Abstract:Polyethylene terephthalate is one of the most used synthetic polymers in industrial fields and daily life due to its excellent durability and plasticity. In the meantime, a large amount of PET waste has caused severe environmental problems, so that researchers are paying more and more attention to the degradability of PET waste. Among all of the methods of plastic degradation, the means of biodegradation is considered to be the most environmental-friendly treatment at present and it will be efficient for large-scale degradation of PET waste. Previous research has indicated that PET degrading enzymes mainly include lipases, esterases and cutinases, but they exhibit poor PET degradation activity. A new PETase discovered recently, which is isolated from Ideonella sakaiensis, has shown high efficacy and specificity to degradation of PET materials. Here, we summarize the progress on structural research of PET degrading enzymes and describe the catalytic mechanism of PETase, to help modify more effective PET degrading enzymes.

Abstract:Chronic hepatitis B virus (HBV) infection is a worldwide public health problem that poses a serious threat to human health. Currently, only a very small fraction of patients can achieve functional cure based on the existing treatment strategies of anti-HBV drugs. The development of more effective drugs against HBV certainly requires a more comprehensive understanding on the roles and mechanisms of each viral component and its related host factors in viral life cycle, and therefore providing scientific clues for further identification of novel therapeutic targets. In vitro cell models supporting HBV replication and infection are important tools for basic researches of HBV life cycle, and play essential roles in the identification of novel anti-HBV targets and efficacy evaluation of drug candidate. In this review, we summarize the recent research advances on the cell culture models supporting HBV infection and replication, and systematically illustrate and discuss their application characteristics and limitations and highlight perspectives for further developments.

Abstract:As a naturally stable disaccharide, the protective function of trehalose on the activity of organisms under adverse conditions has attracted a wide range of research interests, and also has good application value and potential. This paper focuses on the important model microorganisms and industrial applied yeast, combined with the latest progress in the research of omics, and summarizes the new progress in the research of endogenous trehalose from the aspects of trehalose metabolic pathway, trehalose metabolism and transcription characteristics under stress conditions, and the strategy of improving intracellular trehalose.

Abstract:[Objective] The genus Pantoea comprises 26 species that are isolated from a multitude of environments. In this study, 146 strains of Pantoea isolated from rice seeds of 8 different rice genotypes were systematically analyzed to determine their phylogenetic status, diversity, distribution and functional characteristics. [Methods] First, surface of rice seeds was sterilized by ethanol-sodium hypochlorite, and then the surface sterilized seeds were ground into a powder to isolate endophytic bacteria using the standard dilution plating technique. Second, the strains were purified and 16S rRNA gene sequencing and analyzed. Finally, the isolated strains were tested for plant growth-promoting, such as phosphorus solubilization, indoacetic acid (IAA) and siderophore production, antagonistic and hemolysis. Simple sequence repeats molecular marker technology was used to study the genetic polymorphism in five different genotypes of rice, and some agronomic traits such as tiller numbers, plant height, plant weight and rice production were studied. [Results] In total 146 endogenous Pantoea strains were isolated from 8 different genotypes of rice seeds. Pantoea strains were classed to Pantoea dispersa, Pantoea agglomerans, Pantoea cypripedii and Pantoea brenneri based on 16S rRNA gene phylogenetic analysis. Among the 4 species, P. dispersa has the largest size of population and existed in all 8 rice seed samples. Then, 66 strains were selected to test the plant growth-promoting characteristics, 86.3% and 69.7% of the strains were found to have the ability to dissolve phosphorus and produce IAA. 7 strains had the ability to produce siderophore. None of the strains was antagonistic to fungal pathogen Fusarium moniliforme. Another 3 strains had hemolytic activity. Pantoea composition did not correlate with rice phylogeny and agronomic traits. [Conclusion] The species and composition of Pantoea were different in different rice seeds. The seeds could selectively accumulated Pantoea as endophytic bacteria, and most of the strains had growth-promoting characteristics.

Abstract:[Objective] The expression of coenzyme A disulfide reductase in methicillin-resistant Staphylococcus aureus was up-regulated by 2.3 folds when the strain was cultured with oxacillin. Here, the bactericidal effect of oxacillin on coenzyme A disulfide reductase deficient Staphylococcus aureus was studied. [Methods] Homologous recombination mediated double-exchange techniques were used to delete genes in Staphylococcus aureus, whereas pOS1 plasmid was used to construct the complemented strain. Spectrophotometry was used to detect the proliferation ability of the strains in vitro. The bactericidal effect of oxacillin on the strains was studied by time-kill method, and 2',7'-dichlorodihydrofluorescein diacetate was used as a probe to detect intracellular reactive oxygen species levels. [Results] Coenzyme A disulfide reductase gene deleted strain grew more slowly than the parental strain (P<0.05). Under the action of oxacillin, the time-kill curve and intracellular reactive oxygen species level of the knockout strains were slightly different from that of the parental strains at 20 times MIC concentration, and the lethal rate and intracellular reactive oxygen species level of coenzyme A disulfide reductase gene deleted strains were lower than that of the parental strains at 5 times MIC concentrations. [Conclusion] At lower concentrations of oxacillin, the deletion of coenzyme A disulfide reductase gene reduced intracellular ROS levels, and the bactericidal rate, meanwhile the secondary damage was delayed.

Abstract:The effects of heavy metal stress on the community structure of endophytic bacteria in plants are not well understood. To date, few studies have investigated the response of community structure and diversity of endophytic bacteria in hyperaccumulating plants to heavy metals present in soil rhizosphere. [Objective] The objectives of this study were to explore the changes and differences in community structure and diversity of endophytic bacteria in roots, stems, and leaves of hyperaccumulating (HE) and non-hyperaccumulating (NHE) ecotypes of Sedum alfredii under different levels of Cd²⁺ contamination; as well as to explain their differences in ability to tolerate and accumulate the available cadmium by the two ecotypes of Sedum alfredii, based on the mutual relationship between plant and endophyte. [Methods] The community structures of endophytic bacteria in the roots, stems, and leaves of the two ecotypes of Sedum alfredii grown on soils with different Cd²⁺ concentrations were analyzed by Illumina high-throughput sequencing technique. [Results] High concentrations of Cd²⁺ inhibited the growth of NHE Sedum alfredii and decreased the richness and diversity of endophytic bacteria, whereas it promoted the growth of HE Sedum alfredii and increased the richness of endophytic bacteria in stems and roots. Among the three tested soils, the endophytic bacteria in leaves, stems, and roots of two ecotypes of Sedum alfredii were all dominated by Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. For HE Sedum alfredii, the relative abundances of Gammaproteobacteria, Negativicutes, and Clostridia in leaves as well as Alphaproteobacteria in stems increased significantly with the soil Cd²⁺ concentration, whereas the relative abundances of Clostridia decreased significantly. For NHE Sedum alfredii, with the increase in soil Cd²⁺ concentration, the relative abundances for Alphaproteobacteria, Gammaproteobacteria, and Clostridia in leaves did not show significant change, while those for Negativicutes in stems as well as Betaproteobacteria and Clostridia in roots decreased significantly, and those for Negativicutes in roots increased significantly. In highly Cd²⁺-contaminated soils (50 mg/kg), the relative abundances of Sphingomonas in leaves and Veillonella in stems of HE Sedum alfredii were higher than those for NHE Sedum alfredii. Meanwhile, the first, second, and third predominant endophytic bacteria (i.e., Veillonella, Sphingomonas, and Prevotella) that were present in the roots of HE Sedum alfredii did not appear in roots of NHE Sedum alfredii.[Conclusion] The Cd²⁺-contamination level in soils had significant impacts on the community structure of endophytic bacteria in leaves, stems, and roots of the two ecotypes of Sedum alfredii.

Abstract:[Objective] The objective of our study was to compare the soil bacterial communities by using bio-organic fertilizer in disease occurring period, and to excavate the closely related bacterial genera to Watermelon Fusarium wilt.[Methods] The V3-V4 region of the 16S rRNA gene in bacterial DNA was amplified using the polymerase chain reaction technique and sequenced by the Illumina Miseq platform to diversity analysis of the soil microbial community composition after application of bio-organic fertilizer in different years (2016 onset stage and 2017 onset stage).[Results] There was no significant difference in alpha diversity indices among soil microbial community diversity at different years, but the diversity richness increased slightly after applying bio-organic fertilizer by comparison with the control. At phylum level, the Proteobacteria and Firmicutes were the relatively stable bacterial communities in these two periods. However, at genus level, the major dynamic bacteria were Bacillus, Enterococcus, Lactococcus and Mizugakiibacter. Furthermore, the Spearman analysis showed that they were negatively correlated with the watermelon Fusarium wilt disease morbidity.[Conclusion] The application of bio-organic fertilizer plays a certain role in the control of watermelon Fusarium wilt by helping ecological restoration of microbial communities in continuously cropped watermelon-growing soil. Thus, the approach to identify the dynamic changes of soil microbes and find out their relationships between the incidence of watermelon Fusarium wilt provided a new idea of helping control watermelon Fusarium wilt by adjusting soil microbial community structure.

Abstract:A large number of contaminants from rare earth-mines are discharged into the surrounding soil through the floating dust, surface runoff and leachate of the tailings dam, and affect particular fungal communities. [Objective] To investigate the adaption of fungal community structure to the long-term pollution stress by both rare earth and heavy metals, and to characterize the adsorption kinetics of rare earth and heavy metal by fungi isolates. [Methods] Based on the internal transcribed spacer (ITS) gene, Illumina-Hiseq sequencing technique was used to analyze the fungal community structure in five soils from B1 to B5 with increasingly higher contents of rare earth-heavy metal around Baotou rare earth tailings dam, in addition to an uncontaminated control soil about 20 km away from tailings area (sample C). Meanwhile, a total of 6 fungal isolates were obtained by culture-dependent technique, and their absorption kinetics of both rare earth and heavy metals were analyzed.[Results] At the phylum level, Ascomycota was abundant in fungal communities in all samples (13.5%-90.5%). At the class level, Sordariomycetes was apparently higher in B2 (73.1%), B3 (28.4%) and B4 (20.8%) than control C samples (7.4%); and the relative abundance of Dothideomycetes was lower in the B1 (3.5%) than B5 (11.8%). At the genus level, Podospora showed strong adaption to contaminant stress, from the low relative abundance of 0.9% in control soil, to 23.6% in B3. Meanwhile, Aspergillus, unclassified Pleosporales, and unclassified Davidiellaceae were also detected with high relative abundances in B1 (3.0%), B4 (10.5%) and B5 (5.8%), respectively. Intriguingly, Lecanicillium was detected only from the B2 sample and dominated (51.6%). The effect of Zn pollutant on fungal communities was greater than that of rare earth elements, and negative correlation was observed between soil Zn content the relative abundance of dominant unclassified fungi. Six fungal isolates were obtained from contaminated soil, and could be assigned to genera Aspergillus (five isolates) and Fusarium (one isolate). All fungal isolates showed significantly higher adsorption capacity of La³⁺ than that of Zn²⁺, and the Aspergillus sp. B6-3 had the highest adsorption rates for La³⁺ (19.7%) and Zn²⁺ (3.9%). [Conclusion] This study provided a mechanistic basis for the use of fungi to remove rare earths and heavy metals towards the optimization of biosorption process-oriented strategy for environmental bioremediation and protection.

Abstract:[Objective] To detect possible nuclear mitochondrial DNA segments (Numts) within nuclear genomes of Cordyceps militaris and to compare the degree of nucleotide variations and phylogenetic relationships between mitochondrial and nuclear DNAs by employing multiple C. militaris isolates.[Methods] Nuclear genomes of C. militaris were searched for sequence similarities by local BLAST/LAST analyses with its mitochondrial genome as the query. Seven nuclear protein-coding gene fragments were amplified from 10 C. militaris isolates that had available mitochondrial genomes. Nucleotide variations at seven nuclear fragments were compared with those at 14 mitochondrial protein-coding genes. [Results] Five short Numts with a total length of 278 bp were identified in the nuclear genomes of C. militaris. The overall nucleotide variabilities of C. militaris on nuclear fragments were generally higher than that on mitochondrial fragments. The phylogenetic relationships among different C. militaris isolates revealed by nuclear and mitochondrial DNAs were significantly different. [Conclusion] There was no detectable gene transfer of long fragments between mitochondrial and nuclear genomes of C. militaris. The nucleotide variabilities of mitochondrial and nuclear DNAs were different, and the phylogenetic relationships of different C. militaris isolates revealed by them were different either. This study enhanced our understanding of the evolutionary relationship between mitochondrial and nuclear DNAs in C. militaris.

Abstract:[Objective] To screen the key catalytic sites that affect the reverse transcription function of intron-encoded protein (IEP) from Ll.LtrB, and to obtain the IEP mutant without reverse transcription activity. [Methods] The key catalytic sites affecting the reverse transcription of IEP were screened by sequence alignment and homology modeling methods. Then, the screened key catalytic sites were subjected to site-directed mutagenesis. The mutated IEP was combined with the Targetron vector to construct the mutated Targetron targeting system without reverse transcription function. Finally, the function of mutant IEP was verified by using the lacZ gene in Escherichia coli.[Results] The sites C164 and G214 of IEP were screened and mutated, completely inactivated the "retrohoming" function in vivo. [Conclusion] The IEP mutants of Ll.LtrB obtained in our study laid a solid foundation for further research on the structure and mechanism of group II intron.

Abstract:[Objective] To provide a basis for controlling hypertrophy sorosis scleroteniosis, the biological characteristics, the method to induce conidia through mycelia and production pathway of Ciboria shiraiana were studied. [Methods] We observed the morphological characters of conidia in diseased mulberry fruit and artificial culture under microscope. The effects of different temperature and humidity on conidial production by hyphae were determined; the healthy mulberry fruit was inoculated with the conidial suspension in diseased mulberry fruit and artificially induced, and the incidence rate was counted. Also, we used different pathogenic stages of diseased mulberry fruit, mycelium and sclerotium on the PDA medium or induction medium as materials, and the expression level of related genes was detected by qPCR method to reveal the effect of the cAMP pathway on conidial formation.[Results] C. shiraiana produced abundant conidia in artificial cultures at 20-30℃ with 50%-80% relative humidity. The morphology of conidia produced by artificial induction was greatly different from that in diseased mulberry fruits. The conidia suspension in the diseased mulberry fruit infected healthy mulberry, the incidence rate was 37%, and the artificially induced conidia had no infectivity to mulberry. Conidiophores and conidia could be induced using potato slices as the artificial culture medium. Exogenously added cAMP affected the morphology of mycelium and the formation of conidial, but didn't affect the formation of sclerotia. A qPCR analysis showed that the relative transcript levels of adenylate cyclase (AC) content increased rapidly in the second stage, decreased rapidly in the third and fourth stage, and PKA was not expressed in diseased mulberry fruit. [Conclusion] Diseased mulberry fruits of hypertrophy sorosis scleroteniosis can be re-infected by conidia. Conidia formation negatively regulates the expression of AC and PKA in the cAMP pathway. The results of this study can further enhance our understanding of the environmental conditions required for pathogen to infect mulberry fruits, and further improve the infection cycle and conidial formation pathway of C. shiraiana.

Abstract:[Objective] Serine/Threonine Protein Kinases K (PknK) is a eukaryotic-like Ser/Thr protein kinase in Mycobacterium, with important roles in cell growth and signaling transduction. However, its underlying mechanism of action is not completed. [Methods] The pknK knockout strain △pknK was obtained by phage specialized transduction meanwhile the complement strain pMV361-pknK/ΔpknK and the overexpressing strain with pMV261-pknK/BCG was construed for further analysis. The growth curve and resistance of the obtained strains were determined. PknK-interacting proteins were identified by pulldown-MS. [Results] PknK affected Mycobacterium growth, and △pknK had growth advantage over BCG and pMV261-pknK/BCG. Knockout pknK led to increased multiple antibiotic susceptibility. We identified the binding proteins for PnK in BCG using in pulldown combined with Mass Spectrometry. [Conclusion] PknK negatively regulates BCG growth and increases antibiotic susceptibility in mycobacteria. The PknK binding proteins include a Ser/Thr protein kinase PknA, and two component system regulators such as MtrA, MoxR1 and TrcR, which is expected to be a resource for understanding the PknK-mediated signaling pathways in Mycobacterium, thus facilitating new therapeutic strategies for antibiotic-resistant infections.

Abstract:[Objective] Bombyx mori nucleopolyhedrovirus (BmNPV) induced hematogenous sepsis is a common severe disease of silkworm, and there are few effective prevention and control methods. In this study, CVDAR andDaZao (lines with strong resistance to BmNPV) silkworm strains were used as experimental materials. By analyzing the resistance characteristics of CVDAR silkworm, the resistance mechanism of CVDAR to BmNPV was determined. [Methods] In this study, we found that the lethal dose of CVDAR strain to BmNPV infection was more than 10 times higher than that of DaZao through semi lethal dose analysis. Further HE staining was performed to analyze the changes of midgut tissues before and after the infection of CVDAR and DaZao strains, and analyzed the anti-BmNPV mechanism of the resistant CVDAR strain. [Results] The results showed that after 72 hours of infection with BmNPV, the nucleus of large intestinal cells was enlarged and the staining became lighter; after 96 hours, the nuclei continued to increase and tended to fall off. The CVDAR-resistant strains only had enlarged nuclei in part of midgut after 96 hours of infection, but they were well arranged. At the same time by fluorescence quantitative analysis of the CVDAR and DaZao strain proliferation after virus infection, combined with the analysis and comparison of the transcriptional levels of the representative virus genes in each period, no difference in virus copy number and viral genes transcription level between the resistant CVDAR strain and the DaZao strain were found between 0-12 h after BmNPV infection. However, 24 hours after infection, we found that the CVDAR of the resistant strain was significantly lower than that of the control strain, regardless of the number of viral copies or the transcriptional expression level of viral genes. [Conclusion] Our study demonstrated that during the first round of replication after oral addition of CVDAR, the transcription of midgut virus genes was not affected; and later the transcription level was reduced. The critical period for the inhibition of BmNPV proliferation by CVDAR strain was identified to be 24 h after BmNPV infection, laying a foundation for the analysis of the resistance mechanism.

Abstract:[Objective] To know the fungal community composition and diversity of tobacco phyllosphere with different degrees of tobacco pole rot. [Methods] The fungal community composition of both petiole and leaf samples from flue-cured tobacco leaves was analyzed based on Illumina MiSeq high-throughput sequencing platform. [Results] The fungi from severe infected leaf samples (BQ), severe infected petiole samples (BZ), light infected leaf samples (JQ) and light infected petiole samples (JZ), were distributed in 7 phyla (Ascomycota, Basidiomycota, Zygomycota), 27 classes, 58 orders, 104 families, 171 genera and 360 fungal taxa (OTUs). The fungal community composition, relative abundance and dominant fungal taxon of each sample with different pole rot level were all different. When at genus level for sample BQ, the dominant genus was Aspergillus (89.64%), Myrothecium (2.54%), Rhodotorula (2.48%), Gibberella (1.49%). For sample BZ, dominant genus was Aspergillus (96.93%) and Alternaria (1.92%). For sample JQ, dominant genus was Aspergillus (40.13%), Rhodotorula (31.81%), Alternaria (16.75%). For sample JZ, the dominant genus was Aspergillus (62.77%), Alternaria (9.74%), Rhodotorula (5.20%). The diversity of fungal communities is the highest for sample JQ, richness of fungal communities is the highest for sample JZ. The richness and diversity of BQ fungal community were higher than those of BZ. Tobacco pole rot is a common disease in curing barn. This pathogenic fungus is various and widely distributed in tobacco leaf and environment. [Conclusion] The results provided a reference for the prevention and control of tobacco pole rot in flue-cured barn according to the characteristics of fungal community composition in different parts of the disease severity.

Abstract:[Objective] A field experiment and molecular biology techniques were used to study the improvement of rhizosphere microbiome by Bacillus amyloliquefaciens FH-1, in order to understand the promoting mechanism of B. amyloliquefaciens FH-1 in soil. [Methods] The rice field experiments with the inoculation FH-1 treatment (FH) and blank control (CK) were set up. The physiological traits of rice and soil properties were determined. The number of rhizosphere bacteria was quantified by real-time fluorescent quantitative PCR. High throughput sequencing technology of 16S rRNA gene was used to analyze rhizosphere microbiome. The interaction among rice, soil and microbe was analyzed by Pearson correlation analysis. [Results] Compared to the control, the plant height, root length, spike length, panicles per plant, grain number per spike, and thousand-grain weight of rice were significantly increased by FH. There was no significant difference in soil properties and bacteria number between FH and the control. The FH significantly increased γ-Proteobacteria and Chloroflexi while significantly decreased β-Proteobacteria and Actinobacteria in soil. LEfSe analysis showed that there were 19 microorganisms enriched in FH. Correlation analysis showed that Bacillus and enriched species were positively correlated with rice plant. [Conclusions] We speculated that Bacillus amyloliquefaciens FH-1 promoted rice growth by regulating rhizosphere microbial community structure and function.

Abstract:[Objective] This study aims to isolate Clostridium strains from Luzhou-flavor baijiu pit mud and evaluate their metabolic characteristics. [Methods] 16S rRNA gene sequencing was used to analyze the clostridial community structure in pit mud; specific media were predicted according to the sequences of high-abundance OTUs scattered in Clostridia using KOMODO database; volatile compounds of pit mud and the representative isolated Clostridium strains' fermentation broth were detected by headspace solid phase microextraction combined with gas chromatography-mass spectrometry. [Results] Thirty-one strains belonging to 14 species of genus Clostridium were isolated using the 7 media predicted by KOMODO database. Based on the analysis of volatile metabolites, these Clostridium strains could be divided into two clusters, including cluster I, i.e. strains mainly producing acids (C. carboxidivorans, C. sporogenes and C. tyrobutyricum) and cluster II, i.e. strains mainly producing alcohols (C. beijerinckii, C. butyricum and C. sphenoides). [Conclusion] KOMODO method is helpful for the isolation of Clostridium or other bacteria from the pit mud of Luzhou-flavor baijiu, and elucidating their metabolic profiles could be conductive to analyze the function of brewing microbes in baijiu flavor formation.