Abstract:Herpesviruses are a group of enveloped, double-stranded DNA viruses that can infect human and various animals, and induce herpes lesions in skin, mucous membranes and nerve tissues. Their invasion into host cells is the initial step in the virus life cycle, and receptors are the important host factors that mediate virus invasion, including attachment, internalization and membrane fusion. Therefore, to study receptors is important to understand viral invasion and to develop antivirals. We review here the cellular receptors and their mediation of membrane translocation. Current problems and future research needs are also discussed.

Abstract:Microbes own structural diversity and functional diversity, and their ecological behavior is regulated by various signal molecules. Among the signal factors, a most typical one is quorum sensing (QS). QS is a form of intercellular communication within microbial populations and occurs in a density-dependent manner. QS substance can result in down-stream changes in gene regulation and modulation of bioluminescence, virulence, biofilm formation and secondary metabolite production. Corresponding to QS, quorum sensing inhibitors (QSI) has also gradually been noticed in recent years. Previously, QSI is widely applied in medicine, agriculture and environmental sectors. They can exhibit multiple biological functions by regulate QS signal, such as reducing bacterial pathogenicity, interfering biofilm formation, blocking signal receptors and influencing QS-based cascading effects. We reviewed the latest advances of QSI, including their sources, properties, and mechanisms of action. In addition, the ecological applications of QSI in aquaculture, marine antifouling, sewage treatment and marine drug development have also been discussed, to supply novel strategies for developing QSI compounds. We provide herewith a reference for readers to understand QSI roles and using marine source more efficiently.

Abstract:Electroactive microorganisms (EAMs) with capability for extracellular electron uptake are microorganisms that can use electrons from extracellular solid to reduce carbon dioxide or other oxidative substances into extracellular organics, reductive inorganics or intracellular life-supporting organics. The finding of these EAMs broadens our knowledge of microbial diversity. They have significant practical applications in biomass energy synthesis, contaminant treatment and chemical detection. In this review, the substance transformation and electron recovery efficiency of representative EAMs with capability for extracellular electron uptake were introduced. The direct electron uptake mechanism based on membrane-bound proteins and the indirect electron uptake mechanisms based on electron shuttles were summarized. Application potentials in the microbial electrosynthesis system and the microbial sensor were proposed. And future research directions of these EAMs were discussed from the perspectives of mechanism study, biofilm micromechanism and engineering application.

Abstract:[Objective] To study the effect of p-nitrophenol (PNP) on persisters of Escherichia coli and Pseudomonas aeruginosa and to analyze the transcriptome to illustrate the impact of PNP on persister formation.[Methods] Ofloxacin was used to determine the bacteria persisters number. Cell self-digestion assay was done and the effect of respiratory inhibitors carbonyl cyanide chlorobenzene hydrazone (CCCP) on bacteria persisters was studied as well. Based on analysis of bacterial transcriptome, two genes (cyoA and appC) associated with the formation of persistence were screened. Their expressions were confirmed by Real-time Fluorescence Quantitative Polymerase Chain Reaction (PCR). The association between the two genes and the formation of persistence was also checked by antisense oligodeoxynucleotide assay.[Results] PNP inhibited the respiration of E. coli and P. aeruginosa, thus increased the proportion of bacterial persisters. PNP concentration, PNP function time and the bacterial growth period affected the proportion of bacteria persisters. PNP and CCCP inhibited the self-digestion of E. coli and P. aeruginosa, including the changes in dissolved oxygen, protein degradation and cell size, and the integrity of RNA. The results from transcriptome analysis and Real-time fluorescent quantitative PCR showed that PNP addition decreased the expression of cyoA and appC in E. coli and P. aeruginosa. Through the antisense oligodeoxynucleotide inhibitory expression of cyoA and appC was found that bacteria persister proportion increased compared with the original strain.[Conclusion] PNP can increase the proportion of bacteria persisters by inhibiting the cellular respiration.

Abstract:[Objective] We studied the function of glnA, proB and proA genes in the pathway of proline biosynthesis in Bacillus subtilis.[Methods] B. subtilis WB600 was used as the original strain. After a series of intracellular gene deletion and overexpression, we constructed recombinant strain WB601 with proB gene overexpression and WB602 with proA gene overexpression; WB603 with glnA gene deletion. Then, WB604 with proB gene overexpression was constructed. The anabolic key nodes were analyzed by phenotypes of extracellular and intracellular free proline accumulation.[Results] Under non-stress conditions, the extracellular proline content of the recombinant strains WB601 and WB602 were 2.21 times and 2.82 times of that of the original strain, the unit cell extracellular proline yield were 4.09 times and 9.80 times of that of the original strain, and the intracellular free proline content were 1.91 times and 3.34 times of that of the original strain, respectively. The extracellular proline content of the recombinant strain WB603 increased to 1221.43 mg/L, 6.28 times of that of the original strain, and the unit cell extracellular and intracellular free proline yield were 9.13 times and 3.66 times of that of the original strain, respectively. The extracellular proline content of the recombinant strain WB604 up to 1391.65 mg/L, compared with strain WB603, the extracellular proline content and unit cell yield were increased by 13.94% and 14.10%, respectively, and the intracellular free proline content increased by 32.60%. Under 5% NaCl stress, the extracellular proline content of recombinant strains WB601 and WB602 were 1.94 times and 1.54 times of the original strain, and the unit cell yield were 2.15 times and 2.19 times of the original strain, respectively; the extracellular proline content and unit cell yield of recombinant strain WB603 were 4.16 times and 7.29 times of the original strain, respectively; under the same conditions, compared with the recombinant strain WB603, the extracellular proline content and unit cell yield of the recombinant strain WB604 were increased by 32.61% and 5.54%, respectively. In addition, the intracellular free proline content of the experimental groups were higher than that of non stress conditions, and reached a relative equilibrium state.[Conclusion] Overexpression of proB and proA gene can significantly enhance the ability of cells to synthesize proline and enhance the salt tolerance of cells; deletion of glnA could enhance proline de novo synthesis pathway and increase the accumulation of proline; moreover, the positive accumulation of the two effects could further improve the ability of proline synthesis.

Abstract:[Objective] Bartonella quintana, transmitted by the body louse (Pediculus humanis), is the etiologic agent of a variety of disease manifestations in humans including trench fever. To further confirm if macaques can be the natural reservoir of B. quintana, we studied the infections of B. quintana in the experimental macaque populations from the four regions in China and characterized the islolates of B. quintana originated from the macaques.[Methods] The samples of blood and serum collected from 550 macaques were used to isolate strains, detect the specific nucleic acid fragment and test IgG antibody. The molecular taxonomic characterization and nucleotide polymorphisms of the strains were analyzed based on the sequences of the six housekeeping genes using PCR, sequecing and phylogenetic analysis. The fingerprint pattern of random amplified polymorphic DNA (RAPD) were also used to discriminated the stains from the different hosts by the three random primers. The IgG antibody anti-B. quintana were detected using indirect iocmmunofluorescence assay (IFA).[Results] Totally, 8 strains of B. quintana were isolated from 5 females and 3 males from 250 rhesus macaques in Sichuan province, with 3.2% infection rate. Our molecular test, detecting 550 blood samples using qPCR with a total of 8.2% infection rate of B. quintana, showed that the prevalence of macaques from Sichuan was significantly higher than that of the macaques from the other areas. The seropositivity rate was 19.0% in the rhesus macaques which was higher than that of the cynomolgus macaques (5.6%) and there was no difference between males and females and among the different areas. The RAPD-figerprints of the strains from the rhesus macaques in Sichuan province have an identical 8-bands pattern and was discriminated from the pattern of B. henselae. While the fingerprint of the human str. Fuller of B. quintana showed a 6-bands pattern. According to the nucleotide polymorphism analysis, the strains of B. quintana from different hosts show obvious genetic diversity. There was a higher diversity between the monkey strains and the human strains and a smaller difference among the monkey strains.[Conclusion] There are high prevalence of B. quintana in the macaques population in China. The natural infection rate and antibody level in the rhesus macaques were significantly higher than that of the cynomolgus macaques. The genotype of the strains originated from the macaques is different from that of the strains from human. We demonstrated that the macaques are the natural reservoir of B. quintana.

Abstract:[Objective] This study was aimed to explore the effect of different casein hydrolysates on small intestinal bacteria of pigs.[Methods] Luminal and gut wall bacteria from duodenum, jejunum and ileum in Duroc, Landrace and Yorkshire hybridization pigs were used as inocula. Casein acid hydrolysate and casein enzymatic hydrolysate were fermented at 37℃ for 12 h. Samples were collected at 0, 3, 6 and 12 h to measure microbial protein and detect the alteration of bacteria community.[Results] After inoculated with the luminal bacteria, the concentration of microbial protein in duodenum and ileum in casein enzymatic hydrolysate group was higher compared with casein acid hydrolysate group and the casein enzymatic hydrolysate group had higher total bacteria number and Firmicutes in duodenum after 12 h incubation (P<0.05). In the casein enzymatic hydrolysate group, the numbers of total bacteria, Lactobacillus and Escherichia coli were higher than those in casein acid hydrolysate group after 12 h incubation in ileum. For the gut wall bacteria:in duodenum and ileum, after 12 h incubation, the concentration of microbial protein in the casein enzymatic hydrolysate group was higher than that in casein acid hydrolysate group (P<0.05). The number of Lactobacillus and Firmicutes in the casein enzymatic hydrolysate group was higher than those in the casein acid hydrolysate group in duodenum (P<0.05) and the number of Firmicutes was higher in the casein enzymatic hydrolysate group than that in the casein acid hydrolysate group in ileum. (P<0.05).[Conclusion] Compared with casein acid hydrolysate, casein enzymatic hydrolysate can promote the bacterial protein synthesis and stimulate the growth of Firmicutes in duodenum and ileum.

Abstract:[Objective] We studied the immunomodulatory potential of Lactobacillus casei Zhang on preventive and therapeutic effects.[Methods] A murine model of peanut allergy was established and fed with L. casei Zhang. Then we measured the levels of IgE, IgG1 and IgG2a antibodies in blood serum, histamine and IgA antibody in feces, and cytokines and CD4⁺Foxp3⁺ Tregs cells.[Results] Intragastric administration with L. casei Zhang, prior or after allergic sensitization, increased levels of serum allergen-specific IgG2a and fecal total IgA antibodies. Additionally, systemic anaphylactic symptoms and Th2 responses such as total and specific IgE antibodies, histamine release and IL-4 production were decreased. In contrary, L. casei Zhang had no obvious influence on the serum IgG1 antibody and other cytokines (IL-10, IL-12 and IFN-γ). Successful immunomodulation by pre-and post-treatment were further demonstrated by the increase of IFN-γ/IL-4 ratio in vitro and enrichment of CD4⁺Foxp3⁺ Tregs in spleen and mesenteric lymph nodes.[Conclusion] L. casei Zhang could shift the Th2 dominant response to a Th1 response, which finally play a role in preventing and treating peanut allergy.

Abstract:[Objective] Composition and concentration of organic pollutants in cow dung were determined, which were then supplemented in the media for growth and decomposing studies using microbial community in native cow dung fermenting environment.[Methods] Fermentation optimization, strain acclimation, organic acid foulants degradation and rDNA high-throughput sequencing technology were applied to analyze the microbial diversity and relative biomass of beneficial microorganisms in samples and cultures.[Results] The odor in dairy waste was mainly due to short-chain organic acids, and the microbe in cow dung fermenting environment could biodegrade pollutants after experiencing an adaptive lagging phase. The microbial diversity of enrichment culture grown in high concentration of organic acid foulants (W/V, 0.1% to 0.2%) was also investigated. Prokaryotic species predominantly belong to Bacillus or Paenibacillus, and the preponderant fungi are Monascus fuliginosus and Pichia farinose.[Conclusion] These isolated microorganisms can be further exploited for odors removing treatment in cow breeding industry.

Abstract:[Objectives] In order to study the mechanism of heterologous protein expression in Komagataella phaffii, transcriptomics technology was used to study the profile of differentially expressed genes in the recombinant K. phaffii during the methanol-induced expression of phospholipase A₂.[Methods] We used RNA sequencing (RNA-seq) technology to analyze the differentially expressed genes in the recombinant K. phaffii expressing phospholipase A₂ between glycerol and methanol cultivation condition.[Results] In total 5225 transcripts were identified in the recombinant K. phaffii. Compared between glycerol and methanol cultivation condition, 857 genes were significant differentially expressed. According to KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, the differentially expressed genes were mostly related to ribosome composition, methanol metabolism, pentose phosphate pathway, citric acid cycle, glyoxylic acid cycle and protein processing process.[Conclusions] A global effect on the metabolism was observed when changing the carbon source from glycerol to methanol for the induction of heterologous protein. Our results provide rich information for further in-depth studies of the mechanism of protein expression in K. phaffii.

Abstract:[Objective] To establish an efficient method for promoter characterization, the interaction between E. coli promoter and RNA polymerase (RNAp) was studied by atomic force microscope (AFM) based force spectroscopy.[Methods] Protein immobilization was optimized, and the specific promoter/RNAp interaction was verified using core-RNAp (RNAp-C) as control. The force spectrum of promoter Ls2, lack of -10 region of Ls1, was studied.[Results] Based on the established method, the specific interaction between promoter Ls1 and RNAp was studied, and the rupture force was measured as (331.10±5.14) pN. Ls2 showed significantly less binding events towards RNAp compared with Ls1. Using promoter-probe plasmid, the activities of Ls1 and Ls2 were verified by reporter gene-cat, which were (181.73±4.08) U/mg and (0.33±0.21) U/mg, respectively.[Conclusion] A novel promoter analysis method based on AFM force spectroscopy was established. The results demonstrated this method can be applied in quantitative characterization of promoter with high efficacy and reliability.

Abstract:[Objective] We highly expressed the high temperature resistant pectin methylesterase from Talaromyces leycettanus JCM12802 in Pichia pastoris and studied its enzymatic properties. Pectin methylesterase with high catalytic efficiency at high temperature was expected to be widely used in the production of low-methoxyl pectin, for an optimal production process and conversion rate with reduced production cost.[Methods] We cloned the cDNA of pectin methylesterase gene (PmeT) from the total RNA of T. leycettanus JCM12802 as template by RT-PCR, which was inserted into the expression vector pPIC9K and transformed into P. pastoris GS115. We cultivated the high activated positive transformant for high-density fermentation.[Results] The recombinant pectin methylesterase (r-PmeT) expression level reached 428 U/mL. We have identified the enzymatic properties of r-PmeT. The optimum reaction temperature of r-PmeT was 75℃, and its thermostability was below 85℃. The optimum pH was 4.0, and it was stable between pH 2.0 and 7.0.[Conclusion] The recombinant Pichia pastoris could express high level pectin methylesterase from T. leycettanus JCM12802, which shows excellent application potential in its future industrial.

Abstract:[Objective] By testing the virulence of single and multiple GALA mutants generated in Ralstonia solanacearum strain OE1-1, we evaluated the pathogenicity of these effectors in different host plants.[Methods] The deletion mutant of R. solanacearum OE1-1 was constructed through double crossover. Deletion of the target gene was confirmed by colony PCR. Virulence assay and bacterial multiplication were measured by root-cutting and leaf infiltration respectively. Tobacco (Nicotiana tabacum cv. Bright Yellow) and tomato (Lycopersicon esculentum cv. Moneymaker) were adopted as host plants to inoculate.[Results] Deletion mutants of each effector gene barely affected the bacterial virulence in tobacco. A mutant RK7022 with the deletion of all seven gala genes showed 2-days delayed virulence on tobacco compared with the wild type. The bacterial multiplication capacity of deletion mutants correlated with the phenotypes on host plants. Pathogenicity test on tomato among GALA mutants and wild type OE1-1 showed no difference.[Conclusion] GALA effectors contributed collectively to pathogenicity of OE1-1 on tobacco but not on tomato. Some effectors might play more important roles in R. solanacearum pathogenicity.

Abstract:[Objective] The aim of this study is to reveal core microbiota during Chinese liquors fermentation, and to quantify the impact of environmental factors on the variations of the core microbiota.[Methods] The microbial community composition was characterized by high-throughput sequencing. The volatile compounds were analyzed by gas chromatography-mass spectrometry. Core microbiota was defined according to the microbial function and microbial correlation patterns using the microbe-metabolite correlation approach and co-occurrence network analysis. We used redundancy analysis and Monte Carlo permutation test to analyze the influence of environmental factors on the core microbiota.[Results] The core microbiota during the liquor fermentation consisted of Lactobacillus, Saccharomyces, Candida, Rhizopus, Saccharomycopsis, Pichia, Dipodascus, Bacillus, Thermoascus and Lactococcus. The variations of the core microbiota were mainly affected by the chemical factors such as reducing sugar and ethanol. In addition, the interaction between physical and chemical factors was also substantial for the core microbiota variations.[Conclusion] This study disentangled the relationship between microbial communities and metabolites, found out the core microbiota in the liquor fermentation process, and quantified the influence of environmental factors on the variations of core microbiota, hence providing a theoretical perspective for regulating the production of liquors by using synthetic microbial communities or by controlling environmental factors.

Abstract:[Objective] To improve n-butanol tolerance of Escherichia coli by screening and engineering the signal transduction pathways thinvolved in solvent stress response.[Methods] Under butanol stress, expression of response regulator in membrane signal transduction pathways of E. coli was determined and analyzed using RT-PCR. Key regulating components of stress response pathway were deleted and over-expressed in E. coli through red-homologous recombination and one-step clone. Solvent tolerance and membrane hydrophobicity analysis of the deleted and over-expressed strains were conducted against six different organic solvents.[Results] Expression level of cpxR and baeR in two-component stress response pathways Cpx and Bae was increased by 8.3 and 3.3 folds, respectively, after n-butanol (0.8%, V/V) treatment for 10 h. Under the solvent stress of tetrahydrofuran (0.6%, V/V), toluene (0.1%, V/V) and cyclohexane (0.6%, V/V) for 10 h, the OD₆₀₀ of recombinant E. coli JM109/pQE80L-spy and E. coli JM109/pQE80L-nlpE were increased by 0.13-0.17 and 0.05-0.13, respectively, compared with the control (ΔOD₆₀₀ of 0.02-0.04). Organic solvent tolerance of E. coli was improved.[Conclusion] Two component stress response pathways, Bae and Cpx, participate in the response of butanol stress. Overexpression of Spy could effectively improve organic solvent tolerance of E. coli. This study provides theoretical guidance for elucidating the mechanisms of microbial organic solvents tolerance.

Abstract:[Objective] Germination-related proteins in Antrodia camphorata athroconidia were analyzed by two-dimensional gel electrophoresis (2DE), mass spectrum, and real time fluorescent quantitative PCR (RT-qPCR).[Methods] We used 2DE to analyze total proteins of Antrodia camphorata arthroconidia after 0 h and 24 h of incubation. We identified differential proteins by PDQuest software and MALDI-TOF-MS. Then, we obtained germination-related proteins in Antrodia camphorata arthroconidia by matching the amino acid sequences of identified proteins to a local protein database. Finally, we used RT-qPCR to quantify relative expression levels of germination-related genes.[Results] A total of 32 differential expressed proteins, of which 25 up-regulated and 7 down-regulated, existed between non-germinated (0 h) and germinated (24 h) arthroconidia. Among these differential proteins, 24 proteins were successfully identified, and 10 proteins were involved in arthroconidial germination including GerO, Ubc1, Cat-1, Snf1, Cas2, SfaD, Chaperonin, Fad5, Tyrosine-P, and ChiA.[Conclusion] The results provide a theoretical basis for understanding of molecular mechanisms of athroconial germination of Antrodia camphorata.

Abstract:The article summarized proposals that received and funded by National Natural Science Foundation of China from 2008 to 2017 in the field of microbial resources, taxonomy and phylogeny. In view of the prominent problems in the funding and management of the projects, it makes some suggestions on the future work.