 fungal strains were isolated from corals in the South China Sea. The strain SCSIO 40435 with abundant products and multiple antibacterial activities was screened out and identified as Aspergillus sp. SCSIO 40435. Four p-terphenyl compounds were isolated from the crude extract of Aspergillus sp. SCSIO 40435 and identified as dicandidusin A (1), candidusin A (2), terphenyllin (3), and 4″-deoxyterphenyllin (4), among which compound 1 is a new p-terphenyl homodimer. In addition, the single crystal structure of compound 4 was obtained for the first time. [Conclusion] This study demonstrates that corals from the South China Sea are rich in fungal resources and have the potential to produce novel active secondary metabolites, which are expected to become an important source for drug discovery and development.

Abstract:[Objective]This study aims to investigate the genomic characteristics of mink enteritis virus (MEV) in Shandong province, China. [Methods]A total of 109 fecal samples were collected from mink farms for the isolation and identification of MEV strains. Biological characterization of the isolates was carried out based on hemagglutination and hemagglutination inhibition tests, multistep growth curves, and protein structure prediction. The genomic DNA sequences of the isolates were amplified, cloned, and sequenced. MegAlign was employed for multiple sequence alignment and DNAMAN V6 for the prediction of the inverted terminal repeats of 5ʹ-untranslated region (UTR) and 3ʹ-UTR. The neighbor-joining phylogenetic tree was constructed in MEGA V6. [Results] Five strains were isolated and identified as MEV by electron microscopy and indirect immunofluorescence assay (IFA), which were named as MUTQS-1, MUTQS-2, MUTQS-3, MUTQS-4, and MUTQS-5, respectively. The genomic sequences were submitted to GenBank and got the accession numbers OK275645, OK275646, OK275647, OK275648, and OK275649, respectively. The 5ʹ- and 3ʹ-UTRs consisted of long palindromic sequences with a typical stem-loop-like structure at the end of parvovirus genome. The deduced amino acid sequences of NS1 and VP2 genes had several nonsynonymous mutations, among which E/Q545V in NS1 protein and F267Y and Y324I in VP2 protein had not been reported. Biological characterization showed that the above mutations did not significantly alter the hemagglutination and hemagglutination inhibition titers, growth trend, and spatial conformation of viral particles. The phylogenetic tree suggested that the five MEV isolates shared the same clade and were closely related to the Shandong isolate SDNH.[Conclusion] We reported the genomic characteristics of MEV strains containing novel mutation sites and confirmed that the presence of these sites did not alter the hemagglutination, antigenicity, or proliferation in susceptible cells.

Abstract:[Objective] To explore the effects of low-concentration erythromycin on protein expression, cross resistance and capsular polysaccharide of Streptococcus suis, so as to lay a foundation for further studying the effects of low-concentration antibiotic growth promoters in feed on microorganisms in the environment. [Methods] After S.suis was exposed to low-concentration erythromycin, key differentially expressed proteins were screened by iTRAQ. At the same time, we measured the cross resistance of S.suis and the content of capsular polysaccharide.[Results] A total of 181 differentially expressed proteins were identified, accounting for 12% of the total identified proteins. In order to adapt to the selective pressure of erythromycin, S.suis changed the expression of its own proteome. Most differentially expressed proteins were involved in catalytic and metabolic processes, and they belonged to membrane proteins. Among them, 13 ATP-binding cassette transporters, 3 ribosomal proteins and DNA gyrase were up-regulated, while 8 capsular polysaccharide proteins and DNA polymerase Ⅳ were down-regulated. When S.suis was exposed to low-concentration erythromycin, it revealed cross resistance to a variety of antibiotics, whereas there was no significant change in the content of capsular polysaccharide. After elimination of erythromycin, the drug sensitivity recovered. [Conclusion] To adapt to the selective pressure of low-concentration erythromycin, S.suis up-regulated the expression of multidrug resistance efflux pumps to increase the levels of ribosomal protein and decrease the levels of capsular polysaccharide protein.

Abstract:[Objective] Nitrogen-fixing bacteria and ammonifying bacteria are the key initial links of nitrogen cycle to produce bioavailable nitrogen, which directly affect the growth and spread of invasive alien plants. However, the culturable nitrogen-fixing bacteria and ammonifying bacteria in the rhizosphere of typical invasive plant Mikania micrantha H.B.K. have been rarely reported, which restricts our understanding of the efficient nitrogen transformation mechanism therein. [Methods] The culturable nitrogen-fixing bacteria and ammonifying bacteria in the rhizosphere soil of M.micrantha were isolated and purified by traditional culture method, and the inoculation experiment was carried out for verification. [Results] The density and nitrogen transformation rate of nitrogen-fixing bacteria and ammonifying bacteria and their nitrogen fixation efficiency and organic nitrogen mineralization efficiency were all higher than those of two co-occurring native competitors, Persicaria chinensis and Paederia scandens. Phylogenetic tree indicated that the nitrogen-fixing bacterial strains in the rhizosphere of M.micrantha were classified into five genera, including Burkholderia, Enterobacter, Phytobacter, Kosakonia and Rhizobium, and ammonifying bacterial strains fell into seven genera, including Serratia, Acinetobacter, Pseudomonas, Bordetella, Stenotrophomonas, Ochrobactrum and Chryseobacterium. Both Burkholderia and Rhizobium of the nitrogen-fixing bacteria and Serratia of the ammonifying bacteria were the dominant functional bacteria of M.micrantha. The greenhouse pot experiments of inoculation with the culturable functional strains demonstrated that the two groups of bacteria significantly promoted the growth of M.micrantha seedlings. Amongst them Rhizobium YHAzMm-21 of nitrogen-fixing bacteria and Ochrobactrum YHAmMm-14 of ammonifying bacteria had the best growth-promoting effects, which were expected to be developed into microbial fertilizers or engineering microorganisms. [Conclusion]The density and nitrogen transformation efficiency of the culturable nitrogen-fixing bacteria and ammonifying bacteria in the rhizosphere soil of M.micrantha outweighed those of the native species, which promoted the plant growth. These results clarified the contribution of the culturable flora in the rhizosphere soil ofM.micrantha to nitrogen cycle, and provided an ideal material for screening functional strains with high nitrogen cycle rates.

Abstract:[Objective] In this study, the endoribonuclease processing thecip-cel mRNA encoding cellulosome was identified inRuminiclostridium cellulolyticum. [Methods] The activity of four putative endoribonucleases (RNase III, RNase J, RNase G, and RNase Y) in cip-cel mRNA cleavage was analyzed via gene knockout by Clostron, overexpression in vivo, overexpression in vitro, and activity analysis. [Results]Genes (rnc and rnj) encoding RNase III and RNase J were disrupted and the resulted mutants did not affect the processing in intergenic region (IR) of the cip-cel mRNA. Moreover, RNase G and RNase Y were overexpressed and purified in vitro. RNase Y could cleave and degrade the mRNA harboring IR of the cip-cel mRNA in vitro, while RNase G failed to have any effect on that. Furthermore, overexpression of RNase Y in vivo could accelerate the degradation of cip-cel mRNA. [Conclusion]The cip-cel mRNA is potentially processed by RNase Y. The result helps deepen the understanding of the function of RNase Y in Gram-positive bacteria and the enzyme’s regulation of differential gene expression at the post-transcription level.

Abstract:[Objective]Shewanella baltica is a specific spoilage organism commonly foundin seafood during cold storage. To figure out the defense system of S.baltica isolated from large yellow croaker (Pseudosciaena crocea), we analyzed the clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification (R-M) system of this bacterium, aiming to provide a theoretical basis for the research on S.baltica and the spoilage mechanism of seafood. [Methods] The spoilage abilities of S.baltica strains SB-19 and W-3 were determined. The whole-genome sequences of the two strains were compared with 27 Shewanella strains, and their CRISPRs and R-M system were analyzed through comparative genomics tools. Finally, the transcriptomes of SB-19 and W-3 strains in different growth phases were sequenced, and the coevolution of spoilage-related genes in the two strains was analyzed. [Results] The total volatile basic nitrogen and trimethylamine produced in the sterilized fish juice showed that the SB-19 and W-3 had strong and weak spoilage abilities, respectively. The values of average nucleotide identity confirmed that both SB-19 and W-3 were S.baltica, while the phylogenetic tree based on the whole genomes showed genetic differences between them. SB-19 had a complete CRISPR system, while W-3 had an abundant R-M system. Transcriptome sequencing results indicated that SB-19 had more active metabolism and environmental adaptability in the logarithmic phase and stationary phase. The spoilage-related genes in these two strains were relatively conserved during evolution. [Conclusion]The CRISPRs and R-M system in heterotrophic S.baltica provide a good barrier against the invasion of heterologous genetic information and play a role in maintaining the genetic stability of species. However, they are not involved in the evolutionary process of the spoilage ability differences between S.baltica strains.

Abstract:[Objective] To investigate the effect of high-fat diet (HFD) on intestinal microflora and expression levels of inflammatory cytokines in hypothalamic paraventricular nucleus (PVN) of female and male Sprague-Dawley (SD) rats. [Methods] A total of 24 3-week-old SD rats (12 males) were randomized into 4 groups (6 per group). They were given either control diet (male control group, CM; female control group, CF) or the 60% fat diet (high-fat male group, HM; high-fat female group, HF) until 10 weeks old when the fresh feces were collected and total genomic DNA was extracted. For high-throughput sequencing analysis, we amplified the V3+V4 regions of bacterial 16S rDNA by PCR (polymerase chain reaction). The content of short-chain fatty acids (SCFAs) in the caecal contents of rats was analyzed by gas chromatography. Real-time PCR was performed to analyze the mRNA expression of microglial marker CD11b and inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the PVN. [Results]Reduction in quantity and diversity of intestinal microflora in the HM group (P<0.05) and decrease in abundance of the flora in HF group (P<0.05) were observed compared with those in the respective control groups. The acetic acid content in cecum was lower (P< 0.05), and mRNA expression of CD11b in PVN was higher (P<0.05) in HM group than in CM group. [Conclusion] HFD can alter the intestinal microflora of female and male rats, leading to the flora imbalance. However, the effect of HFD on intestinal microflora and brain microglia is different between female and male rats. The HFD-induced variation of intestinal microflora in male rats may be associated with brain microglia activation.

Abstract:[Objective] To investigate the diversity of culturable endophytic actinomyces in the roots of Psammosilene tunicoides in Yunnan province and screen out the strains with high antimicrobial activities.[Methods]The roots of P.tunicoides from four different places in Yunnan province were pretreated at different temperatures, from which the culturable endophytic actinomyces were isolated with 14 media. The antimicrobial activities of 74 isolates against 5 pathogens were evaluated with inverted plates. [Results]Totally 121 strains of endophytic actinomyces were isolated from the roots of P.tunicoides. The results of 16S rRNA gene sequence alignments and phylogenetic analysis showed that the 121 strains could be categorized to 24 genera, 15 families of 10 orders. Streptomyces was the absolutely dominant genus, followed by Nocardia, Micromonospora, and Mycolicibacterium. D4 (0.3 g yeast powder, 0.3 g casein, 0.3 g glycose, 0.3 g bone meal) was the best medium for isolation among all the tested media and 80 ℃ was the optimal pretreatment temperature. Among 74 strains of endophytic actinomyces, 37 strains showed inhibitory activities against at least one indicator pathogen, and the main genus with strong antimicrobial activity was Streptomyces. [Conclusion] The roots of P.tunicoides harbor rich actinomyces and the functional strains with the ability to produce secondary metabolites, which can provide abundant strain resources for pharmaceutical and agricultural production in the future as well as basic data and a theoretical basis for the cultivation, protection, development, and application of P.tunicoides.

Abstract:[Objective] To reveal the impact of soaking seeds with fermentation broth of Pseudomonas alcaliphila Ej2 on the diversity and structure of endophytic community in rice roots under salt stress. [Methods] The total DNA of the endophytic bacteria and fungi of the roots of Ningjing 61 was extracted, followed by high-throughput sequencing of the V5–V7 regions of the bacterial 16S rDNA gene and the ITS1–ITS2 of fungal ITS region. Thereby, the regulatory effects of soaking seeds with Ej2 on endophytic community of rice roots under salt stress were analyzed.[Results] Soaking seeds with Ej2 significantly improved the diversity, richness, and evenness of endophytic community of rice roots under salt stress. To be specific, it increased the number of phyla, classes, families, genera, and operational taxonomic units (OTUs, read number≥5) of the endophytic community. In addition, for bacteria, it raised the relative abundance of Bacteroidetes (by 45.97%) and Actinobacteria (by 9.55%), and lowered the relative abundance of Firmicutes (by 12.22%). As to fungi, it significantly decreased relative abundance of Ascomycota (by 17.96%) and significantly elevated the relative abundance of Mortierellomycota andOlpidiomycota. Among them, Olpidiomycota was only detected after seed soaking with Ej2. After seeding soaking with Ej2, the relative abundance of Pseudomonas,Allorhizobium, Flavobacterium, and Devosia rose, and that of Preussia, Schizothecium, Fusarium, Aspergillus, and Monosporascus decreased. Olpidium was identified only after seed soaking with Ej2. As for the bacterial species, the relative abundance of P.stutzeri, P.alcaliphila, unclassified Pseudomonas, unclassified Rhizobium, and uncultured Rhizobium was up-regulated, and that of Hydrogenophaga flava was decreased after seeding soaking with Ej2. In terms of the fungal species the relative abundance of P.terricola, Neocosmospora rubicola, Gibberella intricans, A.penicillioides and unclassified Fusarium was reduced after seeding soaking with Ej2. PICRUSt2 prediction suggested that seeding soaking with Ej2 increased the abundance of bacterial community involved in various metabolic pathways in rice roots under salt stress, and FUNGuild analysis indicated the increase in the abundance of fungi related to the metabolism of animal pathogenic fungi, mycorrhizal fungi, and endophytic fungi, and reduction in relative abundance of saprophytic fecal community, fungal saprophytes, and plant pathogens.[Conclusion]Soaking seeds with Pseudomonas can significantly increase the diversity of endophytic community of rice roots under salt stress, change the structure of dominant endophytic flora, and improve the microecological environment of rice roots.

Abstract:[Objective]In order to further clarify the functions of profilin (PFN) in filamentous fungi, we explored the effects of point-mutated PFNs on the colony growth and actin polymerization of Neurospora crassa. [Methods] The PFN F78 mutants (F78A and F78D) and V113 mutants (V113E, V113R and V113W) of N.crassa were obtained by techniques of site-directed mutagenesis, homologous recombination, filtration of conidia and PCR. With plate method, race tube assay and microscopy, the phenotypic changes of colonies were observed, and together with polyproline affinity chromatography, fluorescence spectrophotometry and high-speed co-sedimentation, the effect of point mutation of PFN on the actin nucleation and polymerization was analyzed. [Results] Mutants F78A, F78D, V113E, V113R, and V113W all grew slowly compared with the control strain ku70^RIP (P<0.05). Particularly, the colony diameters of F78D and V113W were only 20%–75.7% and 12.7%–39.2% that of the control at 12–48 h. Race tube analysis demonstrated that mycelia of F78D and V113W presented slow growth in comparison with the control. However, both of them exhibited near-normal conidiation rhythms. In addition, the purified wild-type PFN could inhibit the spontaneous nucleation of actin in a concentration-dependent manner, while the inhibition of mutant proteins PFN (F78D) and PFN (V113W) on the actin nucleation was weakened. Wild-type PFN could suppress the polymerization of actin in the concentration range of 0–5 mmol/L, and the suppression was enhanced with the increase of the concentration, causing the monomer actin content in the supernatant to peak at about 82%. However, the inhibitory effect of 5 mmol/L PFN (F78D) and PFN (V113W) on actin polymerization was significantly weakened, as manifested by the decrease of actin monomer content by 12.0% (P<0.01) and 30.7% (P<0.01), respectively, from the control. [Conclusion]This study confirmed that PFN played an important role in N.crassa, and F78 and V113 were important active sites for regulating the polymerization and depolymerization of actin and the growth and development of N.crassa.

Abstract:[Objective]To identify the chemoeffectors recognized by Agrobacterium tumefaciens C58 chemoreceptor MCP₁₉₁₂ (MCP: methyl-accepting chemotaxis protein), and to verify the function of this MCP in regulating the chemotactic response of A.tumefaciens. [Methods] The ligand binding domain (LBD) of MCP₁₉₁₂ was fused with His tag and expressed as an individual recombinant protein (named LBD₁₉₁₂) through heterologous expression. We employed fluorescence-based thermal shift assay (TSA) to screen the potential ligands of LBD₁₉₁₂, and isothermal titration calorimetry (ITC) to test the binding of the potential ligands to LBD₁₉₁₂ and to determine the equilibrium dissociation constant (K_D) of the ligand-LBD₁₉₁₂ complex. We used homologous recombination-based DNA fragment deletion method to construct MCP₁₉₁₂ deletion mutant (ΔMCP₁₉₁₂). We introduced the plasmid expressing MCP₁₉₁₂ into ΔMCP₁₉₁₂ to construct the complemented strain (ΔMCP₁₉₁₂C). Then, we adopted the capillary chemotaxis assay to test the chemotactic response of various A.tumefaciens strains to the potential MCP₁₉₁₂ ligands and to confirm the function of MCP₁₉₁₂ in regulating the chemotactic response of C58. [Results] Data acquired through TSA showed that 5 chemicals, pyruvate, L-lactate, propionic acid, acetic acid, and glycolate, might be the potential ligands of LBD₁₉₁₂. ITC further confirmed that only pyruvate and propionic acid could specifically bind to LBD₁₉₁₂. The binding of pyruvate to LBD₁₉₁₂ is exothermic with K_D of (17.0±0.9) μmol/L, while the binding of propionic acid to LBD₁₉₁₂ is endothermic with K_D of (31.5±5.4) μmol/L. The capillary chemotaxis assay verified that C58 manifested the attractant response to pyruvate and the repellent response to propionic acid. The deficiency of MCP₁₉₁₂ completely eliminated the chemotactic response of A.tumefaciens to pyruvate and propionic acid. The complementation of MCP₁₉₁₂ could restore the chemotactic response of MCP₁₉₁₂-deficient strain to pyruvate and propionic acid. [Conclusion] The chemoeffectors recognized by the chemoreceptor MCP₁₉₁₂ are propionate and propionic acid. MCP₁₉₁₂ mediates the attractant response to pyruvate and the repellent response to propionic acid.