Abstract:Pyoverdine (Pvd)-a diffusible iron-chelating agent produced by bacteria in the genus of Pseudomonas-has recently become a model system for studying sociomicrobiology. The sociobiology framework (specifically, cooperation and cheating) is used to explain the emergence of Pvd nonproducing mutants that are commonly found in natural environments. Pvd producing and nonproducing cells are considered cooperators and cheaters, respectively. Non-producers can receive the benefit of cooperation by taking up Pvd produced by other cells, but they do not pay the full cost of Pvd biosynthesis. Thus, nonproducing mutants are advantageous in direct competition with the producing cells. This review summarizes the multiple evolutionary pathways of Pvd nonproducing mutants, and further discusses the generality of the conclusion that Pvd production is a cooperative behaviour.

Abstract:Bacteria have developed severe resistance to traditional antibiotics, demanding novel antimicrobial agents to overcome resistance. Antimicrobial peptides are promising novel antimicrobial agents. Owing to preferential attack on the cell membrane, antimicrobial peptides will not lead to widespread resistance, although their recognition of specific targets might cause genetic mutations and resistance of the targets. Additionally, some bacteria can overcome endogenous antimicrobial peptides and survive with the host cells. The corresponding defense mechanisms of bacteria also bring cross-resistance to therapeutic antimicrobial peptides. The resistance mentioned above poses a challenge to the development of antimicrobial peptides. In this review, we summarize the research progress in studying the mechanisms of antimicrobial peptides resistance, to provide the reference for developing strategies to fight bacterial resistance.

Abstract:Inorganic phosphate (Pi) plays an important role in the biological processes such as bacterial heredity, energy metabolism and intracellular signaling. Two major bacterial Pi import systems exist in bacteria, Pst and Pit. The Pst system is highly efficient at transporting Pi, particularly in cells growing under low-Pi concentrations. In recent years, studies have found that in addition to regulating metabolism and balance of Pi, Pst system also mediates drug resistance, toxicity and invasion of bacteria. The Pst system is a member of the ABC transporter family, generally consists of five distinct proteins of PstS, PstC, PstA, PstB and PhoU. Among them, PstS and PstB are key proteins in the Pst system. This article reviews the molecular mechanisms of Pi transport and bacterial resistance of PstS and PstB, and the relationship between Pst system and bacterial resistance as well as PstS/PstB-targeted development to manufacture new drug.

Abstract:Over 240 million people worldwide are chronically infected with hepatitis B virus (HBV) although the HBV prophylactic vaccine has dramatically reduced new infections. However, still up to 680000 deaths per year are caused by HBV-related end-stage liver diseases and liver cancer. Currently, strategies for the treatment of Chronic Hepatitis B (CHB) include interferon-alpha (IFNα) and nucleos(t)ide analogues (NAs), but it is difficult to achieve the ideal clinical treatment endpoint, namely hepatitis B surface antigen (HBsAg) negative or serological conversion. Many teams are trying to develop more effective treatments, targeting key steps in the HBV life cycle and potential host factors, aiming to great medical needs for CHB patients that have not yet been met. This article reviews the advances in the research and development of the potential drugs in clinical development and partial preclinical stages.

Abstract:Soybean contains high content of isoflavones such as genistein and daidzein, both can be converted to equol by intestinal microflora in human and animals. Equol may play an important role in human and animal health. Rodent species consistently produce high levels of equol, however, not all humans can produce equol. Equol-producing bacteria may be the key factors for this difference. Equol-producing bacteria may also have different equol-producing mechanism and capacity. In this paper, all equol-producing bacteria and their equol synthesis mechanism are reviewed, for further understanding the factors of equol-producing difference, the transformation of equol-producing ability, equol fermentation in vitro and applying to human clinical studies.

Abstract:Microorganisms-involved oxidation processes of reduced inorganic sulfur compounds (RISCs) are significant components of sulfur geochemical cycle, and could also be applied in biomining industry and acid mine water treatment. Thermophilic archaea are a special group of microorganisms in high temperature environments. They participate in a variety of dissimilatory sulfur oxidation pathways, involving many redox enzymes and sulfur transporter proteins. In this review, the species of thermophilic archaea involved in the metabolic processes of dissimilatory sulfur oxidation, and the sulfur oxidation processes they participate in will be systematically introduced in combination with our research work.

Abstract:[Objective] Bombyx mori nucleopolyhedrovirus (BmNPV) belongs to the family Baculoviridae. It replicates and proliferates using its host cell's energy metabolism. Bombyx mori ADP/ATP translocase (BmANT) is a transporter that interacts with the host energy metabolism factor, heat shock protein 60 (BmHSP60), during BmNPV infection. To determine which of the functional characteristics of the Bmant gene affect the process of BmNPV infection, we here analyze the mechanism by which the baculovirus hijacks host cytokines to promote viral proliferation and replication. [Methods] The structural characteristics of the Bmant gene were predicted, and qRT-PCR was performed to analyze the changes in the Bmant gene after BmNPV infection. The effect of this gene on viral DNA replication and viral protein expression was analyzed using overexpression of the Bmant gene. We further analyzed the relationship between the Bmant and Bmhsp60 genes in transcriptional regulation. The mechanism by which Bmant and Bmhsp60 regulates the proliferation and replication of BmNPV was identified using flow cytometry and Western blotting analysis. [Results] SMART software predicted that BmANT protein contained three mitochondrial vector domains, and the Bmant gene down-regulated expression 24 h after BmNPV infection. Overexpression of the Bmant gene significantly inhibited BmNPV DNA replication and VP39 protein expression. QRT-PCR analysis showed the Bmant and Bmhsp60 genes to have mutual antagonism and to be capable of inhibiting each other's transcription. Co-transfection analysis of the Bmant and Bmhsp60 genes showed that the Bmant gene can significantly inhibit the action of the Bmhsp60 gene and ultimately inhibit the proliferation and replication of BmNPV. [Conclusion] The results indicated that the Bmant gene is a mitochondrial carrier protein with significant antiviral activity that is capable of down-regulating the expression of Bmhsp60 and inhibiting BmNPV proliferation and replication.

Abstract:Zostera japonica is a unique Asian seaweed species and has important ecological value. In recent years, Zostera japonica has been continuing to degenerate, causing widespread concern among researchers. [Objective] We hypothesized that the rhizospheric microorganisms were closely related with the healthy growth of Zostera japonica. We explored the diversity and function of microbial community in root of Zostera japonica at coastal zone of Yellow sea and Bohai sea, and intrinsic relationship with seagrass.[Methods] We collected surface sediment samples of Zostera japonica roots and unvegetated areas from three locations including Dongying, Weihai, and Dalian city. We investigated the rhizosphere microbial community structure by sequencing on Illumina HiSeq300 platform and analyzed the correlation between microbial community structure and environmental parameters. [Results] The most dominated phyla widely distributed in the rhizospheric surface sediments of Zostera japonica included:Proteobacteria (41.1%), Cyanobacteria (15.4%), Bacteroidetes (12.6%), Actinobacteria (9.3%). Comparisons of different regions (Weihai, Dongying, and Dalian) or sample types (rhizosphere vs. non-rhizosphere) showed significant differences in microbial communities, mainly due to the presence of sulfate-reducing bacteria and nitrogen-fixing bacteria found in the rhizosphere. Total nitrogen, total carbon, total organic carbon, clay, were all significantly correlated to rhizospheric bacterial community composition and distribution. [Conclusion] From a functional point of view, the differences between groups was mostly related to sulfur and nitrogen cycling, and sulfate reducing bacteria play a key role in maintaining the ecological health of Zostera japonica.

Abstract:[Objective] To study the phenotypes of Azorhizobium caulinodans ORS571 mutant strain lacking the diguanylate cyclase AZC-2412, and to explore its functional mechanism. [Methods] We constructed the gene knockout system of rhizobia based on the homologous recombination of cre-loxp, and the mutant strain was constructed by the triparental conjugation. Growth rate, chemotactic ability, exopolysaccharide production, biofilm formation and other phenotypes of the wild type and mutant strains were studied. [Results] The mutant strain had almost the same growth rate as the wild type. Compared with the wild type, the mutant strain impaired in exopolysaccharide production, chemotaxis behavior and biofilm formation due to lower levels of c-di-GMP in cells. [Conclusion] The deletion of the diguanylate cyclase AZC-2412 reduces level of c-di-GMP levels. It had a regulatory effect on the exopolysaccharide production, bacterial motility, biofilm formation, cell flocculation of Azorhizobium caulinodans ORS571 and its interaction with host plant.

Abstract:[Objective] To study the bactericidal effect of dielectric barrier discharge low temperature plasma on Listeria monocytogenes and Salmonella Enteritidis under different treatment conditions and the content of antibacterial active substances with treatment time. [Methods] Listeria monocytogenes and Salmonella enteritidis were used as research objects to determine the total number of colonies after treatment with different voltage, time and oxygen concentration. The concentration of O₃ and NO₂ was determined by gas-active substance determination tube with treatment time and fluorescence intensity was utilized. The concentration of free radicals generated in the bacterial liquid was characterized and the changes of·OH, H₂O₂ and ¹O₂ with the treatment time were determined by fluorescence spectrophotometry. [Results] Using 70 kV, 150 s or 80 kV, 120 s could completely kill Salmonella Enteritidis, whereas Listeria monocytogenes could be completely killed only at 80 kV, 120 s. A mixture of 50% O₂+50% N₂ could completely kill the bacteria in 90 s. With the increase of treatment time, The content of intracellular ROS in two bacteria gradually increased. [Conclusion] The voltage, time and oxygen concentration could significantly enhance the bactericidal effect of plasma. Active oxygen radicals such as H₂O₂ and·OH could promote the bactericidal effect on Listeria monocytogenes and Salmonella enteritidis. Moreover, the bactericidal effect of·OH and H₂O₂ was better than that of ¹O₂.

Abstract:[Objective] The aim of this study was to understand the structure variation and assembly of root-microbiota of wild Rhododendron delavay. The spatial resolution of the study distinguished three niches of Rhododendron delavayi root, the endosphere, rhizoplane, and rhizosphere. Here we compared the microbial community structure and composition variation between the three niches. [Methods] A detailed characterization of the root-microbiota of the Rhododendron delavayi by deep sequencing, using 16S rRNA V4 and ITS1 regions of microorganisms. The diversity and composition of microbial community from three niches were compared. In addition, we discussed the relationship between differential microbial genera based on co-occurrence network.[Results] In the constrained unconstrained principal coordinate analyses of Bray-Curtis distances between samples, microbial communities vary significantly between three niches. Moreover, the separation of root-microbiota from rhizosphere to rhizoplane is larger. The bacterial α-diversity between three niches is considered as statistically significant difference. 41 bacterial phyla and 6 fungal phyla abundance in the root-microbiota. The dominant bacterial phyla included Proteobacteria, Acidobacteria and Actinobacteria, which occupied more than 80% of the total abundance of bacterial communities. The dominant fungal phyla included Zygomycota, Basidiomycota and Ascomycota, which account for more than 99% of the total abundance of fungal communities. 589 bacterial genera and 390 fungal genera abundance in the root-microbiota. Moreover, the abundance of 25 bacterial and 10 fungal genera varied between three niches. Furthermore, analyzing the co-occurrence network of the differential genera showed the interaction between fungi and bacteria in the root-microbiota. Eight differential fungi was significantly correlated to differential bacteria, except Waitea. As the core genera in the co-occurrence network, Bryobacter, Nocardia, Rhizomicrobium and Telmatobacter played a very important regulation.[Conclusion] Dynamic changes observed during root-microbiota, as well as compositions of three niches, support a hypothesis for root microbiome assembly correlated with the root. At the same time, co-occurrence network analysis revealed bacteria and fungi interacted with each other among the three niches.

Abstract:[Objective] To improve the thermostability of lipase LipA from Burkholderia sp., a series of cavity-filled lipase LipA mutants were constructed and evaluated. [Methods] Amino acid residuals consisted of cavities in 3D structural model of lipase LipA were predicted using Castp, Voronoia and Cave, and then computational libraries of every amino acid residuals were constructed using software packages FoldX. The following types of mutants were excluded from the computational libraries:(1) volume of the cavity was increased; (2) value of free energy, ΔΔG, was over -0.5 kcal/mol. Total 58 mutants were screened from the computational libraries, and 17 mutants were selected to verify the mutation effect. [Results] Thermostability of lipase mutants (LipA-His¹⁵Pro and LipA-Ala²¹⁰Val) were improved and T₅₀¹² value of lipase LipA-His¹⁵Pro and lipase LipA-Ala²¹⁰Val increased by 4 ℃ and 2 ℃, respectively. Thermostability of the superimposing mutant lipase LipA-His¹⁵Pro/Ala²¹⁰Val was further improved and the half-life (t_1/2) at 55 ℃ increased by 23.1 times. [Conclusion] Cavity-filling mutation was a feasible technique to improve the thermostability of lipase LipA from Burkholderia sp.

Abstract:Monascus pigments (MPs), a kind of natural pigments with diverse biological activities produced in the secondary metabolism of filamentous Monascus, are widely used as colorants in food, cosmetics and health-care products in East Asia. [Objective] In this study, a PigE gene related to MPs biosynthesis in Monascus purpureus Mp-21 (strain Mp-21) was cloned and characterized.[Methods] The PigE gene (one of MPs genes) was knocked out by homologous recombination in strain Mp-21 and the biological characteristics before and after the gene deletion were analyzed with regard to phenotypes, microstructures, growth rates, MPs and citrinin.[Results] The resulting data demonstrated that the disruption of PigE mainly led to the upregulation of yellow pigments in varieties and yields. In comparison to an MPs complex mixture in which the red pigments were dominant in the wild-type strain Mp-21, the △PigE (a PigE gene deletion mutant) lost the ability to produce red pigments and produced at least five new yellow pigments. The MPs production was up to 3548.2 U/g and about 4.82-fold as that in the wild type strain Mp-21 after liquid-state fermentation for 13 d. Additionally, the citrinin production had no significant change, while the productive period was delayed on account of a longer period to adapt to the growing condition. The deletion of PigE blocked the transformation pathway of the yellow pigments to orange pigments, which made the △PigE more favorable to form yellow pigments. As the formation of red pigments required more complex conditions such as amino acids and suitable pH in culture medium, the △PigE prefers to synthesize yellow pigments first and lost the ability to produce red pigments. [Conclusion] This study provides a possible way for the construction of genetic engineering Monascus strains with high yield of yellow pigments.

Abstract:[Objective] The study was to express and characterize β-galactosidase from the fecal microbes of Rhinopithecus bieti. [Methods] A β-galactosidase gene galRBM20_1 was cloned from the fecal microbial metagenome of Rhinopithecus bieti. The pEASY-E2-galRBM20_1 plasmid was constructed and transformed into Escherichia coli BL21 (DE3). The expression was induced by Isopropyl β-D-1-thiogalactopyranoside and the enzymatic properties were studied.[Results] The optimum pH of β-galactosidase galRBM20_1 was 5.0, and the enzyme retained more than 70% activity between pH 4 and 7. The optimum temperature is 45℃ and the enzyme was stable at 37℃ and 45℃, retaining nearly 100% activity after incubation for 1 h. In addition, the enzyme had strong NaCl stability. After 1 h of 1 to 5 mol/L NaCl treatment, the relative enzyme activity maintained 100%; when the concentration of NaCl was 4 mol/L, the activity of β-galactosidase galRBM20_1 was the highest (146%); it still had activity after treatment with 2.5 mol/L NaCl at 45℃ for 24 h. [Conclusion] β-galactosidase galRBM20_1 has good salt tolerance and wide pH range.

Abstract:[Objective] The aim of this study was to reveal the differences in the community structure of ammonia-oxidizing archaea (AOA) between rhizosphere and non-rhizosphere soil, to provide a theoretical basis for further study on the relationship between halophyte rhizosphere soil microorganisms and salt tolerance. [Methods] Using ammonia monooxygenase gene (amoA) as molecular marker, the diversity and community structure of AOA in rhizosphere soil of Reed in different seasons (spring, summer, autumn) in Ebinur Lake Wetland were studied by high-throughput sequencing technique. [Results] The diversity and richness of AOA were different in rhizosphere soil of Reed in different seasons. In summer and autumn, the richness of AOA in rhizosphere soil of Reed was higher than that in non-rhizosphere soil, the diversity was lower than that in non-rhizosphere soil. In spring, the diversity of AOA in rhizosphere soil of Reed was higher than that in non-rhizosphere soil, the richness was lower than that in non-rhizosphere soil. The diversity magnitude of AOA in terms of the season was spring > summer > autumn in rhizosphere soil of Reed. Community composition analysis of AOA showed that the main AOA communities in all soil samples were Crenarchaeota and Thaumarchaeota, and Crenarchaeota were the dominant phylum. RDA analysis showed that soil moisture, soil organic matter, total potassium and pH were the main factors affecting the diversity and richness of AOA community in rhizosphere soil of Reed.[Conclusion] The diversity and richness of AOA in rhizosphere soil of Reed were season dependent, compared with non-rhizosphere soil, AOA was more abundant in rhizosphere soil of Reed.

Abstract:[Objective] The aim was to analyze the gene transcriptional profiles, express the protein in Escherichia coli and analyze functions of the PcF/SCR effector SCR82 from Phytophthora capsici. [Methods] Gene transcripts during the developmental stages (mycelia, sporangia, zoospores and germinated cysts) and infection period (1.5, 3, 6, 12, 24, 36, 72 h post-inoculation) of Nicotiana benthamiana by P. capsici were checked by semi-quantitative RT-PCR. The cDNA full length was cloned into a modified pET28a(+) expression vector in a way that a 6×His-SUMO tag was fused with the protein N-terminal. Protein was induced using the E. coli isolate Rossette 2 at two temperatures (22℃ and 37℃) combined with different IPTG concentrations (0.1, 0.2, 0.5, 1 mmol/L). Protein was purified by Ni²⁺ column affinity chromatography and infiltrated into the leaves of N. benthamiana and Arabidopsis thaliana to test its ability of inducing plant cell death. Meanwhile, the expression changes of three resistance-related marker genes (NbMC1, NbSOD and NbPOX) of N. benthamiana was examined at 12 and 24 h post-infiltration of the recombinant protein. [Results] The expression of scr82 was upregulated during the cyst germination and host infection. The gene was single-copy without any introns and its open reading frame was 249-bp long. The deduced protein was of 82 amino acids consisting of a 21-aa signal peptide and 7 cysteines. It was predicted to be hydrophobic and contain no known domains. The secondary structure consisted of random coil and α-helix. Its homologues existed only in Phytophthora spp. After inducing by 0.2 mmol/L IPTG at 22℃ for about 16 h, the recombinant protein of about 24 kDa was observed on SDS-PAGE gels. About 30 mg/mL of purified protein was obtained. The recombinant did not trigger plant cell death, but caused up-regulated expression of NbMC1, NbSOD and NbPOX genes. [Conclusion] The recombinant could not induce plant cell death, but triggered plant defense reaction.

Abstract:[Objective] The primary goal of this study was to explore the correlation between the diversity and abundance of nitrogen-fixing microbes and the physicochemical factors of rhizosphere and non-rhizosphere soil types during different seasons. We aimed to explore the potential role of nitrogen-fixing microbial communities in restoring ecological functions. This study may provide the theoretical foundation and preliminary data for future studies in this field. [Methods] Illumina HiSeq PE250 sequencing technique was used to analyze the diversity of nitrogen-fixing microorganisms in six different soil samples. RDA analysis was used to explore the correlation between the physicochemical factors and the structure and abundance of nitrogen-fixing microbial communities in these soil samples.[Results] The diversity of nitrogen-fixing bacteria in rhizosphere soil was higher than that in non-rhizosphere soil. Additionally, the diversity observed in July was higher than that in October and April. Bacteria belonging to the genera Geobacter, Pseudomonas, Azotobacter and Bradyrhizobium were predominant in Salicornia europaea rhizosphere and non-rhizosphere soils. The predominant phyla of nitrogen-fixing microorganisms were Proteobacteria and Cyanobacteria, whose relative abundance accounted for 85% and 10%, respectively, while the others accounted for less than 5%. The predominant nitrogen-fixing microflora in the soil was significantly correlated with alkali-hydrolyzed nitrogen (AN), total nitrogen (TN), available potassium (AK), and total phosphorus (TP). [Conclusion] The diversity and community structure of nitrogen-fixing microorganisms in soil samples varied in different seasons. The community structure of nitrogen-fixing microorganisms was different in rhizosphere and non-rhizosphere soil types during the same period. These experimental results may provide the theoretical foundation and preliminary data for the restoration of Ebinur Lake wetland.

Abstract:[Objective] Construction of transgenic strain can effectively improve the insecticidal effect. But the insecticidal toxin cannot be secreted from intracellular to extracellular, so it cannot directly act on the insect body. Therefore, if we can construct an engineered strain with exocrine protein function, it is an effective way to solve these problems.[Methods] In this study, the growth characteristics and resistance of Yersinia pestis (CSLH88) isolated from the intestinal tract and habitat of M. alternatus were determined, and then the molecular modification was carried out. The exocrine expression vectors of HlyA (pGHKW2) and HasA (pGHKW4) were constructed and transformed into CSLH88 strain by electroporation to obtain engineering strain capable of expressing green fluorescent protein. The genetic stability of the two plasmids was tested, and the exocrine function of the proteins was verified by SDS-PAGE and Western blotting techniques. [Results] The strain of CSLH88 could enter logarithmic growth stage after 2-4 hours of culture and had no resistance to kanamycin (Kan). Two exocrine expression vectors, pGHKW2 (GenBank:MK562405) and pGHKW4 (GenBank:MK562404), were successfully constructed. It was found that pGHKW4 plasmid was more suitable for stable inheritance in Y. pestis. The results of SDS-PAGE and Western blot showed that the secretion of extracellular proteins in HlyA system protein was blocked, while the HasA system could play an exocrine expression function in CSLH88 strain. [Conclusion] By studying the exocrine expression systems of HlyA and HasA, HasA heme transport system was selected as the exocrine expression system of CSLH88 strain. It lays a foundation for the construction of exocrine toxin protein strain and the pathogenicity of CSLH88 strain.