Abstract:

Abstract:[Objective] The present study screened the halophilic archaeal strains with flocculation effect and evaluated the flocculation effect of the fermentation liquid, fermentation supernatant, cell suspension, and extracellular polymeric substances of the strains, aiming to develop the microbial flocculants that can adapt to a wide range of salinity and pH for high-salt wastewater treatment. [Methods] The pure culture method was designed to isolate halophilic archaea from the sediment samples of Uyong Brac salt lake in Xinjiang. The 16S rRNA gene sequencing and phylogenetic analysis were performed to evaluate the taxonomic positions of the isolated strains. Furthermore, the flocculation effects of the fermentation liquid, fermentation supernatant, cell suspension, and extracellular polymeric substances of the strains were evaluated. The flocculation stability was evaluated under a wide range of salinity and pH conditions. [Results] A total of 28 strains of halophilic archaea were isolated by pure culture method, among which 16 strains were selected based on the primary screening results. The 16S rRNA sequences and phylogenetic tree suggested that these strains mainly belonged to Natrinema, Halopiger, and Haloterrigena. The fermentation liquid, fermentation supernatant, cell suspension, and extracellular polymeric substances of strains A279-1, A133, RP33, NGA0064, RM-152, and A389 had better flocculation effects than those of other strains. The fermentation liquid and supernatant of strain A389 showcased the flocculation rates reaching 61.06% and 67.92%, respectively. The cell suspensions of all the strains had the flocculation rates over 80%. The extracellular polymeric substances produced by strain RM-152 had the highest flocculation rate of 89.86%, followed by those of strain A389 (81.53%). Strain A389 had the yield of 12.53 g/L of extracellular polymeric substances and it adapted to a wide range of salinity and pH. [Conclusion] There were abundant halophilic archaea in the sediment of Uyong Brac salt lake. The fermentation liquid, fermentation supernatant, cell suspension, and extracellular polymeric substances of halophilic archaea had good flocculation effect. In particular, the extracellular polymeric substances demonstrated good flocculation effect and could withstand a wide range of salinity and pH, being suitable for further industrial recycling and utilization. The isolated strains may serve the subsequent development of functional materials for industrial high-salt wastewater treatment.

Abstract:[Objective] To confirm the taxonomic status of the novel strain KlspL18 with high yield of indole acetic acid (IAA) isolated from the leaves of Oryza rufipogon. [Methods] KlspL18 was identified based on morphological, physiological, and biochemical characteristics, phylogenetic analysis, and whole genome sequencing. The production of IAA was determined by high performance liquid chromatography (HPLC). [Results] KlspL18 was Gram-positive, non-spore-forming, rod-shaped, and catalase-positive. It grew at 15–40 °C (optimum: 28 °C), pH 6.0–12.0 (optimum: 7.0), and in the presence of 0%–15% NaCl (optimum: 1.0%), and could use multiple carbohydrates and organic acids as carbon source. The main amino acid in the cell wall of KlspL18 was ornithine, and the dominant polysaccharides in the cell wall were galactose and mannose. The stain mainly had the polar lipids of diphosphatidyl glycerol, phosphatidyl glycerol, and two unidentified glycolipids, fatty acids of anteiso-C_15:0 (30.33%), iso-C_16:0 (31.53%), and anteiso-C_17:0 (14.32%), and naphthoquinones of MK-10 and MK-11. Phylogenetic analysis of 16S rRNA gene sequence showed the highest similarity (97.64%) to the type strain of Microbacterium proteolyticum RZ36^T. The genome of KlspL18 had the G+C content of 70.2%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between KlspL18 and the closely related strain of Microbacterium were 83.35% and 26.4%, respectively, both below the threshold for species discrimination. HPLC analysis suggested that KlspL18 had high yield of IAA (291.7 mg/L). [Conclusion] KlspL18, a novel high-IAA-producing species of Microbacterium, was named Microbacterium dongxiang sp. nov., which is a model strain KlspL18 (=CCTCC M2022446). It has the potential of improving soil fertility and promoting plant growth in agricultural production.

Abstract:[Objective] Rhizosphere contains a phylogenetically diverse array of microorganisms affecting the growth and health of plants. This study aims to examine the diversity and structure of the microbial community in Brassica juncea var. tumida rhizosphere. The isolated microbial strains can be explored to unleash their chemical potential. [Methods] Roots of B. juncea var. tumida were collected from two villages in Fuling Disctrict of Chongqing. Microbial isolates were then obtained after cultivation on different media. One strain was selected for genome sequencing (Pacbio RS II and Illumina HiSeq) and subsequent bioinformatic analysis. The antiSMASH 6.0 was used for the detection and comparison of biosynthetic gene clusters (BGCs) encoding secondary metabolites. A multiplex amplification-and-ligation strategy was employed to clone the gene cluster responsible for violacein biosynthesis. The gene cluster was then transferred into Streptomyces lividans TK23 by intergeneric conjugation. [Results] A total of 256 microbial isolates were obtained, of which 120 were identified. Duganella sp. BjR8, which produced violacein, was detected. Sequencing of the genome of BjR8 revealed a single circular chromosome of 7 205 593 bp in length with 64.67% G+C content and 6 421 coding sequences. Bioinformatic analysis showed that the strain contained 9 putative secondary metabolite BGCs, of which 7 had low similarities to BGCs encoding known compounds. Heterologous expression of the violacein gene cluster in S. lividans TK23 resulted in the production of deoxyviolacein. [Conclusion] The diversity and structure of the microbial community was examined by phylogenetic analysis of microorganisms isolated from B. juncea var. tumida rhizosphere. Bioinformatic analysis revealed that BjR8 had great biosynthetic potential for novel compounds. The vio gene cluster was cloned from BjR8 and expressed successfully in the surrogate host S. lividans TK23.

Abstract:[Objective] To isolate probiotic Lactobacillus strains from the human reproductive tract and explore the therapeutic effect of the strains on vulvovaginal candidiasis. [Methods] We used the de Man, Rogosa and Sharpe agar containing 1% calcium carbonate to isolate the Lactobacillus strains from the vaginal secretions of asymptomatic women in the childbearing age, screened the strains by using the co-culture method with Candida albicans, and then examined their physiological properties. The inhibitory effect of Lactobacillus was further evaluated in a mouse model of vulvovaginal candidiasis. [Results] Nineteen strains of Lactobacillus sp. were isolated from 53 samples. Among of them, 4 strains, i.e., L. crispatus ZH08, L. fermentum ZH09, L. fermentum ZH11, and L. crispatus ZH17, showed strong inhibitory effect against C. albicans, and grew well in a low-pH environment. L. fermentum ZH09 and ZH11 inhibited the growth of C. albicans, with the inhibition effect over 95% within 24 h. L. crispatus ZH08 and ZH17 had strong aggregation property and demonstrated strong adhesion to epithelial cells. Furthermore, the combination of L. fermentum ZH11 and L. crispatus ZH17 significantly inhibited the growth and the switch from budding to hyphal growth of C. albicans, promoted mucosal repair, and relieved inflammation in a mouse model of vulvovaginal candidiasis. [Conclusion] The screened Lactobacillus strains have probiotic properties and the potential in clinical application.

Abstract:[Objective] To detect the physiological characteristics related to organic acid production and low pH tolerance of the endophytic Saccharomyces cerevisiae 2-2 strain isolated from Usnea sp. in Tibet, China, and explore the molecular mechanism. [Methods] The pH of different strains was compared during liquid fermentation using different initial sugar concentrations, and the growth under stress conditions was monitored. The organic acid production potential and low pH resistance characteristics of S. cerevisiae strains were detected. Comparative genomic analysis was performed to explore the molecular mechanism of acid production and acid tolerance of S. cerevisiae strain 2-2 by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). [Results] S. cerevisiae 2-2 had higher acid-producing potential among the tested yeast strains, which also showed better acid tolerance than that of the model strain S. cerevisiae strain S288C. The transcription levels of PDR15, PDR12 and SUR1 that affect the acid stress tolerance of S. cerevisiae in 2-2 were up-regulated or down-regulated under acid stress conditions, but the trends of these key genes were opposite to that of the strain S288C. [Conclusion] Endophytic S. cerevisiae 2-2 is a strain with potential in organic acid production and shows strong tolerance to low pH. An in-depth analysis of its unique regulatory mechanism will benefit the development of acid-producing yeast strains with improved performance.

Abstract:Foaming is a common problem during fermentation. Inhibiting foaming is of great significance for simplifying operation and reducing fermentation cost. Yarrowia lipolytica is a commonly used starting yeast in synthetic biology and for the synthesis of erythritol and other functional sugar alcohols. However, massive foam is produced in the fermentation for the production of erythritol, which needs to be eliminated by defoamers. [Objective] To develop a Y. lipolytica strain with significantly reduced foaming ability, so as to reduce the addition of defoamer in erythritol production. [Methods] According to the principle that non-homologous end joining (NHEJ) is dominant in Y. lipolytica genome recombination, we randomly inserted an artificial DNA fragment into the genome to produce the mutants with reduced or no foaming ability in the fermentation for erythritol production. [Results] After screening, we obtained a mutant without foaming during the fermentation in flask, 50 L pilot reactor, and industrial 75 m³ fermentor. [Conclusion] This mutant obtained can efficiently synthesize erythritol from high-concentration glucose as does the parental strain while producing less foam. The method to obtain defoaming mutant can provide valuable reference for the engineering of other microorganisms.

Abstract:Fermentation industry, as an important part of biotechnology industry, covers a large proportion in industry in China. However, microorganisms face serious osmotic stress at the late fermentation stage as a result of the accumulation of microbial metabolites, neutralizers, and supplements, which seriously affects the growth of cells and the production of target product and thus results in low fermentation efficiency and low yield of target product. This paper aims to summarize and analyze cell structure, response pathways, genes, proteins, metabolism, and division of microorganisms in the presence of hyperosmotic stress, and to sum up potential measures against osmotic stress based on the characteristics of microorganisms and the current available techniques: strain improvement, addition of protective agent, improvement of neutralizers, removal of osmotic inhibitory factors, and membrane filtration technology. In summary, this study is expected to serve as a reference for raising the production in fermentation industry, saving energy, and reducing waste emission and cost.

Abstract:Lager, produced from bottom-fermenting yeast (Saccharomyces pastorianus) at colder temperatures, was first created in Bavaria, Germany in the 15th century and spread all over the world in the early 19th century. It enjoys the highest production among the alcoholic beverages all over the world. S. pastorianus, a hybrid of ale yeast S. cerevisiae and the wild yeast S. eubayanus, features low-temperature tolerance which is inherited from S. eubayanus. Research in population genetics and genomics has shown that S. eubayanus originated from the Tibetan Plateau and migrated to Europe through the Silk Road. According to the studies in comparative genomics, two distinct genetic groups existed in S. pastorianus taxon: Group Ⅰ (Saaz) and Group Ⅱ (Frohberg) which were prevalent in central Europe and western Europe in the early period, respectively. The former is allotriploid (nearly 2n S. eubayanus+1n S. cerevisiae) and the latter is allotetraploid (nearly 2n S. eubayanus+2n S. cerevisiae). The two groups differ in low temperature tolerance, maltotriose utilization and flavor production abilities. Most of the S. pastorianus strains preserved in the China General Microbiological Culture Collection Center (CGMCC) belong to Group Ⅱ. The discovery of wild S. eubayanus strains provides a new avenue for the creation of new S. eubayanus×S. cerevisiae hybrids and thereby for breeding novel non-transgenic lager yeast strains with unique fermentation properties, which may have significant impact on the future beer brewing. This review briefly introduced the research history of lager yeast, particularly the recent research on the hybrid nature, origin, evolution, and genome characteristics of S. pastorianus, ending with a perspective on research trends in the future.

Abstract:Zearalenone (ZEN) is an estrogen-like mycotoxin that can competitively bind to animal estrogen receptors, resulting in hormonal disorders in the reproductive system of animals. ZEN lactone hydrolase (ZHD) can hydrolyze the lactone bond of ZEN to non-toxic products. [Objective] By bioinformatics analysis and enzymatic property exploration, a ZEN lactone hydrolase with new properties was identified. [Methods] The pET28a-zhd11f expression vector was constructed and introduced into Escherichia coli BL21(DE3). ZHD11F was obtained by purification with Ni-NTA, and its enzymatic properties were analyzed. Furthermore, the mechanism of its low-temperature activity was elucidated by structural simulation and molecular dynamics. [Results] With ZEN as the substrate, the specific activity of ZHD11F was 40.04 U/mg, and its optimum reaction temperature and pH value were 35 °C and 8.0 respectively. The enzyme activity of ZHD11F exceeded 60% in the range of pH 6.0–9.5, and it showed good heat stability below 35 °C and could endure a variety of metal ions. The activity of ZHD11F remained 20% and 40% at 10 °C and 20 °C, respectively. A number of loop regions led to increased structural flexibility, which was the main reason for the poor stability of the enzyme and its high activity at low temperature. [Conclusion] Phialophora attae was a fungus belonging to the genus Phialophora, and the enzymes derived from this fungus were rarely identified. In this study, the ZEN lactone hydrolase ZHD11F from P. attae was successfully expressed in E. coli and pure enzyme was obtained. Characterization analysis showed that this enzyme was the first low-temperature ZEN lactone hydrolase reported so far, which provided reference for studying the cold-tolerance mechanism and wide-temperature range of such enzymes, and also expanded the functional study of enzymes derived from P. attae.

Abstract:Plant biomass is the most abundant renewable bioresource on earth. Many high-value added bio-based products can be produced by biomass biorefinery which needs to use plant-polysaccharide- degrading enzymes (PPDEs), such as cellulase, xylanase and raw-starch-degrading enzymes. Filamentous fungus Penicillium oxalicum can secrete complete PPDEs with high activity, but low yields of PPDEs limit their large-scale production and application. The biosynthesis of PPDEs in P. oxalicum is strictly regulated by many regulators such as transcription factors. In this review, in the biorefinery of sugarcane bagasse and raw cassava starch as feedstocks, some aspects concerning microorganisms were introduced, including the screening and breeding of fungal strains with high production of PPDEs, identification of regulatory genes regulating the biosynthesis of PPDEs and their gene expression in P. oxalicum, and construction of the engineered P. oxalicum strains with improved PPDE production, which would provide theoretical guidance for the exploration and utilization of fungal resources.

Abstract:In light of the continuous discovery of microbial resources and the accumulating microbial genome data, it is an urgent task to screen and identify functional genes associated with important microbial phenotypes from the enormous data, for which high-throughput techniques are indispensible. The techniques are mainly involved in library construction and screening. Library construction refers to the establishment of mutant or interference libraries covering the whole genome of target microorganisms with techniques such as metagenomics, transposon insertion, RNA interference (RNAi), retron library recombineering (RLR), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa). As for the screening, a certain stress is often used to allow the differential growth of microbial individuals of a mutant library and then the causal relationships between specific genes and phenotypic outcomes at genomic level are identified by high-throughput sequencing. In this review, we briefly summarize the current high-throughput techniques used in functional genomics research, hoping to provide a reference to the development and optimization of these techniques in the future.

Abstract:Microbial secondary metabolites are the rich sources of lead compounds. With the booming of genome sequencing techniques, increasing microbial genomes have been sequenced and the corresponding bioinformatics has also developed rapidly. Based on the bioinformatics analysis, a large number of secondary metabolite-biosynthetic gene clusters (SM-BGCs) have been discovered in filamentous microorganisms such as Streptomyces and filamentous fungi. However, most of SM-BGCs are dormant or silent under the conventional culture conditions with their corresponding metabolites difficult to be detected, which are regarded as cryptic or silent gene cluster. Through manipulation of the specific regulatory genes in the cluster or global regulatory genes outside the cluster, reconstruction of the metabolic pathways and heterologous expression in other species can activate the expression of cryptic gene clusters. Through activating expression of the cryptic gene clusters, researchers can discover new structural metabolites with unique bioactivities that cannot be obtained through conventional laboratory culture. Activating the cryptic gene clusters is a key approach to produce lead compounds. However, such activation strategies are heavily dependent on the genetic manipulation of the specific strains. Recently, researchers employ co-culture of specific microbial strains under anaerobic or aerobic conditions to activate the cryptic SM-BGCs by mimicking the microbial interactions occurring in natural environments. This strategy does not rely on the genomic information or the genetic manipulation of target strains, and it has the advantage of easy operation. The co-culture strategy requires that the different microorganisms in the mixed culture have similar growth rates and no antagonistic interaction, which partially limits its application. Emergence of the synthetic microbiomes may overcome this limitation and make the co-culture strategy more widely used in future. Here, we overviewed the co-culture systems and applications, the mining of natural products specifically produced in microbial co-culture, and the possible mechanisms underlying this phenomenon.

Abstract:Microbial fermentation is a process of substrate transformation by cell metabolism. In order to improve the yield of target products, we need to analyze the dynamic characteristics of microbial fermentation in a real-time manner so as to optimize the fermentation process. As a promising inline process analysis technology, Raman spectroscopy can achieve accurate monitoring under the in-situ conditions without causing microbial pollution and help optimize the microbial fermentation process. [Objective] To develop the prediction models with the inline Raman measurement technology and evaluate model performance so as to accurately monitor the concentration changes of glucose, xylose, ethanol, and lactic acid in the whole fermentation process of Zymomonas mobilis. [Methods] Raman spectra for multiple components in the fermentation process of Z. mobilis were collected in situ by the immersion probe, and then the partial least square method was adopted to conduct spectral processing and multivariate data analysis. The multivariate models were developed via the combination of Raman spectra with the HPLC data and then used to predict the concentrations of multiple components in the fermentation broth. [Results] The established models accurately measured the single products in the fermentation broth of Z. mobilis. Then, the prediction models for the concentrations of glucose, ethanol, and lactic acid were developed through multivariate analysis and validated, which can be used for real-time accurate determination of multiple components in the fermentation process. [Conclusion] We successfully established a Raman spectral analysis method for monitoring multiple chemicals in the fermentation broth. This is a promising technology for real-time monitoring of multiple substrates and products in industrial fermentation and can be used for strain development and process optimization of microorganisms.

Abstract:Fungal infections (mycoses) have been on the rise and fungal pathogens have gradually developed drug resistance amid the long-term use of conventional antifungal drugs such as fluconazole. Chinese medicine is superior in anti-fungal infection. Our research group has been carrying out systematic and in-depth research on the in vivo and in vitro effects and mechanisms of antifungal Chinese medicine with the support of a number of national and provincial projects. Therefore, on the basis of the latest research outcomes in Chinese medicine’s anti-fungal infection of our research group and others in China and abroad, we summarize the research on Chinese medicine combating fungi (mainly Candida albicans) in the past five years.

Abstract:Hexavalent chromium [Cr(Ⅵ)] is a carcinogen with the toxicity far greater than that of trivalent chromium. The Cr(Ⅵ) discharged by electroplating and tanning have adverse effects on human health and eco-environment. The Cr(Ⅵ) resistance mechanism and reduction process of Cr(Ⅵ)-reducing bacteria have been discussed, whereas there is a lack of summary of the species, chromate reductase activity, and adsorption mechanism of Cr(Ⅵ)-reducing bacteria. In this review, we reviewed the recently reported Cr(Ⅵ)-reducing bacteria via a phylogenetic tree, elaborated the mechanism of Cr(Ⅵ) reduction by bacteria, summarized the enzymatic parameters and reaction conditions of chromate reductase, and expounded the environmental factors affecting the reduction of Cr(Ⅵ) by bacteria. Further, we clarified the adsorption performance and mechanism of Cr(Ⅵ) by bacteria. Finally, we put forward the research prospects on the mechanism of bacterial bioremediation of Cr(Ⅵ) contamination. With this review, we aim to deepen the understanding about the reduction and biosorption processes of Cr(Ⅵ)-reducing bacteria.

Abstract:The foodborne pathogenic Staphylococcus is common in clinical settings. It contaminates food during the raw material processing, packaging, and transportation, thus causing a variety of serious human infections. However, the drug resistance of this species has been on the rise, posing a huge threat to public health. The methyltransferase encoded by the cfr (chloramphenicol-florfenicol resistance) gene in Staphylococcus can cause methylation of bacterial ribosomal RNA, thus blocking or weakening the binding between multiple antibiotics with different chemical structures and peptidyl transferase center (PTC). This explains the development of multiple drug resistance in this species. Linezolid, an oxazolidone, is regarded as the last line of defense after vancomycin in the treatment of infections caused by drug-resistant Gram-positive bacteria. cfr gene accelerates the spread of linezolid resistance. This gene is ubiquitous in a variety of pathogenic Staphylococcus bacteria and the close relationship of the gene with various mobile elements (plasmids, transposons, integration-related elements, etc.) is the structural basis for its wide spread. In the horizontal transfer of cfr gene, foodborne pathogenic Staphylococcus plays an important role as an intermediary. This study reviews the distribution, resistance mechanism, genetic environment, and transfer mechanism of cfr gene in pathogenic Staphylococcus, which is expected provide a reference for prevention and control of the spread of pathogenic Staphylococcus and the control of further spread of multidrug resistant bacteria.

Abstract:Bacteriophages can serve as an alternative for antibiotics. Tailed phages are the most common phages and can be classified into three families, including Podoviridae, Siphoviridae, and Myoviridae according to the different tail structures. Different phages vary in morphology and host recognition mechanism. It is therefore valuable to explore the host recognition mechanisms of Podoviridae phages with simple structure and short genome, which would be benefit for the research on phage-host co-evolutionary relationship and the phage genetic engineering. We reviewed the taxonomic characteristics and different host recognition mechanisms of Podoviridae phages. Deciphering the host recognition mechanisms can help solve the problems in the application of Podoviridae phages, which will contribute to the use of phages in biological, medical, and food industrial fields.

Abstract:Helicobacter pylori (Hp) plays a role in the pathogenesis of gastric diseases and is one of the key factors leading to gastritis, gastric ulcer, and even gastric cancer. With the continuous increase in the positive detection rate of Hp in patients with gastric diseases, scientists have achieved certain progress in the research on the association between gastric diseases and Hp. The eradication therapy for Hp-positive patients and the antibiotic resistance of Hp have attracted increasing attention. Probiotics, as relatively safe natural microorganisms, have great research potential and significance for their functions of inhibiting Hp and improving gastric health. This review summarizes the pathogenic mechanism of Hp and the pathogenicity of different genotypes, and then elaborates the mechanism of probiotics inhibiting Hp. It is recommended that the treatment of Hp infection should combine probiotics with conventional therapies, which will not only increase the eradication rate of Hp but also reduce the side effects associated with treatment.

Abstract:Antimicrobial peptides are peptides with low molecular weight found in almost all forms of life. They are part of the innate immune response of all classes of life, having broad-spectrum antimicrobial activity and low potential to elicit resistance. Thus, they have unique advantages in combating infections and demonstrate the potential as ideal anti-infective agents. However, some problems such as poor stability and high toxicity limit their application. In recent years, it has been found that artificial intelligence can help develop stable antimicrobial peptides with low toxicity, showing great potential in exploring natural antimicrobial peptides. In this review, we briefly summarized the antimicrobial mechanism and structure modification of antimicrobial peptides as well as the strategy of using artificial intelligence algorithms such as machine learning and deep learning for research and development of antimicrobial peptides. This review is expected to provide new mindset for the structure optimization and development of antimicrobial peptides.

Abstract:[Objective] To investigate the relationship between Akkermansia and pulmonary edema of plateau cattle through the analysis of the gastrointestinal microbiota structure in plateau cattle. [Methods] The fecal samples were collected from healthy Jersey cattle in Shenyang (control), healthy Jersey cattle which had been introduced into Lhasa for half a year, local healthy yellow cattle in Lhasa, and Jersey cattle which had been introduced into Lhasa for six months and suffered from pulmonary edema. Illumina MiSeq was used for sequencing the V3–V4 region of the 16S rRNA gene in the samples. Microbiota structure and abundance were compared among the four fecal samples, thereby elucidating the correlation between Akkermansia and the pulmonary edema. [Results] The content of Akkermansia in the gastrointestinal tract of healthy local Lhasa yellow cattle was significantly higher than that of healthy Jersey cattle which had been introduced into Lhasa for half a year, and the content in Jersey cattle with pulmonary edema was significantly higher than that in healthy Jersey cattle that had been introduced into Lhasa for six months. Specifically, the abundance of Akkermansia in the gastrointestinal microbiota of healthy Jersey cattle from Shenyang, healthy Jersey cattle which had been introduced into Lhasa for half a year, local yellow cattle in Lhasa, and Jersey cattle which had been introduced into Lhasa for half a year with pulmonary edema was 0.07%, 0.09%, 6.62% (dominant genus), and 11.85% (the first dominant genus), respectively. [Conclusion] This paper investigated the relationship between Akkermansia and pulmonary edema of plateau cattle for the first time by Illumina MiSeq, which may provide a reference for using the abundance of Akkermansia as an indicator for the diagnosis of pulmonary edema, although the specific abundance value needs to be further determined.

Abstract:As a new representative flotation reagent, 1-nitroso-2-naphthol is widely used in the mining and metallurgical industry to improve the utilization rate of low-grade mineral resources. It is highly stable, posing a challenge to the treatment of heavy metal pollution and organic smelting agent pollution in mine. Among the crucial techniques for the remediation of polycyclic aromatic hydrocarbons (PAHs), bio-remediation is safe and efficient with low cost and no secondary pollution. [Objective] To screen an efficient 1-nitroso-2-naphthol-degrading strain from the typical non-ferrous metal tailings in the periphery of Hechi city, Guangxi Zhuang autonomous region in China, analyze the degradation characteristics and potential metabolic pathways, and thereby examine the conditions for the microbial remediation of the mine polluted by compound pollutants including PAHs. [Methods] The strain which used 1-nitroso-2-naphthol as the only carbon source was screened and identified by 16S rRNA gene sequencing. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze degradation characteristics of 1-nitroso-2-naphthol and the intermediate metabolites, and the metabolic pathways were predicted. [Results] An efficient strain was screened out and identified as the Gram-negative Pseudomonas putida CUGB-JL11. Under the optimal conditions of 30 ℃ and pH 6-8, the 5-day degradation rate of 40 mg/L 1-nitroso-2-naphthol by the strain was up to 81%. The main intermediate metabolites were the benzodiazepines methyl N-hydroxybenzenecarboximidoate and amphetamine, but the organic substances and most of the intermediates were degraded into small molecules or completely degraded. [Conclusion] CUGB-JL11 boasts high 1-nitroso-2-naphthol-degrading efficiency and strong environmental adaptability. It has a huge potential for the treatment of PAHs. This study lays a theoretical basis and provides microbial resources for bioremediation of nonferrous metal mine polluted by both heavy metals and flotation reagents.

Abstract:[Objective] In the natural environment in ancient buildings, cultural relics are vulnerable to mold, especially in the sweltering summer. Therefore, it is important for protecting cultural relics and visitors there to find out the species of airborne fungi. [Methods] We employed the natural precipitation method and the impacting method to collect samples of six representative sites in the main hall of the Hall of Mental Cultivation in summer and analyzed ITS1 rDNA sequences of the airborne fungi. [Results] A total of 22 species of airborne fungi were yielded with the natural precipitation method, which were dominated by Cladosporium, Aspergillus, and Penicillium. At two sites (the second floor of the Buddha Hall and the West Chamber), the count of airborne fungi exceeded the standard. The impacting method measured over 100 species and a large proportion of them were saprophytes. The dominant taxa were Alternaria, Cladosporium, Trichoderma, Rhizopus, Aspergillus, and Penicillium. At all the six sites, the count of fungal communities was above the standard. As for the correlation between environmental factors and fungal diversity, the abundance of fungi was in close relationship with the temperature, humidity, and suspended particulate matter in the Hall. In June when relative humidity is low, temperature had great impact on abundance. In the instance of high humidity, suspended particulate matter and humidity had greater effect on fungal abundance. The abundance of filamentous fungi was in significantly positive correlation with small suspended particulate matter and relative humidity, while the airborne yeast had a higher correlation with temperature. [Conclusion] This study identifies the species of airborne fungi in the main hall of the Hall of Mental Cultivation and analyzes the correlation with environmental factors, which lays a scientific basis for the prevention, exhibition, and preservation.

Abstract:[Objective] This study aims to predict and analyze the structural characteristics and physicochemical properties of G-protein-coupled receptors (GPCRs) in Aspergillus ochraceus and to explore the clustering of GPCR proteins and their evolutionary relationships with the homologous proteins. The findings are expected to lay a theoretical basis of further research on the locations and functions of GPCRs in A. ochraceus, help inhibit ochratoxin production from the perspective of G protein signaling pathway, and further control mycotoxin contamination in grains. [Methods] Candidate GPCR proteins were screened through BLASTp alignment of A. ochraceus genome against the reported typical GPCR sequences of Aspergillus sp. Conserved domains, especially transmembrane domains, were analyzed by SMART and a variety of software. The physicochemical properties, signal peptides, secondary structures, and subcellular localization of the candidate sequences were further analyzed. Finally, MEGA was used to construct a phylogenetic tree of GPCRs in A. ochraceus and homologous proteins, and the genetic relationship was elucidated. [Results] A total of 15 GPCR proteins with typical seven transmembrane helices were found in A. ochraceus, but they had no signal peptides or transit peptides. They all contained a large proportion of α-helices, and 7 of the 15 proteins were located at the cell membrane. GPCRs in A. ochraceus had close genetic relationship with the homologous sequences in A. flavus and other Aspergillus species. [Conclusion] In this study, the GPCRs in A. ochraceus were predicted for the first time. The structures and physicochemical properties of them and the clustering with homologous proteins were analyzed, laying a theoretical basis for further research on the functions of GPCRs in A. ochraceus.

Abstract:[Objective] Based on the existing problems in the current cell surface display systems, we aimed to establish a novel Saccharomyces cerevisiae spore surface display system with strong universality, better stress resistance, and higher efficiency and stability. [Methods] Firstly, according to the characteristics of immobilized enzymes in S. cerevisiae spores, we searched the potential chitosan-binding modules with high affinity with the chitosan layer of spore wall by referring to literature. Then, the binding module was fused and expressed with green fluorescent protein (GFP) and the affinity ability of the binding module with the spore wall was verified in vitro and in vivo. Finally, we selected α-galactosidase (MEL1) derived from S. cerevisiae AH109 to evaluate the efficacy of the novel display system. [Results] Firstly, we selected a carbohydrate binding module 32 (CBM32) derived from Paenibacillus sp. IK-5 chitosanase as the chitosan-binding module. Next, the fusion protein CBM32-GFP, which was expressed in Escherichia coli and purified, was co-incubated with dit1Δ spores, and the result showed that CBM32 exhibited good affinity ability with the spore wall in vitro by the localization and intensity of GFP fluorescence. Furthermore, the fluorescence localization and binding ability of CBM32-GFP in dit1Δ spores proved that CBM32 was tightly bound to the spore wall in vivo. Finally, we replaced GFP with MEL1 in this display system. Compared with those of spores only expressing MEL1, the activity of spores displaying CBM32-MEL1 was increased by 68.65%, with the highest specific activity reaching 460.59 U/g DCW (dry cell weight), and the reusability was also significantly improved. Moreover, the stability and operability of MEL1 were enhanced. [Conclusion] We constructed a novel S. cerevisiae spore surface display system based on the chitosan-binding module for the first time, which provided a reliable cell surface display platform for the eukaryotic proteins with multi-glycosylation sites and multi-subunit structures. It also provided the theoretical basis for the industrial application of immobilized enzymes in spores.

Abstract:[Objective] To assess the effect of different dilution gradients of dilution-plate method on the number and composition of soil bacteria, and to study the differences in soil bacterial communities under distinct land use scenarios by comparing plate-dilution method and high-throughput sequencing. [Methods] With four types of soil (secondary forest soil, healthy banana soil, diseased banana soil and paddy soil) collected, soil suspensions of five dilution gradients (10^-1–10^-5) were prepared. Classic plate-dilution method was employed to obtain culturable bacteria, followed by colony-counting and DNA extraction. In addition, the total bacterial DNA in background soils was extracted. Then high-throughput sequencing of 16S rRNA gene was performed to study the diversity of bacterial communities at different dilution gradients and in background soils, proportion of culturable bacteria in total soil bacteria, and species difference. [Results] The highest increase in soil respiration was found after the conversion of secondary forest to banana plantations, and both dilution-plate method and real-time fluorescence quantitative PCR found the highest decrease in bacterial counts. However, the results of these two methods were not entirely consistent for other land use changes affecting soil bacterial populations. The Chao 1 index of the soil culturable bacterial community was reduced by 86%–98% compared with that of the total background soil bacteria. Dilutions of 100–100 000 times (10^-2–10^-5) lowered the Chao 1 index of the culturable bacterial community by 35%–60% compared with dilutions of 10 times of soil suspensions. Gradient dilution also reduced the species diversity. Dilution-plate method detected a total of 315–401 microbial genera, with only 21–38 genera common to all dilution gradients, compared with 92–210 genera unique to the 10^-1 gradient and 2–59 genera unique to the 10^-2–10^-5gradients. The proportions of culturable bacteria in the four types of background soils were in the range of 16.1%–47.7% (phylum level) and 7.4%–30.9% (genus level). The number of significantly increased and decreased species detected by plate-dilution method was only 9.7% and 22.9%, respectively of the results by high-throughput sequencing. The two methods identified a number of common divergent species, including Bacillaceae, Micrococcaceae, Microbacterium, Comamonadaceae and Burkholderiales. [Conclusion] The highest percentage of culturable species in total soil bacteria was 47.74% and 30.90% at phylum and genus levels, respectively. The community diversity of culturable species was much lower than that of the background soil bacteria. The proportion and diversity of culturable bacteria was significantly higher in the 10^-1 dilution gradient than in the 10^-2–10^-5 gradients, while there was no remarkable difference between the 10^-2–10^-5 gradients. Although the dilution-plate method greatly underestimated the differences in bacterial communities between land use practices, common differential bacterial taxa were found by the culture-free and culturable methods. Compared to the background abundance of bacteria in the four soils, the solid plates were significantly enriched in Pseudomonas and Flavobacterium, with fold increases of 2 233–5 805 and 43–4 506. Culture-independent molecular techniques and classical culturable methods should be coupled in the future research to deepen the exploration of microbial resources in complex environments.

Abstract:[Objective] To identify a novel type III secreted effector (T3SE) gene in the genome of Xanthomonas campestris pv. campestris (Xcc) strain 8004. [Methods] A Tn5 transposon system integrated with AvrBs1_59-445was constructed for library screening. The mutant library was generated based on avrBs1-deleted mutant and then screened on Capsicum annuum cv. ECW-10R. [Results] Seven clones with visible hypersensitive response (HR) were screened out via large-scale HR assay. In addition to 3 mutants inserted into known T3SE genes, a new locus was identified, which was located between XC_0438 and XC_0439 and un-annotated in Xcc 8004 by plasmid rescue and sequencing. We annotated the new gene designated as XC_0438a according to the bioinformatics analysis results. The translocation assay confirmed that the signal region of XC_0438a could guide the secretion and translocation of the reporter protein AvrBs1 and elicit the HR of ECW-10R. The results of β-glucuronidase (GUS) activity assay demonstrated that the expression of XC_0438a was induced in nutrition sterile medium and activated by the key regulatory proteins HrpG and HrpX. However, in our tested conditions, XC_0438a had no significant contribution to the pathogenesis of Xcc. [Conclusion] In summary, we identified a novel effector gene XC_0438a dependent on the type III secretion system.

Abstract:[Objective] To investigate the drug resistance status of wild birds carrying bacteria and to explore their role in the transmission of bacterial drug resistance. [Methods] Four Klebsiella pneumoniae were isolated from fresh feces of captured Alectoris chukar, Psittacula alexandri, Aratinga solstitialis and Sturnus nigricollis, and were assessed for multidrug resistance phenotypes by micro-broth dilution method. Bacterial genome-wide association analysis and comparative genomics were used to trace the isolates and systematically analyze the association between the multidrug resistance plasmids/genes and their hosts/homologous plasmids. [Results] Four strains of K. pneumoniae showed different drug resistance phenotypes. Specifically, S90-2 from A. chukar was resistant to nine drugs including ampicillin, cefuroxime, cefazolin, ceftriaxone and cefepime; S141 from P. alexandri was resistant to ampicillin, cefuroxime and cefazolin; M911-1 from A. solstitialis was resistant to ampicillin only; S130-1 from S. nigricollis was sensitive to all of the 14 drugs. S90-2 belonged to ST629 type and carried 30 resistance genes including bla_CTX-M-14, fosA6, aac(3)-Iid and bla_SHV-11, and its plasmid pS90-2.3 (IncR) carried resistance genes of mphA, dfrA12, aadA2, qacEdelta1, sul1, tet(A), aph(3')-Ia, sul2 and aph(3')-Ib. S141 belonged to ST1662 type and carried 27 resistance genes including fosA5 and bla_SHV-217, and only plasmid pS141.1 [IncFIB(K)(pCAV1099-114)/repB] carried one resistance gene adeF. M911-1 was a new ST type, carrying 27 resistance genes such as bla_SHV-1 and fosA6, and its plasmid pM911-1.1 (novel) carried three resistance genes qnrS1, bla_LAP-2 and tet(A). S130-1 belonged to ST3753 type, carrying 27 resistance genes such as bla_SHV-11 and fosA6, and its plasmid pS130-1 [IncFIB(K)] carried only one resistance gene tet(A). The plasmids pM911-1.1 and pS90-2.3 failed to perform conjugative transfer, but their resistance gene fragments were derived from multiple homologous chromosomes or plasmids of Enterobacteriaceae strains, and the formation of resistance gene fragments (MDR region) involved interactions between multiple mobile element genes, resulting in a complex and diverse structure of resistance plasmid. The homologous plasmids related to pM911-1.1 and pS90-2.3 were mainly from human bacteria hosts in China, such as K. pneumoniae and Escherichia coli, and the K. pneumoniae ST11 was a major host for the above drug-resistant homologous plasmids. [Conclusion] The multidrug-resistant K. pneumoniae from wild birds in this study had resistance genes mainly from plasmids, which were mediated by transposons, insertion sequences, integrons and prophage and other mobile elements, and these multidrug-resistant plasmids were closely related to the human host bacteria.

Abstract:[Objective] The present study aims to investigate the richness, diversity, and rhythm of lactic acid bacteria in the colon of growing pigs within 24 h. [Methods] The colonic contents from 6 growing pigs with colon fistula were collected every 3 h within 24 h starting from 6:00. The DNA of colonic bacteria was extracted for PCR which was performed with the primers specific to lactic acid bacteria. The abundance and rhythm of intestinal lactic acid bacteria at genus and species levels were analyzed via high-throughput sequencing. [Results] The Chao1 and Simpson indexes of intestinal lactic acid bacteria in growing pigs changed significantly within 24 h (P<0.05). Lactobacillus was the dominant genus in total lactic acid bacteria, with the lowest relative abundance of 94.15% at 6:00 and the highest relative abundance of 97.46% at 18:00. Lactobacillus johnsonii was the most dominant species with the lowest relative abundance of 47.66% at 3:00 and the highest relative abundance of 71.59% at 18:00. Lactobacillus reuteri took the second place in the relative abundance at the species level. Forty-six core OTUs of the lactic acid bacteria in the colon of growing pigs showed circadian rhythm, all of which belonged to Lactobacillus. At the species level, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus sp. KC45a and Lactobacillus reuteri showed circadian rhythm (P<0.05). [Conclusion] The richness and diversity of lactic acid bacteria in the colon of growing pigs changed significantly within 24 h, showing circadian rhythm at the species level. This study enriches our understanding on the circadian rhythm of intestinal microbiota in pigs.

Abstract:[Objective] To improve the comprehensive utilization of salted duck egg white, bioactive proteolytic products were prepared. [Methods] In this study, salted duck egg white wasdesalted and graded by ultrafiltration. The protein fractions of salted duck egg white within different molecular weight ranges were hydrolyzed by pepsin. The bacterial inhibition and antioxidant activitiesofthe hydrolysates were determined in vitro, including the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and cell membrane integrity. [Results] Three fractions of different molecular weights were obtained from salted duck egg white by ultrafiltration, which were fraction A with Mw>50 kDa, fraction B with 50 kDa>Mw>20 kDa and fraction C with 20 kDa>Mw>5 kDa. Among them, the hydrolysates of fractions A and B had inhibitory effects on the growth of Escherichia coli. The MICs of the two fractions were 1 024 μg/mL and 2 048 μg/mL, respectively, and the MBCs were both 2 048 μg/mL. The OD₆₀₀ value of E. coli was about 1.15 after entering the stationary phase. When the hydrolytic peptide was added at a concentration of 1×MIC, the OD₆₀₀ values of the bacterial solution in stationary phase decreased to 0.79 (A) and 0.86 (B), respectively. When the hydrolytic peptide was added at a concentration of 2×MIC, the OD₆₀₀ values decreased to 0.5 (A) and 0.68 (B), respectively. The two hydrolysates had good antibacterial effects, which were attributed to the destruction of E. coli cell membrane. However, Staphylococcus aureus was not sensitive to all three hydrolysates, and the inhibitory effect was poor. Moreover, the hydrolysates of fractions A, B and C all had antioxidant activities, and their scavenging ability against DPPH radicals reached 0.50, 0.67 and 0.38 times Trolox, respectively. [Conclusion] The salted duck egg white could be simultaneously desalted and enriched to obtain different target proteins by ultrafiltration. The hydrolysates of the obtained target proteins possessed antibacterial and antioxidant activities, and could be used as nutritional additives, which improved the added value and utilization of salted duck egg white.

Abstract:[Objective] To evaluate the level of neutralizing antibodies (NA) against foot-and-mouth disease virus (FMDV), a solid-phase blocking enzyme-linked immunosorbent assay (ELISA) based on bovine monoclonal antibodies was developed. [Methods] In this study, the bovine monoclonal antibody (mAb) E32 was used as the capture antibody and a biotinylated bovine mAb C4 as the detection antibody. Both mAbs had been produced in our laboratory. The mAb E32 is a cross-reactive antibody against FMDV and the mAb C4 is an intraserotype broadly neutralizing antibody against FMDV serotype O. With the two mAbs, a solid-phase blocking ELISA for detecting neutralizing antibody (NA-SPBE) against FMDV serotype O was developed. The sensitivity, specificity, repeatability, cross-reactivity, and correlation with virus neutralization test (VNT) of this assay were assessed. [Results] The optimum working concentrations of antibody E32, FMDV-inactivated antigen, and biotinylated C4 were 0.5 μg/mL, 0.25 μg/mL and 0.06 μg/mL, respectively, and the optimum dilution of streptavidin-HRP was 1:40 000. When 1.35 log₁₀ was used as the cut-off value, the sensitivity and specificity of the assay were determined as 97.14% and 98.84%, respectively. There was no cross reactivity with the antibodies specific to bovine viral diarrhea virus (BVDV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), peste des petits ruminant virus (PPRV), or FMDV serotypes A and Asia1. The intra-batch and inter-batch repeatability of the assay showed the coefficient of variation less than 10%. The detection of 160 serum samples demonstrated a correlation (r=0.807 5, P<0.000 1) between the titers obtained by NA-SPBE and VNT. [Conclusion] NA-SPBE can detect the neutralizing antibodies against FMDV serotype O and provides powerful technical support for evaluating the efficacy of FMDV serotype O-inactivated vaccine.

Abstract:Lysobacter capsici X2-3, a plant growth promoting rhizobacteria (PGPR) strain isolated from the wheat rhizosphere, shows strong antimicrobial activity against many plant pathogenic fungi and oomycetes. However, little is known about the antibiotics produced by X2-3 as well as their regulation mechanism. [Objective] Our study aimed to clarify the regulation effect of LC_Clp transcription factor on the antibiotic production of L. capsici X2-3 and to provide reference for deep understanding of its biocontrol mechanism. [Methods] We selected a LC_clp mutant M356 from the mutant bank that was generated by the random insertion of the EZ-Tn5 transposon and certified by plasmid rescue. Additionally, we analyzed the differences of LC_clp gene in antimicrobial activity, secretion of extracellular enzymes, and regulation of gene expression among X2-3, mutant M356 and the complementary strain MCS28. [Results] Antimicrobial activity of mutant M356 against pathogenic fungi and oomycetes was completely lost as compared with that of X2-3 and the antibiotics produced by M356 was undetected by HPLC method. Extracellular enzymes such as protease, cellulose and chitinase that was secreted by mutant M356 decreased significantly. Expression of genes relating transcriptional regulators, secondary metabolism and extracellular enzymes were lowered dramatically in M356 as compared with that of wild-type strain X2-3. However, all the items tested in complementary strain MCS28 were similar with those of wild-type strain X2-3. [Conclusion] As a global regulator, LC_Clp regulates the synthesis of antibiotics, the production of extracellular enzymes and the expression of multiple genes in L. capsici X2-3.

Abstract:[Background] The yeast for biocontrol is characterized by rapid growth, strong stress resistance, and absence of antibiotic production. However, their biocontrol effects are easily influenced by environmental conditions. Brassinosteroid (BR) regulates the balance between plant growth and stress response and efficiently controls the occurrence of gray mold (Botrytis cinerea) on grape berries. [Objective] To investigate the effect and mechanism of Metschnikowia pulcherrima combined with BR in controlling B. cinerea on grape berries, and to provide a theoretical basis for the development and application of new biocontrol agents. [Methods] The berries of ‘Red Globe’ grape were treated with M. pulcherrima P01C004 (Y), P01C004+BR (YBR), and P01C004+brassinazole (YBZ), respectively. The spore suspension of B. cinerea was inoculated on the grape berries 6 h after the Y, YBR, and YBZ treatments. The efficiency of disease control, the activities of antioxidant enzymes, and the content of 13 phenolic compounds were evaluated 7 days after the inoculation of B. cinerea. qRT-PCR was performed to quantify the expression of the genes related to BR signaling pathway and pathogenesis under different treatments. [Results] Compared with Y treatment, YBR increased the control efficiency against B. cinerea by 23.64% and significantly improved the activities of polyphenol oxidase in grape berries. YBR significantly increased the content of chlorogenic acid, protocatechin, caffeic acid, epicatechin, and apigenin, and activated the rapid synthesis of phenols 2 days after inoculation. YBR activated the BR signaling pathway and up-regulated the expression levels of genes VvBZR1 and VvPR1 at the time point of 48 h, which sustained the plant immunity and induced strong defense responses of grape berries to the pathogen. [Conclusion] The combined application of M. pulcherrima and BR triggered multiple defense mechanisms of grape and improved the control efficiency against B. cinerea on grape berries, demonstrating a promising prospect in the postharvest disease control.