Abstract:Pseudomonas chlororaphis is one group of the most studied biocontrol strains. Since the beginning of the 19th century, it was firstly isolated and identified as Pseudomonas by Miguela, and then it was reclassified as P. chlororaphis by Peix in 2007. At present, four subspecies affiliated to this species have been reported, which can produce colored phenazine antibiotics. Most of the strains were isolated from the rhizosphere of plants, which play a protective role in plants against the infection of pathogens and nematodes. In this paper, based on the genomic and functional analysis of a new obtained Pseudomonas chlororaphis strain by our group and related literatures, we systematically summarized the taxonomic characteristics, genomic characteristics, functions of metabolites, and application prospects of Pseudomonas chlororaphis. This review will provide reference for the discovery of active metabolites, gene function research and agricultural application of Pseudomonas chlororaphis strains in the future.

Abstract:Antibiotic killing is a complex physiological process. The downstream metabolic changes of antibiotic-target interactions are closely related to antibiotic efficacy. Bactericidal antibiotics speed up the death process by perturbing the bacterial metabolism, which also influences the antibiotic efficacy. Metabolomics provides comprehensive metabolic information for monitoring the changes of bacterial metabolism under the action of antibiotics. Here we review recent metabolomics-driven studies on the interaction between antibiotics and bacteria, intending to provide a reference for the development of antibiotic adjuvants to enhance the antibiotic efficacy.

Abstract:Lactobacillus kefiranofaciens is Gram-positive Lactobacillus originated from different countries and regions with probiotics characteristics and food safety. At the end of 2020, the China National Health Commission approved L. kefiranofaciens subsp. kefiranofaciens as a novel kind of food ingredients in China. This paper summarized the research progress of L. kefiranofaciens subsp. kefiranofaciens including strain morphology, genomic characteristics, probiotic functions and potential benefit in order to provide references for their future research and industrial applications.

Abstract:Methionine γ-lyase (MGL) catalyzes the cleavage of C-S bond in methionine, which produces equimolar products including α-ketobutyric acid, methanethiol and ammonia. MGL reduces the intracellular concentration of methionine, which significantly inhibits the growth and migration of malignant tumor cells and triggers the antioxidant response in normal cells. Engineering MGL for enhancing its catalytic efficiency is the focus for tumor therapy and anti-aging. MGL widely exists in microorganisms, but not in mammals. MGL is an important target for the development of antimicrobial drugs toward microbial pathogens. Methanethiol and its derivatives are the main components of food aroma, and the composition and concentration of methanethiol and its derivatives determine the formation of food aroma. Elucidating the catalytic mechanism and identifying the key elements control the activity of MGL will promote the precise control of food quality and its stability. In this paper, the latest advances in the discovery, catalytic mechanism and engineering of MGL from microorganisms were summarized. The applications of MGL in tumor therapy, anti-aging, anti-microbial infection and synthesis and manufacturing of food aroma were discussed, and then the development and challenge of MGL were prospected.

Abstract:Candidiasis refers to the acute, subacute or chronic infection caused by Candida, which usually involves the skin and mucosa, as well as internal organs and various system organs, and can cause serious consequences. It is a deep mycosis with the highest incidence at present. Candida albicans is one of the most common opportunistic pathogenic fungi in Candida species. Recently, more and more studies have confirmed that the extracellular vesicles of Candida albicans play an important role in its pathogenicity. They are involved in the transport of important proteins and genetic materials, influence the drug resistance of Candida albicans, and communicate with host cells to mediate the inflammatory and immune responses of the host. Some studies have shown that extracellular vesicles have immune activity and can be used as a vaccine preparation. Therefore, this paper reviews the composition and function of extracellular vesicles of Candida albicans in order to provide reference for further research in the future.

Abstract:Streptococcus pneumoniae causes a range of diseases such as invasive infections (bacteremia of septicemia and meningitis, etc.) and common mucosa infections (pneumonia, otitis media, sinusitis, etc.). According to the structure of surface capsular polysaccharide, it is classified into different serotypes (98 serotypes identified, with 20 highly virulent). A number of related vaccines have been developed, among which 23-valent pneumococcal polysaccharide vaccine (PPV23) and 13-valent pneumococcal protein-conjugate vaccine (PCV13) are commonly used. However, natural polysaccharide antigens face some challenges, such as difficulty in purification and uneven composition. Therefore, synthetic oligosaccharide-based glycoconjugate vaccines have become a promising alternative. After vaccination, serotype substitution occurs and the pathogenicity rate of certain serotypes (such as serotype 3 and 19A) increases. Thus, serum-independent protein vaccines and whole-cell vaccines have become a new research focus. This review took S. pneumoniae serotype 3 vaccine as an example to summarize the mechanism of different types of vaccines and their research progress.

Abstract:Type V secretion system, one of the key pathways employed by Gram-negative bacteria to secrete virulence factors, is classified into five subtypes (Va-Ve). Among them, type Va (also known as the classical monomeric autotransporters) is extensively used to export virulence and adhesion factors to the bacterial cell surface or into their surroundings, a process combining the passage across the inner and the outer membrane in two consecutive steps that are facilitated by the Sec translocon and the BAM (β-barrel assembly machinery) complex, respectively. It is believed that type Va is the simplest secretion system for the translocation of proteins across membranes, which is therefore considered as an ideal biotechnological tool to display recombinant proteins on bacterial cell surface. In this review, we first summarized the subtypes, structural domains, and possible secretion mechanisms of autotransporters to enhance the understanding of them as a biotechnological tool for various surface display purposes, particularly vaccine development. Then, we explored the potential application of autodisplay in pathogen detection to further expand its application as a biotechnological tool.

Abstract:Flaviviruses are a group of single-stranded positive-sense RNA viruses with envelope, which are transmitted by mosquitoes. They are the major pathogens of emerging infectious diseases which threat human health in the world. Although the diseases caused by different flaviviruses vary, the clinical symptoms are similar to some extent. Fever, especially high fever, is the most common symptom after flavivirus infection. Studies have demonstrated that caspase-1-dependent inflammatory response occurs in the infections with Zika virus and Japanese encephalitis virus, and this process is partially consistent with the mechanism of pyroptosis. Pyroptosis is a type of programmed death of inflammatory cells dependent on caspases, characterized by gasdermin-mediated pore formation, cell swelling, rupture, and release of inflammatory cytokines. In this paper, the flavivirus-caused macrophage pyroptosis in innate immunity was reviewed. The molecular mechanism of pyroptosis and the important components involved in pyroptosis were summarized, and the relationship between pyroptosis and representative flaviviruses was analyzed. This study will provide reference for the follow-up research on the role of pyroptosis in the pathogenesis of flaviviruses and shed light on the antiviral treatment.

Abstract:Mangrove forest is an important ecosystem in the sea-land interface zone and a hot zone for pollutant accumulation and transformation. Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants widely distributed in mangrove wetlands, threatening ecosystem health. The degradation and transformation of PAHs have attracted increasing attention of researchers in recent years. This paper reviews the available studies about microbial degradation of PAHs in mangrove wetlands from the perspectives of biodegradation rules, functional microorganisms, and influencing factors of PAH degradation in mangrove habitats. We found that the PAHs in mangrove forests had higher content than those in the beach outside the forests, and they accumulated in the sediments at a depth of 10-20 cm. Sulfates were the main electron acceptors (EAs) for anaerobic degradation of PAHs, as they had the highest concentration and infiltrated deeper into the sediments. EAs nitrate, bicarbonate, Fe(III), and Mn(IV) were also distributed in mangrove sediments and played roles on PAH-degradation. PAH-degrading bacteria had high diversity, among which Sphingomonas, Bacillus, Novosphingobium, and Sphingobium were reported frequently. The aerobic-anaerobic alternation condition, radial oxygen loss, root exudates, and exogenous biostimulators were the main factors affecting the biodegradation of PAHs in mangrove habitats. We suggest that researchers can focus on the application technologies of PAH-degrading microorganisms to meet the actual needs of wetland remediation.

Abstract:Converting lignocellulose into fermentable sugars for the production of biofuels and bio-based chemicals is one of the effective ways to achieve carbon neutrality. Lignocellulose-degrading enzymes play an important role in this process. Trichoderma reesei is the most widely used strain for the production of cellulases and hemicellulases. For a long time, T. reesei has been considered as the asexual form of Hypocrea jecorina, which can only reproduce asexually. The strain improvement is mainly based on traditional mutagenesis and gene manipulation. The sexual cycle of T. reesei has been discovered over the last few years, which provides the possibility for the genetic crossbreeding of T. reesei. Combined with previous work in genetic breeding of T. reesei from our laboratory, this article comprehensively describes the sexual reproduction process, characteristics, and regulation mechanism of T. reesei and prospects the genetic improvement strategy, providing a reference for the further development and application of T. reesei as a cellulase- and recombinant protein-expressing host.

Abstract:[Objective] In this study, we compared three room-temperature preservation methods with −80℃ preservation and investigated the effects of these methods on the sequencing results of microbiota in the fecal samples, thence providing guidelines for large-scale and standardized sampling. [Methods] Fresh fecal samples from five healthy volunteers were collected and subjected to four different preservation methods:room-temperature preservation methods (DETs, GITC, and RNAlater) and storage at −80℃ without stabilizers. High-throughput sequencing was deployed to sequence the V3−V4 region of the 16S rRNA genes in the samples being stored for 0, 1, 3, 7, 14 and 28 days. The effects of preservation stabilizers and time on the microbiota composition and species diversity were determined by comparison between different treatments.[Results] A total of 489 operational taxonomic units (OTUs) were obtained from the five groups of samples (fresh samples, three room-temperature samples, and −80℃ preserved samples), among which 488 OTUs were shared by these five groups. No significant difference was observed in the alpha diversity of microbiota amory preservation methods. The relative abundance of Bacteroides in the samples preserved with DETs was higher than that in fresh samples, and some other OTUs in the samples preserved with DETs changed considerably after 3-day storage. No significant difference in the relative abundance of microbial genera was observed amory other groups apart from the DETs group. The microbiota diversity and abundance showed no significant difference between the samples preserved with the four methods and the fresh samples after the storage for different days. The samples stored at −80℃ had the highest similarity to the fresh samples. However, the similarity of the samples from each volunteer varied, and such variation tended to increase with the prolonging of storage time. Nevertheless, DTEs and GITC methods exhibited outstanding stability, especially after prolonged storage time. Cluster analysis revealed that the variation associated with preservation methods and time was neglectable. [Conclusion] Subject to the availability, storage at −80℃ is considered the gold standard for fecal samples, and storage at room temperature with the addition of GITC shows comparable performance to low temperature preservation.

Abstract:[Objective] Bacillus subtilis ComQ is a kind of isoprene biosynthesis enzyme. We used bioinformatics to predict and analyze the biological characteristics of ComQ. In addition, we also constructed comQ overexpression and knockout mutants to verify their effect on biofilm morphology. [Methods] Firstly, we obtained the amino acid sequence of ComQ protein from the Protein database in the NCBI website. Secondly, we predicted and analyzed the biological characteristics including physical and chemical properties, signal peptide, domain and spatial structure of ComQ by online bioinformatics software. Thirdly, we constructed comQ overexpression and knockout strain of Bacillus subtilis, and observe their growth characteristics of biofilm by plate fermentation. [Results] ComQ protein was composed of 299 amino acids with a theoretical molecular weight of about 34 204.08 Da. It was a stable intracellular protein without signal peptide and transmembrane region. Colony PCR verified that Bacillus subtilis BS168 mutants were constructed successfully. The biofilm morphology of the two mutants was different from that of the original strain BS168. [Conclusion] Through the prediction and analysis of the basic properties, key amino acid sites and protein structure of ComQ protein, the mutant strain was constructed to verify the morphological changes of biofilm, which laid a foundation for the subsequent study of ComQ on the growth and metabolism of Bacillus subtilis.

Abstract:[Objective] DNA phosphorothioation (PT) is a novel DNA modification in which a non-bridging oxygen atom on the phosphodiester bond is replaced with sulfur atom. PT modification serves as a constitute element of bacterial restriction-modification (R-M) defensive system, but more biological functions of PT modification are awaiting exploration. The methods currently used for the identification of PT-DNA are complicated, time-consuming and labor-intensive. Enzyme-linked immunosorbent assay (ELISA) has been known for its merits of easy handling, low cost and time-saving, which could be used for new PT-DNA identification method development. The homologous protein of PT-dependent REase ScoMcrA from Streptomyces coelicolor can be found in Streptomyces gancidicus, which suggested that this homologous protein presumably encodes the sulfur-binding domain (SBD) to specifically recognize sulfur atom on PT-DNA. In this study, the PT-DNA binding activity of SBD from S. gancidicus (Sg-SBD) was characterized qualitatively and quantitatively, respectively, which served as an assessment of its potential for the PT-DNA ELISA method development. [ Methods] Sequence alignment of ScoMcrA-SBD and Sg-SBD was conducted. The homology modeling was used to compare the possible structure of Sg-SBD with that of ScoMcrA-SBD. ScoMcrA-SBD and Sg-SBD were heterologously expressed in E. coli and purified. Using the purified Sg-SBD, the electrophoresis mobility shift assays (EMSA) was conducted firstly. Then, the biolayer interferometry (BLI) analysis was used to confirm the binding affinity of Sg-SBD for PT-DNA quantitatively. [Results] Bioinformatics analysis revealed that Sg-SBD contains conserved amino acid residues that bind to sulfur atoms, and the structure of Sg-SBD was similar with that of ScoMcrA-SBD. The EMSA showed that Sg-SBD can bind PT-DNA and form an obvious migration band, suggesting its PT-DNA binding activity. BLI data further confirmed that Sg-SBD possessed higher binding affinity to PT-DNA than that of ScoMcrA-SBD. Moreover, Sg-SBD exhibited no affinity to non-PT-DNA. [Conclusion] Sg-SBD could specifically bind PT-DNA, and the binding affinity could be comparable to that of antigen and antibody, which suggested the potent potential for ELISA method development for rapid detection of PT-DNA.

Abstract:[Objective] To provide biochemical support for studying the potential function during DNA repair via systematically characterizing the enzymatic properties of the DHH superfamily nuclease (Saci0542) of Sulfolobus acidocaldarius. [Methods] Saci0542 was recombinantly expressed in Escherichia coli and then purified by affinity chromatography. The enzymatic properties of Saci0542 were characterized in vitro with fluorescence-labeled oligonucleotides as substrates by urea-denaturing polyacrylamide gel electrophoresis. [Results] The recombinant Saci0542 had a typical 3'-5' exonuclease activity on ssDNA. Its activity was dependent on the divalent metal ion Mn²⁺ and inhibited by the divalent metal ions such as Ca²⁺, Mg²⁺, and Zn²⁺. Saci0542 showed high activity in a wide range of pH 5.5-10. NaCl above 50 mmol/L strongly inhibited the activity of this enzyme, and the optimum reaction temperature was 50-55℃. The terminal phosphate group inhibited the 3'-5' exonuclease activity. [Conclusion] This study confirmed that Saci0542 was a Mn²⁺-dependent 3'-5' exonuclease with the activity similar to that of NrnA nuclease, which may be responsible for DNA repair or RNA degradation and recycling in the cell.

Abstract:[Objective] To isolate and identify two endophytic nonconventional yeast strains from Usnea lichen samples collected in Tibet, and investigate their biological characteristics and application potential through genome mining, which was further experimentally validated. [Methods] We isolated two cold-tolerant yeast strains from the Usnea samples under 15℃ culture condition. These two strains were further identified by physiological tests, biochemical tests, 26S D1/D2 sequence analysis, and ITS sequence analysis. Then we performed genome sequencing and analysis, and further tested lipid production capability by Nile red dye staining. [Results] We identified two strains of cold-tolerant yeast as Curvibasidium rogersii, which grow well at 10℃, and the optimum temperature for growth was determined to be 20℃. The growth was poor or not observed at 25℃ and the above temperature. Sequencing and analysis of the genome revealed that the genome is the most similar to that of Leucosporidium creatinivorum, which has been reported as a microbial lipid producer. We found that these two strains can accumulate lipid. Moreover, a putative xylose reductase protein encoded gene was also identified in the genome, and we also confirmed growth of the two strains using xylose. [Conclusion] We isolated two cold-adaptive C. rogersii strains which were identified for the first time from Usnea lichen. Our results provide a basis for further exploration of microbial resources from Usnea species and other lichens, as well as using novel xylose-utilizing yeast strains for single cell lipid production.

Abstract:[Objective] In this study donkey strangles samples were collected from Xinjiang donkey farm, suspicious pathogenic bacteria were isolated and identified. Then we performed genotyping and evolution analysis of these isolates. [Methods] First, bacteria isolation and culture were conducted using the samples collected from the abscess of submandibular lymph node in donkey farm in Xinjiang and two isolates (HT111, HT321) were identified. Then the morphological observation, physiological and biochemical analysis, drug sensitivity test, 16S rRNA sequencing and phylogenetic tree construction were performed. Besides, the seven housekeeping genes were amplified by PCR and the sequences of genes were analyzed through multiple locus sequence typing (MLST). [Results] Two isolates (HT111, HT321) were identified as S. equi ssp. zooepidemicus (S. zooepidemicus) from the samples. The HT111 isolate was resistant to penicillin, charithromycin, and tetracycline and HT321 was resistant to eight drugs, amoxicillin, cefuroxime, ceftiofuroxime, penicillin, clarithromycin, clindamycin, oxytetracycline and tetracycline. MLST result showed that ST179 (HT111), ST420 (HT321) were found and ST420 (HT321) was the first identified type. [Conclusion] Two donkey S. zooepidemicus strains were isolated and one novel genotype ST420 was identified, which could provide some data for the epidemic investigation and the prevention and control of donkey strangle in the future.

Abstract:[Objective] The aims of this study were to clarify the spatial pattern of the nitrogen-fixing microbial community in the rhizosphere of Kudzu (Pueraria lobata), and to explore the relationship between environmental factors and the spatial pattern. [Methods] A total of 17 rhizosphere soil samples were collected from Hunan, Jiangxi, and Guangxi province, where Kudzu was widely distributed. The physical and chemical parameters of the samples were measured by routine methods, and the composition and diversity of nitrogen-fixing microbial community was performed by high-throughput sequencing of nifH gene. Multivariate statistical analysis was used to explore the relationship between the composition of the nitrogen-fixing microbial community and environmental factors. [Results] The α diversity of nitrogen-fixing microorganisms were not significantly different among three sampling regions. The composition of nitrogen-fixing microbial communities were similar in the same sampling region, while were quite different among three sampling regions. It indicated that biogeographical pattern of nitrogen-fixing microbial community was existed. The predominant phylum of nitrogen fixing microorganisms was Proteobacteria, whose relative abundance accounted for more than 60%. At the order level, the species that could be annotated mainly belonged to Rhizobiales and Burkholderiales. The relative abundance of Rhizobiales was highest in Jiangxi, while the relative abundance of Burkholderiales was highest in Hunan. pH, exchangeable calcium, total potassium, elevation and annual mean temperature were the main factors that influenced the composition of nitrogen fixing community. Burkholderiales was positively correlated with pH, exchangeable calcium and total potassium; while was negatively correlated with the ratio of carbon to nitrogen. Rhizobiales was positively correlated with total potassium and the content of sand, while was negatively correlated with pH, exchangeable calcium, elevation and annual mean temperature. [Conclusion] There was no significant difference in the α diversity of nitrogen-fixing microorganisms in the Kudzu rhizosphere soil across different geographical regions; while a clear biogeographical pattern of nitrogen-fixing microorganisms was existed. The spatial pattern of the community was jointly affected by the geographic and soil factors; in addition, the effects of the same environmental factor were distinct for different nitrogen-fixing microbial populations.

Abstract:[Objectives] To provide theoretical basis and technical support for exploiting and utilizing beneficial microbes and their functions, the mechanism of color formation in tomato fruits related to endophytic bacteria in stems of different tomato varieties were analyzed. [Methods] Based on MiSeq high-throughput sequencing technology, informatics analysis of the endophytic bacteria in stems of tomatoes between red and yellow varieties were conducted. [Results] At the genus level, Brachybacterium, unclassified_f_Enterobacteriaceae, Lactobacillus, Corynebacterium, Pseudomaonas, Microbacterium, norank_f_norank_o_Gaiellales, Chujaibacter, Sphigomonas were the specific dominant endophytic bacteria in stems of red tomato varieties; And Curtobacterium, norank_f_Mitochondria, Amycolatopsis and Trichococcus were the specific dominant endophytic bacteria in stems of yellow tomato varieties. Meanwhile, the abundance of endophytic bacteria and their functions in stems of red tomato varieties were higher than those of yellow tomato varieties. [Conclusions] The composition of endophytic bacteria in stems between with red and yellow fruits of tomato varieties formed specific dominant endophytic bacteria, respectively. And higher abundance of specific endophytic bacterial genera and gene functions were found in red fruit tomato varieties than those of the yellow variety. It suggests that the fruit color formations were significantly related to the composition and function of endophytic bacteria in plant.

Abstract:[Objective] We used a super-bright variant of GFP, superfolder GFP (SfGFP), to label the Bacillus velezensis FZB42 strain, so as to establish a universal GFP labeling method for B. velezensis. This helps to develop a viable technique to study the colonization mechanism of beneficial rhizobacteria. [Methods] A mutant strain was generated by integrating SfGFP into a neutral locus called xkdB in the genome of FZB42, and then the fluorescence intensities of the mutant strains carrying different GFPs were detected to evaluate the influence of xkdB knockout. [Results] In this study, a marker strain labeled with SfGFP for FZB42 was constructed, whose fluorescence intensity was more than 5 times that of the gfp+ variant marker strain. The knockout of xkdB did not affect the growth rate, carbon source utilization, motility or biofilm formation, which confirmed the feasibility of using xkdB as a neutral locus for exogenous gene expression. [Conclusion] In this study, we confirmed a new locus suitable for the expression of foreign genes in the FZB42 genome, and exogenously expressed SfGFP at this locus, thus labeling FZB42 with fluorescence. This study provides a reference for the labeling of similar strains.

Abstract:[Background] Autoinducer-2 (AI-2), ubiquitous in Gram-negative bacteria and Gram-positive bacteria, can mediate intraspecific and interspecific communication and regulate a variety of physiological processes. However, whether Pseudomonas putida KT2440 can sense AI-2 signal has not been reported yet. [Objective] To explore the chemotactic receptor mediating the chemotaxis of KT2440 to AI-2, and to detect the regulatory role of AI-2 in the biofilm formation of KT2440. [Methods] Firstly, soft agar plate assay and quantitative capillary assay were employed to detect the chemotactic response of KT2440 to AI-2. Then, the ligand binding domain (LBD) of the methyl-accepting chemotaxis protein McpU which had a high homology with that of the known AI-2 receptor TlpQ in Pseudomonas aeruginosa was expressed and purified. Vibrio harveyi MM32 bioluminescence and isothermal titration calorimetry (ITC) were adopted to detect the interaction between McpU-LBD and AI-2. Soft agar assay and quantitative capillary assay were carried out to evaluate the chemotaxis of the mcpU knockout strain (ΔmcpU) to AI-2. The effect of AI-2 on the biofilm formation of KT2440 and ΔmcpU was detected by crystal violet staining. [Results] KT2440 exhibited chemotaxis to AI-2. The bioluminescence and ITC assays showed that AI-2 bound to McpU-LBD with high affinity. The chemotaxis of KT2440 to AI-2 was mediated by McpU, and AI-2 significantly enhanced the biofilm formation in KT2440 via its receptor McpU (P<0.05). [Conclusion] McpU mediates the chemotaxis of P. putida KT2440 to AI-2, and AI-2 significantly enhances the biofilm formation of P. putida KT2440 by engaging McpU.

Abstract:[Objective] We studied the selenium tolerance, organic selenium content, and biological characteristics of the domesticated Armillaria mellea strains, so as to screen out the strains with strong biological activity. [Methods] Na₂SeO₃ was used as the inorganic selenium reagent to domesticate A. mellea. The selenium content and inorganic selenium content of A. mellea was determined by hydride atomic fluorescence spectrometry and hot water bath extraction method, respectively. Excellent A. mellea strains were identified based on morphological, physiological, and biochemical characteristics. [Results] A. mellea showed a good ability to accumulate inorganic selenium. The tolerance thresholds of strains J-234, M-H, A-10, and YN-2 to Na₂SeO₃ were 100, 60, 60, 20 mg/L, respectively. The selenium content in strains J-234, M-H, A-10, and YN-2 was 8.378 1, 7.249 4, 1.926 9, 3.606 1 µg/g, respectively. Strain J-234 had the highest biological activity after domestication, with the xylanase activity of 92 522.46 U/L, amylase activity of 11 951.49 U/L, cellulase activity of 8 439.47 U/L, laccase activity of 1.25 U/L, and polypeptide content of 0.087 g/L.[Conclusion] The addition of exogenous selenium enhances the biological activity of A. mellea, and this finding provides reference for the selenium domestication of edible and medicinal fungi.

Abstract:[Objective] The emergence of the multidrug resistant bacterial pathogens is posing great threats to food safety. Phages are the important bactericidal factors different from antibiotics. The evidence of phage application can be provided by analyzing the phage biological characteristics and genome. [Methods] In this study, one lytic phage phiP4-7 specific to the strain Plesiomonas shigelloides P4-7 was investigated on its biological characteristics, genome annotations, and taxonomic analysis. [Results] The images of transmission electron microscopy (TEM) showed that the phage has an icosahedral head with a diameter of (50.59±1.68) nm, a noncontractile tail with a length of (76.53±4.90) nm, belonging to the family of Siphoviridae. One-step growth curve suggested that phiP4-7 has a latent period of 25 min and its burst size was about 63 PFU/infection center. Additionally, phiP4-7 can effectively inhibit the growth of the host bacteria. The genome of phiP4-7 has been sequenced, assembled, and curated. It has a total of 39 825 bp with the GC content of 48.67%. Bioinformatic tools reveals that the phiP4-7 genome has 72 protein encoding genes, and no tRNA genes, antibiotic resistance genes, or virulence genes were identified. Comparative genomic and taxonomic analyses indicated that the genome of phiP4-7 showed little homology to any known viruses, and it can be classified as a member of a new genus of the Siphoviridae family. [Conclusion] A new Plesiomonas shigelloides phage was isolated, and its biological characteristics and genome were analyzed.

Abstract:Bioremediation in petroleum pollution has attracted wide attention due to its advantages including no secondary pollution and low cost. However, due to the large input of petroleum hydrocarbons into the environment, the relative shortage of nitrogen sources in the environment has become one of the key factors that restricted the efficiency of bioremediation. Therefore, screening microorganisms that can grow in the oligotrophic-nitrogen environments has important ecological significance. [Objective] To screen microorganisms that can grow on nitrogen-free conditions from the Liaohe Oilfield reservoir, and to provide candidate strains for the bioremediation of petroleum- contaminated environments. [Methods] The strains were isolated using modified nitrogen-free medium, followed by the analysis of 16S rRNA gene sequence and nitrogenase activity, and amplication of nitrogen fixation genes and petroleum degradation genes. [Results] 21 microorganisms were screened, belonging to 16 different genera, among which Pseudomonas accounted for the highest proportion. The analysis of nitrogenase activity showed that 8 microorganisms could detect acetylene reduction activity, 3 microorganisms successfully amplified the nitrogen-fixing gene nifH, and the remaining 13 microorganisms defined them as oligotrophic-nitrogen bacteria. Amplification of petroleum degradation-related genes in these 21 strains revealed that 5 strains have alkB monooxygenase genes or cytochrome P450 genes in their genomes. [Conclusion] The bacteria isolated from the reservoir water of the Liaohe Oilfield in this study can adapt to the oligo-nitrogen environment, coupled with the potential to degrade oil. This study enrichs the microbial diversity in oil-contaminated areas and provide bacterial foundation for microbial remediation.

Abstract:[Objective] To explore whether porcine deltacoronavirus (PDCoV) can infect and proliferate in different animal species-derived cell lines. [Methods] The Sichuan isolate CHN-SC2015 of PDCoV was inoculated in twelve cell lines derived from hamster, poultry, monkey, human and swine. After at least five blindly passages in each cell line, the virus was identified by RT-PCR, RT-qPCR, indirect immunofluorescence assay (IFA), and sequencing. [Results] PDCoV caused distinct cytopathic effect (CPE) in Vero, PAM, PK15, ST, and LLC-PK1 cells at the 1st passage (P1) and proliferated to various degrees in PAM, PK15, ST, and LLC-PK1 cells, while the CPE gradually disappeared during subsequent passages in Vero and PAM cells. Except that in the three susceptible cell lines (PK15, LLC-PK1, and ST), the viral copies of the infected cell lines gradually decreased with the increase in passages, and PDCoV could not be detected at P4 or P5 of DEF, Marc-145, HEK-293, ZYM-SIEC02, and PAM cells. PCR results showed that PDCoV could be detected only in CEF and Vero cells at P5. The IFA results showed that PDCoV could infect other cell lines except BHK-21 and ZYM-SIEC02, and specific immunofluorescence was observed in PK15, LLC-PK1, and ST cells at P1, P3, and P9. Therefore, only three cell lines (PK15, LLC-PK1, and ST) were suitable for serial passage, with the virus titers up to 10^7.11, 10^7.00, and 10^7.37 TCID₅₀/mL at P9, respectively. After passage in different cell lines, CHN-SC2015 accumulated 14 nucleotide mutations corresponding to 12 amino acid mutations. [Conclusion] This study indicates that PDCoV can infect a variety of cells in vitro, suggesting that it may have the potential of cross-species transmission.

Abstract:[Objective] Ligustrum lucidum Ait., a plant with landscaping and medical values as well as an excellent host plant for the resource insect Ericerus pela, is widely distributed in China. This study aims to investigate the community structure, species composition, and diversity of bacteria in four niches (leaf, stem, root, and rhizosphere soil) of L. lucidum, and to lay a foundation for the interaction between L. lucidum and its associated microbiota, so as to provide a scientific basis for exploiting and utilizing endophytic and rhizospheric bacterial resources. [Methods] The leaf, stem, root, and rhizosphere soil samples of L. lucidum were collected, and the V5-V7 region of bacterial 16S rRNA gene was sequenced with the Illumina MiSeq platform. The bacterial community structure, species composition, and function in different niches were compared with bioinformatics tools. [Results] The high-throughput sequencing generated 168 229 valid sequences for the four samples of L. lucidum. A total of 977 OTUs were annotated, belonging to 23 phyla, 54 classes, 138 orders, 227 families, and 399 genera. The bacterial community diversity decreased gradually in the order of rhizosphere soil, root, stem, and leaf, with the Shannon indexes of 4.514, 3.856, 2.704, and 1.908, respectively. The β-diversity analysis indicated that there were significant differences in bacterial community structure among four niches (R=0.842 6, P<0.05). The bacterial community structure was similar between leaf and stem (R=0.481 5, P>0.05) as well as between root and rhizosphere soil (R=0.888 9, P>0.05). At the level of phylum, Proteobacteria and Actinobacteriota were dominant in leaf, stem, and root, while Chloroflexi had the highest relative abundance in rhizosphere soil. The dominant genera mainly included Rhodococcus, Massilia, Sphingomonas, Burkholderia, Bradyrhizobium, and Acidothermus, some of which, like Bradyrhizobium and Burkholderia, were plant probiotics. Chemoheterotrophy was the predominant function in each niche. The bacterial community in the aboveground part (leaf and stem) mainly played a role in carbon and hydrogen cycle, while that in the underground part (root and rhizosphere soil) mainly played a role in nitrogen cycle. [Conclusion] The bacterial community diversity in different niches of L. lucidum showed a selective filtration mechanism from bottom to top. The niche differentiation of bacterial community structure was obvious in different samples, and plant compartment specificity existed in species composition. There were a variety of plant probiotics in L. lucidum-associated microbiota, which were worthy of further study and utilization.

Abstract:[Objective] Endophyte and rhizosphere microorganisms of tea plant not only play an important role in the fermentation of Pu-erh tea, but also promote the growth of tea plants and induce disease resistance of tea plants. Studying the community structure and their relationship can provide a theoretical basis for the development and utilization of microbial resources. [Methods] In this study, we sequenced the 16S ribosomal RNA (16S rRNA) gene of bacteria in tea leaves and rhizosphere soil by high-throughput sequencing technology, analysed the community structure of bacteria in tea leaves and rhizosphere soil. [Results] The results showed that endophytic bacteria in tea leaves were mainly composed of 76 genera, belonging to 9 phyla. Among them, the phyla with higher relative abundance were Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. At the genus level, the genera with high relative abundance were Lactobacillus, Blautia, Faecalibacterium, Pseudomonas, Rhodococcus, Bacteroide, and Prevotella. Rhizosphere soil bacteria were mainly composed of 198 genera, belonging to 10 phyla. The phyla with higher relative abundance were Proteobacteria, Chloroflexi, Actinobacteria, and Acidobacteria. At the genus level, the high abundance genera of bacteria in rhizosphere soil were the same as those in tea leaves, but the relative abundance of the same genus was different in the two. The abundance and diversity of endophytic bacteria in tea leaves were significantly lower than those in rhizosphere soil, but more than 50% of the endophytic bacteria were also identified in the rhizosphere soil. [Conclusion] Our findings would provide a basis for the bacterium-based strategies in bacterial manure and Pu-erh tea quality.

Abstract:[Objective] To study the transcriptional regulation of calR gene by the master quorum sensing (QS) regulators AphA and OpaR in Vibrio parahaemolyticus, and to investigate the regulatory actions of CalR on the transcription of type VI secretion system 1 (T6SS1) genes. [Methods] Total RNAs were extracted from the wild-type (WT) strain and the regulatory gene mutants. Quantitative real-time PCR was carried out to calculate the transcriptional variation of each target gene between WT and the mutants. Primer extension assay was employed to detect the transcription start site and the expression variation of each target gene between WT and the mutants. The promoter region of each target gene was cloned into the restriction endonuclease sites of pHRP309, which harbors a gentamicin resistance gene and a promoterless lacZ reporter gene. Thereafter, each recombinant pHRP309 plasmid was transferred into WT and the mutants. The transformants were cultured and then lysed, and a β-Galactosidase Enzyme Assay System (Promega) was used for quantifying the β-galactosidase activity in cell lysates. The over-expressed His-recombinant regulatory proteins were purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay (EMSA) and DNase I footprinting were adopted to analyze the binding activity of each His-recombinant protein to its target promoter DNA region in vitro. [Results] The expression level of calR gene was up-regulated gradiently with the increase in OD₆₀₀ value from 0.05 to 1.20. AphA operating at a low cell density had no regulatory activity on calR gene transcription, whereas OpaR operating at a high cell density indirectly activated the transcription of calR gene. Moreover, CalR bound to the promoter regions of the T6SS1 operons vp1388-1390, vp1393-1406, vp1400-1406, and vp1409-1407 to activate their transcription under the non-inducing growth condition. [Conclusion] The transcription of calR gene was indirectly activated by OpaR. CalR was required for the basal transcription of T6SS1 genes under the non-inducing growth condition.

Abstract:[Objective] We evaluated the immunoenhancement effect and mechanism of pig spleen transfer factor (TF) on Newcastle disease virus (NDV) La Sota strain attenuated vaccine to provide a theoretical basis for the prevention and control of Newcastle disease (ND). [Methods] We inoculated La Sota strain alone and La Sota combined with TF into specific pathogen free (SPF) chickens at four different doses, and challenged SPF chickens by reference virulent NDV strain F₄₈E₉ (10^4.7 ELD₅₀) 14 days post vaccination. In addition, we determined the concentrations of IL-6, IL-10, IL-16, and IL-21 in chicken peripheral blood by protein chip technology, the hemagglutination inhibition (HI) antibody titer by HI assay, and the viremia level of F₄₈E₉ by RT-PCR. [Results] The challenge test showed that the protection rates of vaccination with La Sota strain alone at the doses of 10^5.17, 10^4.17, 10^3.17, and 10^2.17 EID₅₀ were 100%, 55%, 0%, and 0%, respectively, and the median protective dose (PD₅₀) was 12 023 EID₅₀. The protection rates of vaccination with La Sota strain combined with TF at the corresponding doses were 100%, 75%, 0%, and 0%, respectively, and the PD₅₀ was 6 918 EID₅₀. The mortality of control group (non-immunized but challenged) and blank group (non-immunized and non-challenged) were 100% and 0%. Upon vaccination at the dose of 10^4.17 EID₅₀, the IL-6, IL-10, IL-21, and ND HI antibody titer (log₂x) of the chicken exposed to combined vaccination increased at most by 254.95 pg/mL, 62.10 pg/mL, 1.51 pg/mL, and 2.6 (P<0.01), respectively, compared with those in the chicken vaccinated with La Sota strain alone. After challenge, the IL-10, IL-16, IL-21, and the viremia levels in the chicken under combined vaccination decreased at most by 428.61 pg/mL, 167.81 pg/mL (P<0.01), 1.48 pg/mL (P<0.01), and 20%, respectively, compared with those in the chicken vaccinated with La Sota strain alone. [Conclusion] TF improves the immune protection rate of NDV attenuated La Sota strain vaccine, the immune response mediated by IL-6, IL-10, and IL-21, and the ND HI antibody titer. In addition, TF reduces the PD₅₀, the inflammatory responses mediated by IL-16 and IL-21, the inhibition of immune response mediated by IL-10, and the viremia after challenge. It is clear that TF has a good immunoenhancement effect on the La Sota strain attenuated vaccine, which will provide a reference for research and development of vaccine adjuvant as well as prevention and control of ND.

Abstract:[Objective] This study was conducted to explore the diversity of dietary patterns and gut microbiota among local and immigrant pre-adolescent children in Guangdong province. [Methods] 48 local children and 34 immigrant children in Shenzhen city were randomly selected for food frequency questionnaire survey and morning feces collection. In addition, differences concerning the frequency of dietary intake between local and immigrant children were analyzed by the Mann-Whitney U test and used the Illumina Miseq high-throughput sequencing platform to analyze the gut microbiota. [Results] Among all children, the dietary patterns were mostly rice as the staple food, with vegetables, meat, and fruits. However, the significantly higher intakes of carbohydrates enriched foods, vegetables and seafood in the local group, while significantly higher intakes of fruits, low-fat milk and yogurt in the immigrant group. The immigrant group was associated with increased α-diversity assessed by the Chao1 (P<0.001) and Ace (P<0.001) index. Furthermore, at the genus level, a significant increase in Prevotella and Parabacteroides was observed in the local group (P<0.05). The principal coordinate analysis biplots (ANOSIM, P>0.05) showed no significant changes among the two groups. In addition, the linear discriminant analysis effect size analysis indicated Prevotellaceae (family) and Prevotella (genus) were markedly higher in the local group. The result of redundancy analysis indicated that the intake of vegetables and low-fat milk significantly affected the distribution of intestinal flora in local children, and suggested that the dietary habits of local children may affect the abundance of Prevotella and Parabacteroides. [Conclusion] This study revealed that the differences in diet among local and immigrant children. A significant correlation between the intake of vegetables and low-fat milk and gut microbiota in local children was observed, which provided a new theoretical foundation for understanding the effects of diet on gut microbiota.

Abstract:[Objective] This study aims to reveal the effect of the long non-coding RNA (lncRNA) CTO-S on the replication of pseudorabies virus (PRV). [Methods] We employed 3'RACE and 5'RACE to identify the full-length sequence of CTO-S, and verified whether CTO-S had the ability to encode polypeptides. After constructing the CTO-S deletion and complementation strains with the Red recombination method, we analyzed the differences in the replication of wild-type, deletion, and complementation strains. We used RNA pull down combined with mass spectrometry to screen out the host protein interacting with CTO-S, and then adopted RNA immunoprecipitation (RIP) method to verify the interaction between the screened protein and CTO-S. Finally, the methyl tetrazolium (MTT) method was used to detect the effects of wild-type, deletion, and complementation strains on cell apoptosis. [Results] CTO-S had a full length of 267 bp and did not have the ability to encode polypeptides. The deletion of CTO-S did not affect the replication of PRV. CTO-S interacted with cytochrome b-c1 complex subunit 1, a component of ubiquinol-cytochrome c reductase, and induced apoptosis. [Conclusion] CTO-S is not essential for the replication of PRV. It interacts with cytochrome b-c1 complex subunit 1, a component of ubiquinol-cytochrome c reductase, and induces cell apoptosis.