Abstract:Syntrophic metabolism is one of the important interspecies relationships among microbes. Syntrophic microorganisms not only distribute in soils, freshwater, marine anoxic sediments, anaerobic digestion and gastrointestinal tract of animals, but also present in extreme environments such as subsurface oil reservoirs. They play essential roles in anaerobic degradation of organic compounds to methane and carbon dioxide. Study on the syntrophic metabolisms of syntrophic microorganisms through culture-dependent methods, would help understand the biogeochemical cycle of elements in anoxic environments, and deal with the global energy crisis and global warming problems. However, it is difficult to isolate syntrophic microorganisms for their slow-growing and oxygen-sensitive properties. This review summarizes the recent studies on the isolation strategies of syntrophic microorganisms, and their physiological and biochemical properties. Furthermore, the future development trend of culture techniques including high throughput screening and targeted isolation of syntrophic microorganisms were discussed.

Abstract:Autophagy (macroautophagy) is an essential and highly conserved protein degradation mechanism in eukaryotes. In this process, cytoplasmic organelles, old proteins and other macromolecules are sequestered into a double membrane vesicle called autophagosome and delivered to a degradative organelle for degradation and recycling. Extensive studies have revealed that autophagy plays an essential role in the cellular processes such as cell differentiation, nutrient homeostasis and pathogenicity in pathogenic fungi. In this review, we introduce the process of autophagy and describe the role of autophagy in regulation of the fungal sexual reproduction, using the human fungal pathogen Cryptococcus neoformans as an example. Furthermore, we summarize the autophagy related genes studied so far and the deduced physiological functions of autophagy for proper asexual and/or sexual reproduction in model pathogenic fungi. We also discuss perspectives on autophagy function in fungal reproduction.

Abstract:[Objective] wblA disruption mutant of Streptomyces ansochromogenes fails to produce nikkomycin.RNA-seq and transcriptional analysis revealed that the san7324 could be transcribed normally in S.ansochromogenes(wild-type),whereas not transcribed in the wblA(a global regulatory gene) disruption mutant (ΔwblA).This research aims to reveal the function of san7324 in nikkomycin production.[Methods] To investigate the role of san7324 in nikkomycin biosynthesis,a san7324 disruption mutant was constructed via homologous recombination,and was subsequently confirmed by genetic complementation.The transcriptional levels of selected genes in the nikkomycin biosynthetic cluster were analyzed.[Results] The mutant (Δsan7324) lost ability to produce nikkomycin under the same culture conditions compared with the wild-type strain.Disruption of san7324L(a san7324 homologous gene) caused the decrease of nikkomycin production.However,when san7324 and san7324L were disrupted,the double mutant (Δsan7324-san7324L) failed to form grey spores or spore chains but conferred a white phenotype of aerial hyphae,and no nikkomycin production was observed in comparison with wild-type strain.In addition,the nikkomycin production and morphological differentiation could be restored by the complementation of san7324-san7324L in Δsan7324-san7324L.Further studies preliminarily indicated that disruption of san7324 and san7324L mainly affected the transcriptional level of pathway-specific regulatory gene sanG in nik cluster,affected the morphological differentiation and nikkomycin biosynthesis in S.ansochromogenes.[Conclusion] These results provided more evidence for investigating the relationship between morphological differentiation and physiological metabolism in Streptomyces,and lay the foundation for elucidating the regulatory mechanism of the pleiotropic regulatory gene wblA.

Abstract:[Objective] To isolate Vta1 of Spodoptera frugiperda and to detect the requirement of Vta1 in replication of Autographa californica multiple nucleopolyhedrovirus(AcMNPV).[Methods] Vta1 was isolated from Sf9 cells using reverse-transcription PCR.Two mutations of Vta1,which removed the first or second microtubule-interacting and transport domain (MIT) were transiently expressed.The interaction of Vta1 and its mutants with Vps4,Vps46 and Vps60 was detected with bimolecular fluorescence complementation (BiFC).Using a viral complementation assay,the effect of Vta1 mutants on replication of AcMNPV was determined.[Results] We obtained Vta1 of S.frugiperda.The amino acid identities between Vta1 of insect and yeast or between Vta1 of insect and human are about 20% or 50%.Western blotting analysis showed GFP-tagged Vta1 and its mutants were expressed in Sf9 cells.BiFC analysis revealed that deletion of MIT1 or MIT2 significantly reduced the interaction of Vta1 mutants with Vps4,Vps46 or Vps60.Overexpression of Vta1 mutants significantly decreased the infectious AcMNPV budded virions production but had no effect on the expression of the reporter genes LacZ and GUS,which separately controlled by AcMNPV ie1 and p6.9 early or late promoter.[Conclusion] Vta1 might be involved in assembly and/or budding of progeny virions of AcMNPV.

Abstract:[Objective] To test the regulation of spo0A transcription by the regulatory protein Sigma H (σ^H) from Bacillus thuringiensis (Bt) HD73;Expressing and purifying Sigma H in Escherichia coli to verify its direct binding to the promoter of spo0A;To detect the effect of deletion of sigH on production of spores and crystal proteins in B.thuringiensis HD73.[Methods] The transcription level of spo0A was determined by measuring the β-galactosidase activities directed by the promoter of spo0A in Bt HD73 and sigH deletion mutant.The open reading frame of the sigH was amplified by PCR from Bt HD73 genome,and then ligated into the vector pET21b to generate pETsigH.The pETsigH was transformed into BL21(DE3) to obtain the recombinant strain BL21(pETsigH).The Sigma H protein was extracted and purified by Ni Sepharose 6 Fast Flow column purification and anion exchange purification.The electrophoretic mobility shift assay (EMSA) was carried out to verify the direct binding of Sigma H and the promoter of spo0A.The phenotypic characteristics of the sigH deletion mutant strain (HDΔsigH) were analyzed by microscopic observation and sporulation efficiency determination. [Results] The deletion of sigH decreased the transcription level of spo0A.The 28 kDa-Sigma H-His was expressed and purified from E.coli strain.EMSA results showed that the Sigma H-His could bind to the promoter of spo0A.Microscopic observation and sporulation efficiency determination demonstrated that the HDΔsigH failed to produce spores and crystal proteins.[Conclusion] The Sigma H-His protein directly regulates the expression of spo0A by binding to the promoter of spo0A.Deletion of the sigH blocks the spores and crystal proteins production in Bt strains.

Abstract:[Objective] The aim of this research was to analyze the change of diversity and species abundance of bacterial communities in male Bactrocera dorsalis (Hendel) after ¹³⁷Cs radiation treatment.[Methods] We used Illumina HiSeq high-throughput sequencing technology to sequence 6 intestinal samples of Bactrocera dorsalis.[Results] Proteobacteria (60.38%) and Firmicutes (24.33%) were the dominant phylum and subdominant phylum in the intestinal microflora of Bactrocera dorsalis respectively, and Enterobacteriaceae (60.38%) and Enterococcaceae (15.69%) were the dominant family and subdominant family. Moellerella, Morganella, Cosenzaea, unidentified-Streptococcaceae and Acidothermus have significant difference after irradiation.[Conclusion] Irradiation treatment significantly reduced the diversity and abundance of intestinal microflora in Bactrocera dorsalis. And we found significant differential species in different taxonomic level. This result provides a theoretical basis to improve the irradiated-induced damage to Bactrocera dorsalis.

Abstract:[Objective] To identify the mitochondrial genome (mitogenome) of Hirsutella rhossiliensis OWVT-1 strain, verify erroneous sequences present in the published USA-87-5 mitogenome, and perform comparative mitogenomic analyses of three Hirsutella species.[Methods] The OWVT-1 mitogenome was assembled based on high-throughput DNA sequencing data and Sanger sequencing. PCR assays were performed to verify 3 long varying regions observed during alignment between the published USA-87-5 mitogenome and OWVT-1 mitogenome assembled in this study. Bioinformatics analyses were used to annotate the mitogenome sequence of OWVT-1.[Results] We detected several sequence errors in the published USA-87-5 mitogenome, including long sequence insertions/deletions and small indels. The mitogenomes of OWVT-1 and USA-87-5 were identical without any nucleotide difference. The complete mitogenome of H. rhossiliensis was 62949 bp in length, with 13 introns in seven genes. Several introns and intergenic regions seemed to have degenerated. The mitogenomes of H. rhossiliensis, H. vermicola, and H. minnesotensis showed a high synteny. Except free-standing ORFs, the gene order of core protein-encoding genes, rRNA genes, and tRNA genes were highly conserved among the three Hirsutella mitogenomes. The length at intergenic regions was a main factor affecting mitogenome sizes of different Hirsutella species.[Conclusion] There were erroneous sequences in the published USA-87-5 mitogenome. We reported the authentic mitogenome sequence of H. rhossiliensis using the highly virulent strain OWVT-1.

Abstract:[Objective] Nitrification has been thought to be as a two-step process, where ammonia-oxidizing bacteria and archaea (AOB and AOA) first oxidize ammonia to nitrite, which nitrite-oxidizing bacteria (NOB) subsequently transfer to nitrate. Recently, the ability to oxidize ammonia has also been discovered in members of the genus Nitrospira, which were formerly supposed to only be capable of nitrite oxidation. The discovery of these bacteria that oxidize ammonia to nitrate (complete ammonia oxidizing bacteria, Comammox bacteria), refuted the dogma that the oxidation of ammonia and nitrite requires two distinct groups of microorganisms. The discovery of one-step nitrification and Comammox Nitrospira triggered many important scientific issues of the global nitrogen cycle, but relevant studies were still in the preliminary stage.[Methods] Using real-time quantitative PCR (Q-PCR), we characterized the distribution patterns of functional amoA gene abundances of Comammox (Clade A and Clade B), ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) at 5, 10, 20, and 40 cm soil depths (up-down error within 1 cm) of flooded paddy field (FPF) in Beibei, Chongqing in 2017.[Results] Quantitative PCR (Q-PCR) revealed that soil depth had a significant effect on different nitrifiers. Comammox Nitrospira were ecologically widespread and numerically abundant in all depths of the standard profile. The Comammox Clade A amoA gene copies were higher at deeper layers, while Comammox Clade B didn't show the same trends and its abundances varied between 1.85×10⁶ copies/g and 3.26×10⁶ copies/g in different depths of the standard profile. However, the abundance of Comammox Clade A was about to an order of magnitude more abundant than Comammox Clade B in paddy soils. On the contrary, the amoA gene abundances of AOA and AOB significantly decreased with increasing depth (P<0.05), both AOA and AOB amoA gene abundances were highest in the top layer (5 cm, 1.23×10⁷ copies/g and 1.83×10⁵ copies/g, respectively). The ratio of abundances of Comammox amoA genes to those of AOA and AOB increased significantly with the increase of soil depth, ranged from 10 to 2000.[Conclusion] The complete ammonia oxidizing bacteria (Comammox bacteria) are widely distributed in all soil depths of paddy soil and their abundances were significantly higher than "incomplete ammonia oxidizers (AOA, AOB)", which implicated that Comammox cannot be neglected in rice soil ecosystem.

Abstract:[Objective] To better understand how antigen density on the surface of hepatitis B virus core protein (HBc) virus-like particles (VLPs) affects their antibody response, we generated HBc VLPs with different antigen densities, and tested their antibody response in mice.[Methods] First, we prepared the recombinant antigen domain 4 (AD-4) of human cytomegalovirus (HCMV) as a model antigen, which contains three glycine molecules at its N-terminus, for Sortase A-mediated ligation onto the HBc VLPs. Displaying different densities of antigens onto the surface of VLPs was achieved by using a serial diluted recombinant AD-4 in ligation reactions. After that, HBc-AD-4 VLPs with different antigen densities were applied to 6-8 weeks old BALB/c mice. Each group was inoculated three times at 2-week intervals and the AD-4-specific IgG was detected by indirect ELISA.[Results] When the reaction concentration ratio between HBc and AD-4 is 1:0.5, which HBc surface antigen density is 44.4%, VLPs couldn't induce a high antibody titer. When the reaction concentration ratio between HBc and AD-4 is 1:1, which HBc surface antigen density is 64.2%, VLPs could induce similar highest humoral immune response compared to 100% antigen density HBc VLPs. When HBc surface antigen density is greater than 64.2%, no further enhancement of antibody response was observed by further increasing the antigen density.[Conclusion] In conclusion, we found that antigen density on HBc VLPs is positively correlated with the antibody response. However, it reaches a peak at 64% antigen density, and no further enhancement of antibody response was observed by further increasing the antigen density.

Abstract:[Objective] Extracting the metabolites of hydrothermal Bacillus sp. SY27F from deep-sea hydrothermal vents, the antibacterial and antitumor activity of metabolites were assayed.[Methods] The antibacterial activity of the secondary metabolites was determined by the method of paper disk and microbroth dilution method, and the anti-tumor activity of the secondary metabolites was evaluated by CCK-8 method.[Results] The bacteriostatic experiments showed that the metabolites of thermophilic Bacillus sp. SY27F have antibacterial effect on Escherichia coli and Staphylococcus aureus, with MICs of 1.56 mg/mL and 3.13 mg/mL, respectively. Cell cytotoxicity assay showed that the metabolites have the effect of inhibiting cell growth in tumor cells (A549, HepG2, HeLa and MCF-7), with IC₅₀ values of 0.390 mg/mL, 0.451 mg/mL, 0.704 mg/mL and 1.105 mg/mL, respectively. Compared with HepG2, the normal liver cells (L02) showed good biocompatibility.[Conclusion] The metabolites of thermophilic Bacillus sp. SY27F have antibacterial and antitumor activity, and can provide resources for the search of novel compound of antibacterial and antitumor activity.

Abstract:[Objective] A method of sorting single cells of Cryptococcus neoformans by flow cytometry was established to determine factors influencing proliferation of single C. neoformans cells.[Methods] The Moflo XDP flow cytometric sorter was used. The clonal recovery rate of C. neoformans cell was determined under various conditions.[Results] Single C. neoformans cells sorting process by cytometry was established. Single C. neoformans cells could recover growth which was affected by nutritional conditions and strains. Under nutrient-rich conditions, the clonal recovery rates of C. neoformans stains JEC21 and H99 were 74% and 88%, respectively. Under oligotrophic conditions, the clonal recovery rate of JEC21 was 37%; while the clonal recovery rate of H99 was 80%. The clonal recovery rate of JEC21 increased with increasing initial number of cells. When the cell number of JEC21 strain was 100, the clonal recovery rate was 55%; when the cell number was 1000, the clonal recovery rate was 97%.[Conclusion] The single C. neofroman cells sorted by flow cytometry had the capacity of recovery to growth, and the capacity was affected by nutritional conditions and strains.

Abstract:[Objective] The objective of this study was to elucidate the phylogenetic group and diversity of structure composition of culturable yeasts isolated from supraglacial cryoconites and subglacial sediments of the Glacier No.1 in Tianshan Mountains and to analyze the physiological and biochemical characteristics of different low temperature yeasts.[Methods] Culturable yeasts were detected on rose Bengal agar (RB), clichloran 18% glycerol agar (DG18), malt extract yeast extract soytone agar (MYP) and yeast-malt extract agar (YMP) media. The phylogenetic position of yeasts was determined by internal transcribed spacer (ITS) gene sequence. The physiological and biochemical tests included optimal growth temperature, salinity tolerance and enzymatic activities.[Results] A total of 152 culturable yeasts were isolated from supraglacial cryoconites and subglacial sediments. The results of National Center of Biotechnology Information (NCBI) alignment of internal transcribed spacer (ITS) rRNA gene sequence and Rep-PCR fingerprinting showed that the yeast groups included Basidiomycota and Ascomycota, belonging to 14 genera and 26 species. Among them, 88 strains were Pucciniomycotina, 24 strains were Agariomycotina, and 40 strains were Ascomycota. Vishniacozyma victoriae was the dominant strain (21.84%). The optimum growth temperature for 17 yeasts was at 15℃, 2 strains at 10℃ and 6 strains at 20℃. The analysis of lipase, protease and amylase activity showed that 11 of the 25 low temperature yeasts produced lipase and 11 produced amylase and 5 produced protease, 6 strains did not produce any enzymes.[Conclusion] The results of this study provide valuable data for the research of community structure and physiological characteristics of cryogenic yeasts in glaciers.

Abstract:[Objective] Comparative genomics analysis of the fully sequenced plasmid p1512-KPC from the multi-drug resistant clinical Klebsiella pneumoniae 1512 isolate.[Methods] We used 16S rRNA gene sequencing to identify the bacterium.Seven housekeeping genes (gapA,infB,mdh,pgi, phoE,rpoB,and tonB) were used for the multilocus sequence typing (MLST) scheme.Resistance genes were screened by PCR amplification.The plasmid was transferred into recipient Escherichia coli EC600 using conjugal transfers and electroporation experiments.The activity and type of carbapenemase were detected using Carba NP,and the minimum inhibitory concentration (MIC) was determined using VITEK 2 compact system.Sequencing and bioinformatics analysis were used to characterize the plasmid p1512-KPC.[Results] The 1512 isolate was an ST11 multi-drug resistant K.pneumoniae strain showing Ambler class A carbapenemase.PCR demonstrated strain 1512 harbored bla_KPC-2,dfrA1 and sul1 genes.The bla_KPC-2 gene was located in non-self-transmissible plasmid p1512-KPC.Sequencing and bioinformatics analysis revealed that p1512-KPC,117.69 kb in length,harbored IncFⅡ replicon and replicon repB belonging to Rep_3 family unknown incompatibility groups.In addition,p1512-KPC contained bla_KPC-2,bla_CTX-M-65, bla_TEM-1 and rmtB genes,among which bla_KPC-2,bla_CTX-M-65 and bla_TEM-1 were carried by ΔTn6296,Tn6367 and ΔTn2 respectively.[Conclusion] The resistance of K.pneumoniae 1512 to penicillin,cephalosporin,carbapenem,monobactam and aminoglycoside were mediated by non-self-transmissible plasmid p1512-KPC.Moreover,the comparative genomics analysis provided a deeper insight into the diversification and evolution of those plasmids that harbored IncFⅡ replicon and replicon repB belonging to Rep_3 family unknown incompatibility groups.

Abstract:[Objective] To provide scientific data for studying the ecology of bacteriophage in natural environments, the genetic diversity and distribution patterns of g23 of T4-type bacteriophage in wetland sediments were revealed.[Methods] Degenerate primers MZIA1bis and MZIA6 were used to PCR amplification of g23 gene of T4-type bacteriophages, and the PCR products were cloned and sequenced to reveal the diversity of g23 sequences in 6 different wetland sediment samples obtained from northeast China. In addition, the Unifrac analysis was used to compare the T4-type bacteriophage community structures in wetland sediments with those in other environments.[Results] In total, 262 different g23 clones of T4-type bacteriophage were obtained. Neighbor-joining phylogenetic tree showed that the distribution of g23 genes in wetland sediments was similar to those in aquatic ecosystems such as oceans and lakes and paddy ecosystems, but distinctly different from those in upland black soils. In addition, the T4-type bacteriophage community as evaluated by g23 gene assembles was remarkably different among different wetlands.[Conclusion] The composition of T4-type bacteriophage community was complex and diverse in wetland ecosystem, and the novel g23 groups of T4-type bacteriophage existed in wetland sediments in northeast China.

Abstract:[Objective] Urea ABC-transporter permease subunit UrtB may be involved in urea metabolism and branched-chain amino acid transport. This study aimed to obtain experimental evidence to clarify the effect of urtB gene on the nodulation and nitrogen fixation of Mesorhizobium huakuii and provide scientific basis for further study of its functional mechanism.[Methods] The structure characteristics and biological functions of UrtB protein were analyzed by bioinformatics, the expression pattern of urtB gene under free-living growth and symbiosis conditions were detected by fluorescent quantitative real-time PCR, and in situ expression of urtB gene in nodule tissue during nodulation process were detected by promoter fusion with β-glucuronidase activity. The urtB gene mutant was constructed by site-directed insertion, pot plant experiments and in combination with nitrogen addition treatment were carried out to identify symbiotic phenotypes and restoration.[Results] It showed that the urtB gene was anticipated to be involved in nitrogen transport of rhizobia. The expression level of urtB gene under symbiotic conditions was significantly up-regulated compared to that under free-living conditions, and urtB gene was highly expressed in the nitrogen-fixing zone of the mature nodules; an urtB mutant of 7653R was screened and obtained. Compared with the wild-type strain 7653R, the 7653R△urtB mutant inoculated with host plant resulted in an abnormal development of the nodule, and the above ground biomass of plants and nodule nitrogenase activity were significantly decreased. Adding nitrogen in the pot experiment could restore its symbiotic defective phenotype.[Conclusion] The urtB gene of Mesorhizobium huakuii may affect nitrogen transport or assimilation in root nodules, and therefore it plays an important role in root nodule development and symbiotic nitrogen fixation.

Abstract:[Objective] To investigate the expression and subcellular localization of post-synaptic density-95, Drosophilia tumor suppressor protein diskslarge-1, the tight junction protein zonula occludentes 1 signal protein encoded by Rv3194c gene from Mycobacterium tuberculosis, and to serve the identification of the cellular binding proteins of Rv3194c protein.[Methods] The gene encoding tRv3194c (Rv3194c 1-234 aa) with T2A and EGFP sequence was cloned by PCR from the genomic H37Rv, then inserted into the eukaryotic expression vector to construct pcDNA3.1-tRv3194c-T2A-EGFP. Indirect immune fluorescence assay, flow cytometry sorting and Western blotting assay were used to observe subcellular localization, when L929 cells were transfected with constructs before infection with the recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase.[Results] The eukaryotic expression vector pcDNA3.1-tRv3194c-T2A-EGFP was constructed correctly. After the transient transfection with the plasmid, localization of fusion protein tRv3194 in mitochondria was observed by immune fluorescence assay. The dramatically enhanced expression level by co-infection with vTF7-3 before transfection was detected by flow cytometry sorting and Western-blotting assay.[Conclusion] Post-synaptic density domain in Rv3194c protein can bind to its ligand protein which located in mitochondrial outer membrane, which provides a key clue to understand its pathogenic mechanism in intracellular.

Abstract:[Objective] We isolated bacterial strain that can antagonize the aflatoxin B1 (AFB1) producing fungus Aspergillus flavus in maize seed.[Methods] The plate method and in vivo screening were performed for samples from various sources. The positive bacterial strains were screened by their ability to degrade AFB1 in culture media.[Results] The inhibitory rate of bacterial strain to Aspergillus flavus was 79.2% and its degradation rate was 68.39% in 1 μg/mL AFB1. During storage period, the inhibition rate of the bacterial strain to Aspergillus flavus was inversely proportional to the water content of maize. When the water content in the maize was 15%, 92.46% inhibition rate was recorded, while the inhibition rate of 19.41% was noted with 30% water content in maize. When the maize water content was 28%, the control effect on Aspergillus flavus was 36.39%.[Conclusion] An AFB1-degrading strain not only could inhibit the growth of Aspergillus flavus, but also may control the contamination of AFB1 to maize.