Abstract:Anaerobic fungi are among the most effective lignocelluloses-degrading microbes in nature. Recently, increasing number of the co-cultures of anaerobic fungi with methanogens have been isolated. In the co-culture, methanogens utilize metabolites produced by anaerobic fungi, enhancing the lignocelluloses-degrading ability of anaerobic fungi; anaerobic fungi provide methanogens with energy and nutrients, with which methanogens rapidly generate substantial methane. A comprehensive and in-depth understanding of the mutual interaction and the lignocelluloses-degrading methane-producing characteristics of the co-culture will be conducive to investigating the regulation of the lignocellulosic degradation and methane production. Thus, in this review, we summarized the isolation and identification, the diversity and the mutual interaction of the co-culture, and the decomposition of lignocellulosic material by the co-culture.

Abstract:Gut microbiota affects human nutrition, metabolism, immune development and functionality, and other physiological activities. Recent findings have revealed the roles of gut microbiota in susceptibility and prognosis in virus infection diseases. Here, we summarize the relationship, interaction between gut microbiota and enteric virus infection diseases, as well as non-enteric virus infection diseases. In addition, the potential therapies for modulating microbiota to prevent or treat virus infection diseases are discussed. This review will provide new ideas for further related research on prevention and control of virus infection diseases.

Abstract:Elemental sulfur (sulfur globules) is an intermediate product of biological oxidation of sulfide. Elemental sulfur can be the main metabolite of sulfur-oxidizing bacteria (SOB) according to stoichiometric O/S ratio. According to the distribution of elemental sulfur, they can be divided into intracellular sulfur globules and extracellular sulfur globules. The transmembrane transport of elemental sulfur from the inside to outside of cell is an important physiological characteristic of sulfur-secreting SOB. Recovery of elemental sulfur from biodesulfurization system is of great significance to the realization of waste recycling. In this paper, the formation process, occurrence form, metabolic characteristics, transport mechanism of elemental sulfur in SOB and the recovery of elemental sulfur from biodesulfurization system were reviewed in order to provide a reference for the research and development of a new biodesulfurization process.

Abstract:In the past decade, metallo-β-lactamases, represented by New Delhi metallo-β-lactamases (NDM-1), have been widely spread around the world, threatening public health and safety. Especially in recent years, the emergence of mutants of these enzymes has caused more complex and difficult challenges to human health. At present, there is still a lack of effective treatment drugs and means. The key to solve this problem is to develop effective broad-spectrum inhibitors of the enzyme. Therefore, we review the three-dimensional structure analysis of NDM-1 and its related inhibitor complexes, to give some inspiration and to help relevant researchers from the perspective of biological mechanism research.

Abstract:The needs to increase vegetation coverage and plants’ immunity in ecological restoration are important topics of current research. Microbe-assisted phytoremediation has been considered as the most promising technology for the remediation of ecology. Arbuscular mycorrhizal fungi (AMF) and dark septate endophyte (DSE) are soil-borne beneficial fungi that form root-fungus association with plant roots and provide a variety of benefits to the host plants. Moreover, AMF and DSE generally help plants uptake nutrient better and can enhance plant tolerance to many biotic and abiotic stresses, and thus they can be used to control fungal diseases and promote plant growth in extreme environment and polluted soil. The purpose of the present research is to investigate the function and application of AMF and DSE in ecological restoration and provide a basis in studying synergic mechanisms by these fungi. The possible problems and prospects of AMF and DSE in ecological restoration are involved.

Abstract:Xanthomonas represents a range of bacterial species that cause diseases in a variety of crops. They produce and use DSF (Diffusible signaling factor)-family quorum sensing (QS) signal to sense cell population to regulate the expression of virulence genes. At post-logic growth phase, the levels of DSF-family signal decreased rapidly, indicating a naturally occurring signal turnover phenomenon in Xanthomonas. The expression levels of rpfB that encodes a putative fatty acyl CoA ligase, significantly increased at logarithmic growth phase. Deletion of rpfB significantly increased the production of DSF-family signal. Therefore, RpfB is involved in the degradation of DSF-family signals, including Xcc to exit from the quorum sensing phase at post-QS phase. DSF-family signal negatively regulates rpfB expression via rpfC/rpfG two-component signaling system, cyclic di-GMP and the global regulator Clp. The RpfB-dependent naturally DSF turnover system is present in a range of bacterial species, representing a conserved mechanism for QS signal turnover. However, the RpfB-regulated biological roles differ from species to species.

Abstract:[Objective] Isolation of strains with high-efficiency of phosphorus-dissolving and growth-promoting functions from sugarcane leaf compost has provided available microbial resources for bio-fertilizers production. [Methods] Phosphorus-dissolving strains were screened from insoluble inorganic phosphorus media containing Ca₃(PO₄)₂ or Zn₃(PO₄)₂, and identified by morphological characteristics and ITS rDNA sequence analysis. The phosphorus-dissolving ability of the strains was determined by the halo zone and molybdenum antimony colorimetric methods, and the growth-promoting ability of the strain was analyzed by pot experiment of pepper. [Results] Strain DC30-2-P1 was screened from the compost and identified as Aspergillus awamori. The diameter of the colony of the strains (d) was 55.33 mm and 45.00 mm, the halo zone (D) was 65.33 mm and 67.67 mm, and the D/d value was 1.16 and 1.50 on the medium with Ca₃(PO₄)₂ and Zn₃(PO₄)₂, respectively. After 96 hours of cultivation in a liquid medium with Ca₃(PO₄)₂ as the phosphorus source, the available phosphorus content reached 2974 mg/L. The results indicated that strain DC30-2-P1 had a significant effect on dissolving phosphorus. The results of the pot experiment show that the inoculation of DC30-2-P1 promoted the growth of pepper. Compared with the treatment group without phosphate solubilizing fungi, the above-ground fresh and dry weight increased by 18.6% and 43.5%, the under-ground fresh and dry weight increased by 30.2% and 25%, plant height increased by 13.6%, and chlorophyll content increased by 44.9%, both plant total phosphorus content and soil available phosphorus content increased by more than 80%. [Conclusion] Strain DC30-2-P1 from sugarcane leaf compost has a high efficiency on dissolving insoluble phosphorus compounds, promoting the absorption of phosphorus and improving crop yield.

Abstract:[Objective] To establish a rapid, simple and efficient Agrobacterium-mediated genetic transformation system for Chlamydomonas reinhardtiii, we used the model organism C. reinhardtiii as the receptor material and optimized the Agrobacterium-mediated transformation system of C. reinhardtiii from two aspects: transformation method and transformants identification method. [Methods] We compared the effect of solid co-culture and liquid co-culture on the transformation efficiency of C. reinhardtii CC425 mediated by A. tumefaciens LBA4404. Besides, we analyzed the optimal reaction conditions and amplification efficiency of (1) two-step PCR after TE cleavage, and (2) one-step PCR without TE cleavage. [Results] The highest transformation efficiency was achieved by a 5-day liquid-medium co-culture of Agrobacterium and Chlamydomonas. The transformation rate was 43.33±1.67 transformants/10⁶ algal cells. The optimal reaction conditions were: amplification with high fidelity DNA polymerase Taq 1; the cell density involved in PCR was 5×10³–5×10⁶ cells/mL; before amplification, cells were boiled in TE lysis buffer for 20 min (two-step PCR method), or initial denaturation for 15 min (one-step direct PCR method). The amplification efficiency of two-step PCR is better than that of one-step PCR, but the latter is more concise. [Conclusion] Agrobacterium-mediated transformation system of C. reinhardtii was established and optimized, through which rapid genetic transformation can be fulfilled and the workload could be reduced.

Abstract:[Objective] Three lactobacillus strains and four prebiotics for symbiotic combination were screened and their effects on in vitro colonic fermentation characteristics were investigated. [Methods] Lactobacillus reuteri L45, Lactobacillus plantarum L47 and Lactobacillus reuteri L63 were separately added to the medium with inulin, fructo-oligosaccharide, galacto-oligosaccharide or lactulose as the sole carbon source; the growth activity and acid production characteristics of the strains for 24 h were used to select the optimal combination; in vitro fermentation was used to investigate the effects of symbiotics on microorganisms and fermentation characteristics. [Results] The growth curves of L45 and L63 fermentation with fructo-oligosaccharide and lactulose as carbon sources were similar to glucose respectively; the OD₆₀₀ of inulin and fructo-oligosaccharide with L47 was significantly higher than glucose (P<0.05), and the lactic acid concentration of inulin was 1.20 times higher than glucose; L47 with fructo-oligosaccharide and L47 with Inulin have better composite effects. In vitro fermentation results showed that the combination of two synbiotics (L47+fructo-oligosaccharide and L47+Inulin) significantly increased the relative abundance of Lactobacillus and Bifidobacterium compare to the control (P<0.05); L47+FOS and L47+Inulin increased the concentration of total short chain fatty acid (P<0.05). [Conclusion] L47+fructo-oligosaccharide and L47+Inulin had favorable combination effects, implying that they had the development potential as symbiotics. The effect of both combinations in vivo remains to further investigated.

Abstract:[Objective] To isolate, identify and evaluate the safety of Clostridium butyricum from two breeding hen farms in Shandong province. [Methods] Anaerobic culture was used to isolate anaerobic bacterial strains from both Luhua chicken and SPF bird droppings originated in Shandong Province. Suspicious colonies were selected for mass spectrometry and then identified by 16S rRNA gene sequencing. The 16S rRNA sequencing results were analyzed for homology with 16S rRNA sequence of Clostridium butyricum in NCBI nucleotide data. Meanwhile, all isolates were tested for susceptibility to 9 drugs, such as ofloxacin and cefepime. PCR was used for the determination of 23 antimicrobial resistance genes such as mefA. Four clostridium toxin genes including alpha and four botulinum toxin genes including typeA were determined based on probiotic safety requirements. [Results] A total of 24 strains of Clostridium butyricum were identified, and they were sensitive to 7 antibiotics such as ofloxacin. L-1, L-6 and L-12 were moderated only for neomycin and L-19 was moderated only for cefepime. All strains of 16 drug-resistant genes were negative, and 3 drug-resistant genes of sul2, flor and bla_TEM were positive, tetC carrying rate is 79.2%, cmlA carrying rate is 45.8%, bla_OXA carrying rate is 37.5%, aadB carrying rate is 12.5%, qnrA carrying rate is 4.2%. PCR results showed that the alpha, beta, epsilon, iota clostridium toxin gene carrying rates of all isolated strains were 0%. The carrying rate of botulinum toxin genes typeA, typeB, typeE and typeF of all isolates was 0%. [Conclusion] The 24 isolates of Clostridium butyricum from tested chickens never fed with antibiotics and Clostridium butyricum met the expected safety requirements and could be used as a screening reference strain for probiotic added bacteria.

Abstract:[Objective] Characterization of the aflatoxin B1 (AFB1) degradation roles of the laccases screened from Stenotrophomonas acidaminiphila CW117. [Methods] Two laccase genes lc1 and lc2 from strain CW117 genome were screened, and their AFB1 degrading activity was examined in vitro by heterologous expressed proteins of rLC1 and rLC2 in E. coli BL21. On the basis of in vitro test, two laccase-deficient strains CW117^△lc1 and CW117^△lc1-lc2 were constructed by homologous recombination method by using suicide plasmid pK18mobsacB, and the laccases (lc1 and lc2) AFB1 degradation role on strain CW117 was validation in vivo. [Results] Laccase rLC1 showed the AFB1 degradation activity in vitro, but rLC2 did not show degradation activity. Degradation activity of rLC1 was improved by redox mediators of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), acetosyringone or syringaldehyde. The degrading activity of mutants CW117^△lc1 and CW117^△lc1-lc2 showed similar degradation activity to the wild-type strain CW117 in most evaluation time-points. [Conclusion] Laccase LC1 from S. acidaminiphila showed AFB1 degradation activity, and the degradation activity could be enhanced by redox mediators as previous study. However, the laccases’ contribution to AFB1 degradation in strain CW117 was minimal, and other degradation pathways existed in the strain.

Abstract:[Objective] Colletotrichum fructicola is an important pathogen that results in yield loss of Camellia oleifera. We studied the functions of the mitogen-activated protein kinase gene CfMKK1 in C. fructicola for analyzing the pathogenic mechanism of oil-tea tree anthracnose.[Methods] The homologous recombination method was used to construct the CfMKK1 gene-deleted fragment, which was transformed into the protoplasts generated by using PEG-mediated method to obtain the mutant strain △Cfmkk1. The PCR-amplified CfMKK1 gene-containing complement of the promoter of C. fructicola was taken to construct a complementary vector pYF11::CfMKK1, then the complementary vector was transformed into the mutant protoplasts by using PEG-mediated method to screen the complementary strain △Cfmkk1-C. The biological phenotypes of the wild-type strain CFLH16, the mutant strain △Cfmkk1, and the complementary strain △Cfmkk1-C were measured in vegetative growth, appressorium formation, stress response and pathogenicity. [Results] The mycelial growth rate of △Cfmkk1 was significant slowed down compared to the wild-type strain CFLH16 and the complementary strain △Cfmkk1-C, more sensitive to Congo red, lost the ability to infect hosts and was unable to form appressoria.[Conclusion] CfMkk1 is involved in regulating the growth, response to external stress and appressorium formation of C. fructicola, affecting the pathogenicity.

Abstract:[Objective] Lycium ruthenicum Murr. is an important medicinal and halophyte plant of Solanaceae family in desert regions of China. Our study aims to reveal the diversity and distribution of endophytic bacterial community in different tissues of Lycium ruthenicum Murr.. [Methods] Through high-throughput sequencing technology, sequences at V5-V7 regions of endophytic bacteria 16S rRNA gene in different tissues of Lycium ruthenicum Murr. were amplified, and community composition, diversity and function were analyzed by bioinformatics. [Results] The diversity and function of endophytic bacterial community in different tissues of Lycium ruthenicum Murr. were significantly different. The OTUs (operational taxonomic units) of flower, leaf, fruit, stem and root were 182, 173, 119, 187 and 254, respectively. The community diversity index showed that root > flower > fruit, stem > leaf. At the level of phylum, Proteobacteria was the dominant group, which was simultaneously found in different tissues. The relative abundances of flower, leaf, fruit, stem and root were 87.66%, 41.51%, 81.76%, 97.67% and 61.85%, respectively. At genus level, the distribution of endophytic bacteria displayed difference in different tissues. Serratia and Acinetobacter were the dominant groups in flower, with relative abundances of 11.57% and 8.55%, respectively. Rhodococcus and Bradyrhizobium were the dominant groups in leaf, with relative abundances of 29.68% and 5.53%, respectively. Pantoea，Rhodococcus and Serratia were the dominant groups in fruit, with relative abundances of 23.12%, 5.52% and 4.29%, respectively. Serratia and Pseudomonas were the dominant groups in stem, with relative abundances of 12.03% and 17.71%, respectively. Halomonas, Fodinicurvata and Lipingzhangella were the dominant groups in roots, with relative abundances of 24.18%, 5.16% and 4.86%, respectively. Some dominant microbial groups, such as Halomonas, Serratia, Acinetobacter, Rhodococcus, Pantoea etc., exhibited functional characteristics related to salt tolerance, promoting growth, biological control, degradation of toxic and harmful substances and anti-oxidation etc. PICRUSt function prediction analysis showed that the functions of endophytic bacteria in different tissues were involved in the biosynthesis of secondary metabolites such as polysaccharides, terpenes, ketones, enzymes and vitamins etc. [Conclusion] The endophytic bacteria of Lycium ruthenicum Murr. had rich community and functional diversity. They also contained a variety of probiotics and functional information related to human and plant metabolism. The dominant bacteria genera and functional information of different tissues were different, among which the endophytic bacteria species in the root were the most abundant, and the bacteria involved in various metabolic functions in the flower and stem were the highest. In addition, there were a large number of unknown species and genera in tissues, which provided a broad development space to explore functional utilization of endophytic bacteria and find new microbial resources.

Abstract:[Objective] Tomato is a vegetable that is largely affected by continuous cropping. We investigated the effect of the rotation of different crops and tomato on soil microbial diversity, enzyme activity, and soil physico-chemical properties, with the aim to select rotation crops suitable for tomato and thus to provide a theoretical basis for alleviating or avoiding continuous cropping obstacles in tomato cultivation.[Methods] Together with tomatoes, cultivated over 2 consecutive years, we planted six crops, namely Chinese cabbage (A), cucumber (B), pepper (C), eggplant (E), okra (F), and zucchini (G); treatments with continuous tomato cultivation (D) and temporary non-crop soil (H) were used as controls. Using 16S rRNA and fungal ITS region sequencing, soil enzyme activity, pH value, organic matter content, and available nitrogen, phosphorus, and potassium levels were determined. This enabled us to investigated the effects of the rotation of different vegetable crops and tomato on soil microbial diversity, enzyme activity, and soil physicochemical properties. [Results] The diversity index values of bacteria and fungi in soil from treatments A, B, C, E, F, and G were significantly higher than that in H. The community structure of bacterial phyla in D soil and in A, B, C, E, F, and G soils was relatively stable, albeit with differences in species abundance. Fungi were more sensitive to environmental changes than bacteria, and the differences in fungal community structures were greater than those in bacterial communities. The abundance of nitrifying bacteria (Nitrosomonas and Nitrosospira) in soil was significantly increased by rotation of eggplant and Chinese cabbage in the tomato field. Principal components analysis showed that the community structure of bacteria and fungi in E and H soil was significantly different from that of other treatments. Soil enzyme activity of different crops in rotation soils (A, B, C, E, F and G) was significantly different, but the change law was not obvious. Catalase activity in A, B, and H soil was significantly higher than that in other soils. According to the results of the soil fertility analysis, soil pH, organic matter, available nitrogen, and available phosphorus were the main contributing factors. [Conclusion] Crop cultivation can significantly increase the soil microbial diversity; rotation of eggplant and Chinese cabbage in the tomato field can significantly increase the abundance of nitrifying soil bacteria, which is beneficial to nitrogen metabolism and use. Rotation of cucumber and Chinese cabbage in the tomato field can significantly increase soil catalase activity, which is conducive to reducing the self-toxic effect produced by tomato in continuous cropping mode. Based on our results, eggplant, cucumber, and Chinese cabbage are potential dominant crops for avoiding or alleviating the tomato continuous cropping obstacle.

Abstract:[Objective] In order to investigate the target genes in Escherichia coli, we used the CRISPR-Cas9 tool combining with homologous recombination to develop a two-step strategy for point mutation. [Methods] First, we assembled the up- and down-stream homologous arms of a gene and the amp gene with the pKOV plasmid to construct the pKOV-HR plasmid. Then the pKOV-HR plasmid was transformed into E. coli and the gene knock-out mutants were obtained by the RecA recombination system of E. coli itself. Subsequently, we applied two plasmids system (pSGKP-km and pCasKP-apr) to handle the point mutations of the target gene. Firstly, we designed a spacer sequence of the amp gene which was able to guide Cas9 proteins. The overlap PCR was used to produce the homologous arm containing point mutations of the target gene. Then the spacer and homologous arm were connected by pSGKP-km plasmid and the plasmid pSGKP-km-spacer-HR was obtained. Next, we transformed this plasmid and pCasKP-apr plasmid into the above-mentioned knock-out mutants. Finally, we got the point mutants by the DNA-cleaving of Cas9 proteins and λ-Red recombinant proteins which were produced by the two plasmids. [Results] By this two-step strategy, we obtained yjjW knockout strains named D7ΔyjjW::amp^R, and point mutants named D7yjjW-24 (from T to C the 24th base) successfully. [Conclusion] The study established an efficient method for point mutations of target genes in E. coli. The insertion of amp gene provided an effective screening marker. The recombinant plasmid pSGKP-km-spacer established in this process can be applied to point mutations of other target genes, which will greatly shorten experimental processes. It contributes the scientific theory and practice for the development of gene editing technology.

Abstract:[Objective] As a negative regulator of G protein signal transduction pathway, G protein signal regulator factor plays an important role in the pathogenicity asexual reproduction regulation of plant pathogens. To identify the relationship between RGS protein types and their physicochemical properties in fungi. [Methods] We analyzed 229 of previously identified RGS protein sequences in 49 fungi including model organism, pathogenic bacteria and non-pathogenic bacteria, and identified 6 proteins according to their conserved domains, such as DEP-RGS, RGS-TM, PXA-RGS-PX, RGS, RGS-PAS-PAC and TM-RGS. We used the protein database, ProtComp v9.0, PHD and MEME to analyze the physicochemical properties, subcellular localization, secondary structure and motif of the above RGS proteins. [Results] The above-mentioned different types of RGS proteins had obvious characteristic futures, and following common characteristics: theoretical isoelectric point was between 6.01 and 7.00, instability coefficients were between 40.01 and 60.00, more than 95% of RGS proteins were hydrophilic protein, the strongest hydrophilic amino acid residues were arginine, aspartic acid, glutamic acid, glutamine, asparagine, the strongest hydrophobic amino acid residues were leucine, alanine, valine, phenylalanine, isoleucine, the secondary structure was characterized by less Beta strand, situation for the transit peptide situation is unclear, and subcellular location is more concentrated in the nucleus. [Conclusion] The physicochemical properties of the six RGS proteins have some common characteristics, but different types of RGS proteins also have obvious category characteristics, mainly in the conserved domain, secondary structure, isoelectric point, transport peptide and subcellular localization.

Abstract:[Objective] In order to screen a safe and efficient thermotolerant Trichoderma strain to be used in tropical areas to effectively promote banana production, Trichoderma strains that can survive under a wide temperature range were isolated from agricultural soils collected from tropical areas. Then the biofertilizers containing Trichoderma isolates were developed and their effects on banana Fusarium wilt disease, crop yield and fruit quality were explored. [Methods] Trichoderma strains were screened by series-dilution separation method, and then re-screened according to the growth conditions of Trichoderma strains at different temperatures. Further antagonistic abilities against Fusarium oxysporum f. sp. cubense tropical race 4 (FOC 4) and the produced enzyme activities of Trichoderma strains were tested to isolate efficient thermotolerant Trichoderma strain. Then the effects of the biofertilizers developed containing the above selected Trichoderma strains on banana Fusarium wilt disease, crop yield and fruit quality were studied under field condition. Finally, the selected strain with best effect on improving crop production was identified using ITS and tef1 sequencing. [Results] Four thermotolerant Trichoderma strains were isolated from the soil collected from Hainan province, and all of these four strains could grow under between 20 ℃ and 40 ℃. Among them, strain JS84 grew fastest at 40 ℃, displayed the highest inhibition rate against FOC 4 and showed highest enzyme activities. Field experiment showed that application of biofertilizer containing Trichoderma JS84 (JS84) significantly suppressed banana Fusarium wilt disease incidence, improved the quality of banana fruit and soil nutrients condition. Strain JS84 was identified as Trichoderma citrinoviride based on both ITS and tef sequence. [Conclusion] Strain JS84 classified as Trichoderma citrinoviride with a wide growth temperature was successfully isolated from tropic area in China. Biofertilizer developed based on this strain exhibited an excellent ability in suppressing banana soil-borne Fusarium wilt disease and improving crop yield.

Abstract:[Objective] To clone, express and purify the RecJ nuclease gene (AF_0699) from Archaeoglobus fulgidus, identify and analyze its nuclease activity. [Methods] Recombinant RecJ nuclease (AfuRecJ) was expressed in E. coli and purified by affinity chromatography. The enzymatic properties of AfuRecJ nuclease were characterized in vitro using synthesized oligo (deoxy) nucleotides as substrate. [Results] AfuRecJ nuclease has a single-stranded DNA specific 3'–5' exonuclease activity, which depends on the divalent metal ions Mn²⁺ or Mg²⁺, and the catalytic efficiency of Mn²⁺ is significantly higher than that of Mg²⁺. The optimal reaction temperature is between 55 and 65 ℃. The presence of high concentration of NaCl decreases the exonuclease activity of AfuRecJ, and cleavage percentage greatly reduces at 200 mmol/L of NaCl. AfuRecJ shows 3'–5' exonuclease activity on single-stranded RNA, higher than that of single-stranded DNA. The 3' terminal phosphate group inhibits the AfuRecJ nuclease. AfuRecJ has different preferences for substrate length. It can effectively hydrolyze ssRNA≥4 nt, and ssDNA≥12 nt. Moreover, the hydrolysis activity of AfuRecJ on dsDNA with special structure (3' overhang and 3' fork) is similar to that of ssDNA. [Conclusion] The nuclease activity of AfuRecJ depends on the Mn²⁺ and can cleave ssDNA and ssRNA with a preference for RNA substrate.