Abstract:Small open reading frames (sORFs) are ubiquitous in the genomes of living organisms, which are generally not fully annotated and studied due to their short length and difficulty in detection of their encoded small proteins (also termed microproteins or miniproteins). With the advancement of high-throughput sequencing, translatomics and mass spectrometry, increasing sORFs have been identified in different living organisms. At the same time, specific bioinformatic tools have also contribute to the mining of sORFs. Small proteins encoded by sORFs as well as translational regulation mediated by sORFs have been applied in medicinal development and exploration of mechanisms underlying plant disease resistance. However, the studies and applications of sORFs in microorganisms remain limited. This review summarizes the latest progress in the identification of small proteins encoded by sORFs, the annotation of upstream open reading frames (uORFs), as well as uORF-mediated regulation on mRNA translation. In particular, we reviewed the identification and functional analysis of sORFs in microbial genomes, especially budding yeast and filamentous fungi. This review provides a basis for understanding the function and mechanism of sORFs, as well as studies on microproteins and translation regulation in other organisms including plans and animals.

Abstract:As biotechnology advances, biodegradation of polyethylene terephthalate (PET) has emerged as an attractive strategy, promising to reduce the environmental burden caused by waste PET. Among many PET hydrolases, PETase from Ideonella sakaiensis has become a hot research topic owing to high specificity to PET substrate. Based on the structure and function of PETase, this review summarized modifications of this enzyme in recent years to improve the degradation activity, thermostability, and adsorption of the enzyme. Moreover, we also introduced the secretory expression systems and the cell surface display of PETase, as well as the application of the PETase together with MHETase. Finally, we summed up the challenges in and possible solutions to plastic biodegradation. This study is expected to provide a reference for promoting the practical application of polymer biodegradation.

Abstract:Aromatic hydrocarbons, a class of organic compounds with one or more benzene rings, are ubiquitous in the natural environment. They are difficult be degraded naturally and thus easy to be bioaccumulated, posing a huge threat to the environment. Biological degradation seems to be the mainstream method for the transformation of organic compounds, and electroactive microorganisms have great potential in the removal of aromatic hydrocarbons because of their unique extracellular electron transport ability and physiological metabolism mode. They can finally achieve the degradation and mineralization of aromatic hydrocarbon pollutants by combining reductive dehalogenation and denitrification and oxidative ring cleavage. In this paper, we focused on the main reduction/oxidation reaction mechanisms in the degradation of aromatic hydrocarbon pollutants by electroactive microorganisms, summarized the key enzyme activities, metabolic pathways, and transformation mechanism of electroactive microorganisms in the reductive dehalogenation and denitrification, analyzed the ring cleavage modes and metabolic pathways of electroactive microorganisms under different oxygen-containing conditions, and improved the extracellular electron transport process of the microorganisms by regulating microbial extracellular polymers and adding conductive materials. Moreover, we discussed the influence of electrode potential, electrode materials, and environmental factors such as electrolyte properties and temperature on the degradation of aromatic hydrocarbon compounds, and the feasibility of enhanced biodegradation strategies for aromatic hydrocarbon pollutants. Finally, in order to provide theoretical and technical reference for accelerating the engineering application of bioelectrochemical systems, we summed up the directions of future potential research in the related fields of electroactive microbial degradation technology.

Abstract:The increase in people’s life pressure makes many men in the middle age suffer from hair loss, even alopecia areata or alopecia totalis, in which androgenetic alopecia is the most common. The occurrence of androgenetic alopecia is mainly due to the high content of testosterone (T) and its metabolite dihydrotestosterone (DHT) in the scalp. The 5α-reductase in the hair follicle converts free T to active DHT, resulting in excessive androgen which can make hair follicles more microscopic and shorten the hair growth cycle to cause hair loss. A growing number of studies based on the gut-skin axis theory have demonstrated that probiotics can alleviate or treat hair loss. Probiotics can regulate the balance of intestinal flora and promote the expression of hair growth factors and the growth of hair follicle cells to alleviate the hair loss caused by excessive pressure or androgen. This paper mainly discusses the association between probiotics and hair growth. Focusing on the regulatory effects of probiotics on nervous system, immune system, and endocrine system, we reviewed the functions and action mechanism of probiotics, aiming to provide a theoretical basis for the research and development of food and drugs alleviating hair loss.

Abstract:Type 2 diabetes (T2D) is a metabolic disorder characterized by the imbalance in the levels of blood glucose and lipid, threatening human health. Therefore, it is an urgent task to find a safe therapy with no side effects. Probiotics top the microbial therapies against T2D, which exert the anti-diabetes effect by regulating different tissues and metabolic pathways. To be specific, they modulate blood glucose by reducing chronic low-grade inflammation, alleviating oxidative stress, regulating intestinal flora, and increasing the content of short-chain fatty acids. They regulate blood lipids by enhancing the coprecipitation of cholesterol and bile salt and the transformation of cholesterol into fecal sterol in the gastrointestinal tract, reducing the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the liver, lowering the expression of cholesterol transporter, and regulating adipocytes. In this review, we summarized the current status of probiotics against diabetes and the anti-diabetes mechanisms based on intestinal microecology, hoping to lay a theoretical basis for developing hypoglycemic and lipid-lowering products with probiotics.

Abstract:Anaerobic ammonium oxidation (anammox) is an important reaction in microbiology, geology, and environmental science, and anaerobic ammonium-oxidizing bacteria (AnAOB) are usually regarded as the driver of anammox. Demonstrating the biological characteristics of AnAOB is a vital issue for the application of anammox. Crateriform structure is a special microstructure and has been identified as one of the key features of AnAOB. However, little is known about the crateriform structure of AnAOB due to the failure to isolate AnAOB. In this study, the research advances about the crateriform structure of AnAOB and Planctomycetes were reviewed in terms of morphological characteristics, physiological functions, and ecological effects. Conclusions can be drawn as follows: (1) The crateriform structure of AnAOB is uniformly distributed over the cell surface, with a diameter of about 5 nm. (2) The crateriform structure of AnAOB may connect cytoplasmic membrane, outer membrane, and anammoxosome membrane, and play a role in substrate transport. (3) The crateriform structure has genetic stability, and its formation may be related to flagellum degeneration. (4) The crateriform structure may serve to maintain the niche stability of AnAOB by promoting material exchange and communication between microorganisms.

Abstract:Aging is characterized by hypofunction of tissues and organs and increased risk of related diseases, which poses a series of challenges to the health and longevity. Despite extensive basic research on aging, the progress is limited. There is a growing awareness that the structure and function of the gut microbiota are involved in the aging. Disturbed gut microbiota manifests itself in the aging of many age-related extraintestinal organ axes. Gut microbiota can be modulated, suggesting anti-aging can be realized through gut microbiota. This study summarizes the dynamic succession of gut microbiota in different age groups. This dynamic gut microbiota develops rapidly from the fetus through birth and infancy, changes rapidly from weaning to early childhood, and then establishes a stable adult microbiota until it finally declines with age. Moreover, the research on the gut microbiota and aging-related diseases of the extraintestinal organ axis (brain, heart, liver, pancreas, muscle, skin, and bone), and targeted anti-aging by modulating the gut microbiota through diet, fecal microbiota transplantation, and microecologics is also summed up. This review is expected to provide a reference for research on anti-aging via gut microbiota.

Abstract:Peptidoglycan is the most important component of bacterial cell wall, as it is crucial for the maintenance of cell morphology, cell size and cell survival. Meanwhile, peptidoglycan is the target of many commonly used antibiotics. The synthesis and hydrolysis of peptidoglycan occur simultaneously in bacteria under normal growth conditions. For the sake of cell wall integrity, the biosynthesis of peptidoglycan needs to be spatiotemporally controlled. Peptidoglycan biosynthesis and the regulatory mechanism are among the most fundamental research topics in microbiology. In recent years, researchers around the world have made remarkable progress in this field. On this basis, this review summarizes the de novo synthesis of peptidoglycan and peptidoglycan recycling pathway, and emphasizes the research advances in peptidoglycan synthases (key enzymes involved in peptidoglycan synthesis) and their regulatory mechanisms. Finally, this review puts forward the questions to be addressed in the future.

Abstract:In recent years, microbial therapy based on the fact that microorganisms can provide health benefits has created new opportunities for the diagnosis and treatment of diseases. Studies have demonstrated that oral administration of Lactobacillus lactis, Escherichia coli, and Bifidobacterium can assist in the treatment of a variety of diseases. In microbial therapy, engineered bacteria have attracted much attention owing to their specific functions. They can either assist in diagnosis by sensing specific signaling molecules in the disease environment, or target the specific signals of the disease site and initiate their expression systems to release specific molecules for precise treatment. This paper reviewed the sensing systems of engineered bacteria for different signaling molecules and the application of the sensing systems in biomedicine.

Abstract:Insects lack the acquired immune system in higher animals and rely on the innate immune system (humoral immunity and cellular immunity) to combat pathogen infection. Humoral immunity is a major antiviral mechanism in insects, involving Toll-like receptor (TLR), immunodeficiency (IMD), nuclear factor kappa B (NF-kB), Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathways. These pathways play an essential role in the antiviral invasion. Additionally, DNA damage response (DDR) is also an effective mechanism of antiviral defense in insects. It can block a series of cell signaling pathways by initiating cell cycle arrest, triggering cell apoptosis or aging in the host. We summarized the roles of JAK-STAT signaling pathway and DDR in the antiviral invasion of insects. Understanding the interactions between pathogens and insects can help us to not only understand the molecular mechanism of insects blocking viral invasion but also lay a foundation for us to find new targets for antiviral invasion.

Abstract:The terrestrial Cymbidium goeringii is of high ornamental and economic value. With the habitats being destructed, most terrestrial orchids are endangered. The roots of Orchidaceae plants maintain a symbiotic relationship with microbes from seed germination to flowering and fruiting. Therefore, the endophytic microbes of the roots play an important part in the life cycle of terrestrial orchids.[Objective] In this study, we analyzed the composition and potential functions of endophytic microbes in the roots of wild C. goeringii, hoping to provide a reference for artificial conservation of this species. [Methods] Metagenomic sequencing of wild C. goeringii roots collected from Kunming city, Baoshan city, and Dali city in Yunnan province was performed, and the species and functions of species were analyzed. [Results] The abundance and diversity of microbes in roots of C. goeringii collected from Baoshan city were higher than those in samples from Dali city and Kunming city. The dominant phyla of endophytic fungi were Basidiomycota, Ascomycota, and Glomeromycota and the dominant families were Glomeraceae, Polyporaceae, and Ceratobasidiaceae, among which Ceratobasidiaceae fungi were the major orchid mycorrhizal fungi (OMF). The endophytic bacteria were dominated by Firmicutes, Proteobacteria, and Bacteroidetes, as well as Erysipelotrichaceae, Cyclobacteriaceae, and Acetobacteraceae. As for the endophytic archaea, the dominant phyla and family were Euryarchaeota, Thaumarchaeota, and Natrialbaceae. Caulimoviridae dominated the endophytic viruses. These microbes were mainly involved in Kyoto encyclopedia of genes and genomes (KEGG) pathways of metabolism and genetic information processing. The annotation of pathogen-host interactions (PHI) database suggested that endophytic Fusarium/Gibberella, Magnaporthe, and Aspergillus may be the potential pathogens of wild C. goeringii. [Conclusion] In this study, the main groups of endophytic microbes in the roots of wild C. goeringii in three regions of Yunnan province were identified, and the colonization of Glomeraceae was found for the first time in C. goeringii. Moreover, the functions of the endophytic microbes were analyzed. The result is expected to lay a theoretical basis for the conservation of wild C. goeringii, mycorrhization of artificial seedlings, and disease control.

Abstract:[Objective] A metabolic pathway was designed in Saccharomyces cerevisiae so that it could produce ethanol from cellobiose, a hydrolysate of cellulose. [Methods] First, with the total DNA of Escherichia coli DH5α as template, the LacY gene encoding lactose permease was cloned. With plasmid YEplac181 as the vector, the strong promoter PGK1p of S. cerevisiae was added in front of the LacY, and the CYC1t terminator was added behind LacY to construct the plasmid YEplac181-PGK1p-LacY-CYC1t. Then, the cellobiose transporter (LacY) expression plasmid and β-glucosidase expression plasmid pRS316-PGK1p-gh1-1-CYC1t were successively transferred into wild-type S. cerevisiae W303-1A. The engineered S. cerevisiae W303-1A GL was constructed by heterologous expression of cellobiose transporter LacY and β-glucosidase GH1-1 in W303-1A. Finally, the cellobiose utilization and ethanol yield of W303-1A GL were determined based on fermentation, and the cellobiose activity in the cellobiose metabolic pathway was detected. [Results] W303-1A GL which expressed LacY and GH1-1 was developed, which can use cellobiose to produce ethanol. The ethanol yield of W303-1A GL was up to 3.25 g/L at 24 h, and the production rate was 0.325 g ethanol/g cellobiose, 64% of the theoretical production rate from glucose (0.511 g ethanol/g cellobiose). The highest cell density was OD₆₀₀=10.84 at 54 h, and the activity of intracellular β-glucosidase peaked (0.51 U/mg) at 72 h. [Conclusion] W303-1A GL which can effectively utilize cellobiose is developed. This study is expected to serve as a reference for improving the production of cellulosic ethanol at lower cost.

Abstract:[Objective] To isolate and identify a heavy metal-resistant strain in the polluted soil and explore the factors influencing the adsorption of the strain for lead and cadmium under different conditions. [Methods] The strain was identified based on physiological and biochemical characteristics and ITS sequence. The streak plate method was employed to determine the maximum lead and cadmium tolerance of the strain and to explore the optimal conditions for the adsorption of lead and cadmium. The adsorption process was studied by pseudo-second-order kinetics, Langmuir and Freundlich models, and infrared spectroscopy. [Results] Strain JB16 was isolated and identified as Aureobasidium pullulans, which can tolerate the maximum lead concentration of 1 500 mg/L, the maximum cadmium concentration of 750 mg/L, and the maximum lead-cadmium mixture concentration of 1 500 mg/L and 300 mg/L, respectively. The single factor experiments (temperature, time, cell age, pH, wet mycelial concentration, and initial heavy metal concentration) optimized the lead adsorption conditions as follows: 30 ℃, 2 h, cell age of 72 h, pH 6, wet mycelial concentration of 5 g/L, and initial lead concentration of 150 mg/L, under which the adsorption rate of lead was 88.5%. The optimum conditions for cadmium adsorption: 30 ℃, 1 h, cell age of 96 h, pH 6, wet mycelial concentration of 5 g/L, and initial cadmium concentration of 20 mg/L, under which the adsorption rate of cadmium was 59.4%. The adsorption process of lead and cadmium by the strain conformed to the Langmuir model and the pseudo-second-order kinetic model, which indicated surface monolayer adsorption. The results of scanning electron microscopy and infrared spectroscopy demonstrated that the heavy metal ions changed the morphology of the strain, and the hydroxyl, carboxyl, saturated C-H bonds, and amide groups on the cell surface participated in the adsorption. [Conclusion] Strain JB16 can adsorb lead and cadmium, which enriches the strain resources and provides data support for the remediation of water and soil contaminated by lead and cadmium.

Abstract:[Objective] CalA3 subunit catalyzes the release of the polyketide chain in the biosynthesis of calcimycin. This study aims to obtain CalA3 proteins with stable biochemical properties and homogeneous structure for cryo-electron microscopy, to understand the mechanism of polyketide chain releasing by the module of the polyketide synthase, and to explore the selectivity of CalA3 to polyketide substrates with different structures, thereby providing biochemical materials for the complexes of CalA3 with small ligands and a reference for further exploring the catalytic potential of CalA3. [Methods] The medium for culturing the heterologous expression strain and the biochemical conditions for CalA3 protein purification were optimized. Then, the CalA3 proteins were analyzed by negative-stain transmission electron microscopy. The reaction products of S-(2-acetamidoethyl)3-(1H-pyrrol-2-yl) propanethioate (SANC-C3), N-(2-(pentylthio) ethyl) acetamide (SNAC-C5), lauroyl-CoA and 3-hydroxy anthranilic acid (3HA) catalyzed by CalA3 were identified by high performance liquid chromatography and high-resolution mass spectrometry (LC-MS). [Results] The strains cultured in the optimized medium PGTY, expressed more CalA3 proteins which exhibited featured structures under negative-stain transmission electron microscope. The purified CalA3 proteins were prepared for cryo-electron microscopy for structural determination. CalA3 catalyzed the aminolysis reaction of SNAC-C5 and SNAC-C3 with 3HA. However, the products of lauroyl-CoA and 3HA catalyzed by CalA3 were not detected. [Conclusion] The results of protein purification and negative-stain transmission electron micrographs show that the conditions for culturing Escherichia coli for heterologous expression and purifying CalA3 proteins are established. The results of in vitro catalytic experiments demonstrate that CalA3 has broad selectivity for the structure of polyketide substrates. These findings provide a reference for further exploring the structure, functions, and application of polyketide synthases.

Abstract:[Objective] To study the changes in microbial communities and metabolites during the vegetable fermentation exposed to oxytetracycline residue and further provide a theoretical basis for evaluating the effects of antibiotic residue on vegetable fermentation. [Methods] In this study, we determined the oxytetracycline residue by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), organic acids by high performance liquid chromatography (HPLC), volatile components by the electronic nose and gas chromatography mass spectrometry (GC-MS), microbial species by high-throughput sequencing. [Results] The oxytetracycline residue decreased from 4.00 mg/L to 2.53 mg/L in spontaneous vegetable fermentation. The vegetable without oxytetracycline residue experienced homolatic and heterolactic fermentation, while that with oxytetracycline residue only experienced homolatic fermentation. Meantime, the characteristic microorganisms changed from Lab. pentosus and Lab. plantarum to L. paratarrginis, Lab. buchneri, and Lab.kisonensis. Furthermore, oxytetracycline residue significantly affected the content of lactic acid, citric acid, acetic acid, citronellol, 3-octanol, allylsenevol, geranyl acetate, and phenylethyl isothiocyanate. [Conclusion] Oxytetracycline residue affected the type of vegetable lactic acid fermentation, the succession of microbial communities, and the production of organic acids and volatile compounds. Therefore, the antibiotic residue should be listed as a quality control indicator of raw vegetable materials for fermentation.

Abstract:[Objective] Our study investigated the inhibitory effect and pathogenicity of Lactobacillus plantarum culture supernatant (LPC) on Salmonella cholerae (SC), Salmonella enteritidis (SC) and Salmonella pullorum (SP). [Methods] Three serotypes of Salmonella enterica were co-cultured with 2% LPC. Salmonella growth and antibacterial compounds were identified using turbidimetry and the Oxford cup antimicrobial zone. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of pathogenicity-related genes in Salmonella, and crystal violet staining was used to identify the biofilm of Salmonella. [Results] 2% LPC reduced the development of three Salmonella strains considerably, with an efficacy comparable to gentamicin (GM) and even better than SE. Additionally, the main antibacterial substance was organic acids. 2% LPC markedly suppressed the expressions of the main virulence genes encoded by SPI-1 (InvA, InvF, SopE, SopB, SipB, HilA and SipA), SPI-2 (SopD2), pili related genes (FliF, LpfA, SefA and FimF) and flagellum related genes (FlhD, FlIC and FliD) and the inhibitory effect was similar to GM. 2% LPC had significant inhibitory effect on biofilm formation of the three Salmonella strains, the inhibition rate was 40%–50% at 12 h and 60%–80% at 24 h. Compared with SC and SP, 2% LPC was superior to inhibit SE. [Conclusion] 2% LPC has significant inhibitory effects on the growth and pathogenicity of Salmonella serotypes, particlarly SE.

Abstract:[Objective] To reveal the microbiota structure and diversity in different parts of Herzensteinia microcephalus and predict the functions of the microbiota.[Methods] High-throughput sequencing of 16S rRNA gene was performed to reveal the microbiota structure. Tax4Fun was employed to predict the functions of the microbiota. [Results] The microbiota in skin mucosa had the highest alpha diversity, with the Shannon index higher than that of the microbiota in intestinal mucosa (P<0.05) and intestinal content (P<0.001). Moreover, the microbiota in skin mucosa can be significantly distinguished from that in intestinal mucosa and intestinal content by principal co-ordinates analysis. Actinobacteria, Proteobacteria, Firmicutes, Chloroflexi, and Cyanobacteria were the top 5 phyla accounting for more than 75% of the total microbiota. Among them, Actinobacteria in intestinal content had higher relative abundance (46.53%) than that in intestinal mucosa (29.23%, P<0.05) and skin mucosa (25.83%, P<0.01). The relative abundance of Proteobacteria in intestinal mucosa (40.33%) was higher than in intestinal content (26.10%, P<0.05). The top 10 families and genera in each part were further analyzed, which showed that skin mucosa and intestinal content had more different microorganisms, including 6 families (e.g., Microbacteriaceae, Burkholderiaceae, and JG30-KF-CM45) and 9 different genera (e.g., Cryobacterium, Carnobacterium, and Arthrobacter). The genera with higher abundance in skin mucosa were associated with organic material degradation and bacterial inhibition. The microbiota in the three parts showed different functional characteristics on pathway level 3 as predicted by Tax4Fun. Specifically, the microbiota in skin mucosa were mainly involved in ABC transporters and histidine metabolism pathway, that in intestinal mucosa in a two-component system and glycerophospholipid metabolism pathway, and that in intestinal content in fatty acid biosynthesis and terpenoid backbone biosynthesis pathway.[Conclusion] The microbiota structures in different parts of H. microcephalus were related to the environment. The skin mucosa had the highest microbial diversity among the three parts, and the intestinal mucosa and intestinal content had similar microbial diversity. In addition, the microorganisms related to the environment, those associated with the host’s physiological characteristics, immunity, and digestion were identified. Revealing the characteristics of microbial distribution in different parts will provide basic data and a scientific basis for the protection and the living environment improvement of fish in the plateau regions.

Abstract:[Objective] Gut microbiota plays an important role in the environmental adaptation of insects. This study aims to explore the diversity of gut microbiota in Locusta migratoria manilensis Meyen from different regions of China. [Methods] The Illumina NovaSeq platform was used to sequence the 16S rRna gene of gut microbiota in L. migratoria manilensis Meyen from four representative locust regions in China. [Results] Geographical location, development stage, and gender affected the structure of gut microbiota, among which geographical location had the most significant effect, followed by gender and development stage. The L. migratoria manilensis Meyen from Qingyuan City, Guangdong Province and Cangzhou City, Hebei Province showed the largest difference in the diversity of gut microbiota. Mean temperature and precipitation among the eight selected environmental factors may influence the gut microbiota diversity. [Conclusion] This study analyzed the environmental adaptability of L. migratoria manilensis Meyen from the perspective of symbiotic microorganisms and environmental factors, which provided a basis for the development of technology for tracing geographical origin and microbial preparations.

Abstract:[Objective] To explore the role of the virulence gene ompA in apoptosis of chicken trachea epithelium cells (CTECs) induced by outer membrane vesicles (OMV) of avian pathogenic Escherichia coli (APEC), so as to lay a foundation for further research on the pathogenesis of APEC-OMV. [Methods] On the basis of the wild-type APEC strain AE17, the ompA-deleted strain was constructed via the CRISPR/Cas9 system and the ompA-overexpressing strain via the expression plasmid pET-28a. OMV was respectively extracted from the wild-type strain, the ompA-deleted strain, the empty plasmid-transformed strain, and the ompA-overexpressing strain. The role of ompA in the apoptosis of CTECs induced by APEC-OMV was explored by transmission electron microscopy, nanoparticle tracking analysis, annexin V-FITC/PI double staining assay, and ultramicroscopic pathological sections. [Results] The ompA-deleted strain AE17△ompA, the empty plasmid-transformed strain AE17-pET-28a, and the ompA-overexpressing strain AE17-pET-28a-OmpA were successfully constructed. Compared with those of AE17-OMV, the particle density and size of OMV decreased in AE17△ompA-OMV (P<0.05) and increased in AE17-pET-28a-OmpA-OMV (P<0.05). Compared with the CTECs induced by AE17-OMV, those induced by AE17△ompA-OMV showcased reduced apoptosis rate and damage degree, with slightly swelling of only some mitochondria. However, the CTECs infected by AE17-pET-28a-OmpA-OMV presented increased apoptosis rate and damage degree as manifested by the matrix fading of some mitochondrion and disappearance or even transformation into vacuolated structures of mitochondrial cristae. [Conclusion] The virulence gene ompA positively regulated the particle density and size of APEC-OMV and promoted the apoptosis of CTECs induced by APEC-OMV.

Abstract:[Objective] A polycyclic aromatic hydrocarbon (PAH)-degrading strain was isolated from the soil contaminated by PAHs. This strain was mixed with Pseudomonas aeruginosa B6-2 to construct a mixed bacterial system for remediation of the soil or liquid co-contaminated by phenanthrene and cadmium. We investigated the remediation efficiency and the response characteristics of microbial metabolism to Cd at different concentrations, aiming to provide bacterial resources and technology reference for the bioremediation of combined pollution. [Methods] The PAH-degrading strain was screened via enrichment, domestication, isolation, and purification and then identified by physio-biochemical tests and 16S rRNA gene sequence analysis. High performance liquid chromatography and inductively coupled plasma mass spectrometry were employed to evaluate the removal efficiency of phenanthrene and cadmium at different cadmium concentrations. Furthermore, the effects of cadmium stress on phenanthrene biodegradation were analyzed on the basis of cell morphology observed by scanning electron microscopy and metabolic activity. [Results] A novel strain SZ-3 with heavy metal tolerance and high PAH-degrading ability was screened out and identified as Arthrobacter. The mixed system (M) of the two degrading bacteria had high phenanthrene-degrading efficiency and resistance to cadmium stress. The removal rates of M for phenanthrene and cadmium were higher than 85% and 80%, respectively, at the cadmium concentrations of 0.5 mg/L and 10 mg/L. When the cadmium concentrations were 25 mg/L and 50 mg/L, M showed the removal rates higher than 65% for the two pollutants. Cadmium stress increased the surface roughness, led to different degrees of cell deformation, and strengthened the adhesion and aggregation between cells. Both catechol 1,2-dioxygenase activity and electron transport system activity decreased with the increase in cadmium concentration during the reaction cycle, which was consistent with the variation of phenanthrene degradation rate. [Conclusion] Arthrobacter sp. SZ-3 is a strain with high PAH-degrading efficiency, which, together with P. aeruginosa B6-2, can efficiently remediate the soil co-contaminated by phenanthrene and cadmium. The two strains demonstrated significant synergistic effect on the removal of target pollutants.

Abstract:[Objective] Soil microorganisms are essential for the long-term sustainability of agricultural ecosystems. This study aims to explore the effects of continuous cropping of hot pepper on the structure and function of soil bacterial community. [Methods] We employed high-throughput sequencing of 16S rRNA and PICRUSt-based functional prediction to investigate the bacterial abundance, diversity, community composition, and potential functional component in the soils with continuous cropping of hot pepper for 1, 3, 5, and 10 years (1Y, 3Y, 5Y, and 10Y, respectively). [Results] The Shannon index and co-occurrence network complexity of soil bacteria decreased with the extension of continuous cropping years. The bacterial community structure was markedly affected by the years of continuous cropping, and different bacterial populations had varied responses to continuous cropping. Long-term continuous cropping increased the relative abundance of Proteobacteria and Bacteroidetes, while decreasing that of Chloroflexi, Acidobacteria, Firmicutes, and Patescibacteria. Furthermore, PICRUSt-based functional prediction showed that the continuous cropping changed the overall nitrogen and phosphorus metabolism of soil bacteria. After long-term continuous cropping, the functional genes in energy metabolism, amino acid metabolism, and carbohydrate metabolism showed reduced relative abundance, while those involved in folding, classification and degradation, replication and repair, membrane transport, and cell growth and death had increased relative abundance. The results of redundancy analysis indicated that soil organic matter and available phosphorus were the key factors associated with the migration and functional changes of soil bacteria. [Conclusion] After long-term continuous cropping of hot pepper, the change of community structure and the decline of diversity to the dysfunction of soil microbial community, which may be one of the reasons for the continuous cropping obstacles of hot pepper.

Abstract:[Objective] To characterize the Loigolactobacillus coryniformis Lc7 isolated from feces of healthy adults and evaluate its probiotic effect. [Methods] Phylogenetic tree was constructed based on the 16S rRNA gene and the core genes of the genome for taxonomic identification of the strain. The tolerance to acid, bile salt tolerance, adhesion, antioxidant activity, and antibacterial activity were tested, and the hemolysis, gelatinase activity, and antimicrobial susceptibility of Lc7 were examined to evaluate the probiotic property. In addition, ulcerative colitis was induced in mice to assess the in vivo anti-inflammatory potential of the strain. [Results] Lc7 was identified as L. coryniformis. The survival rate of this strain was up to 70.17% at pH 3.0 and in the presence of 0.3% bile salt. The adhesion index of Lc7 to HT-29 cells was 56.33 CFU/cell while self-aggregation and hydrophobicity were 80% and 40%, respectively. Lc7 inhibited the growth of seven common pathogens including Shigella flexneri and Salmonella typhimurium. Moreover, it scavenged 91.70% and 48.53% of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and hydroxyl radicals (·OH), respectively. Lc7 showed no hemolysis or gelatinase activity and was sensitive to the antibiotics tested. In the experiment on colitis mice, Lc7 group had longer colons than the model group (P<0.01). In addition, Lc7 alleviated colonic lesions (P<0.01), decreased serum pro-inflammatory cytokines TNF-α and IL-1β, and increased serum anti-inflammatory cytokines IL-10 (P<0.01). [Conclusion] Lc7 has probiotic and anti-inflammatory properties, which can be further developed as a probiotic.

Abstract:[Objective] We explored the regulatory effects of heat shock protein 27 (HSP27) on various signaling pathways during the proliferation of encephalomyocarditis virus (EMCV) in vitro, aiming to lay a foundation for deciphering the mechanism of other host factors in regulating EMCV proliferation in vitro and provide evidence for comprehensively revealing the pathogenic mechanism of EMCV. [Methods] Real-time quantitative PCR (RT-qPCR), Western blotting, indirect immunofluorescence, and nucleocytoplasmic separation were employed to evaluate the effects of the knockdown and overexpression of HSP27 on the EMCV infection-induced autophagy, eukaryotic translation initiation factor 2α subunit 1 (EIF2S1)-activating transcription factor 4 (ATF4) signaling pathway, and myeloid differentiation factor 88 (MyD88)/nuclear transcription factor kappa B (NF-κB) signaling pathway.[Results] Interfering with the expression of HSP27 in host cells promoted autophagy and activated EIF2S1-ATF4 signaling pathway, and inhibited EMCV-induced activation of MyD88/NF-κB signaling pathway. On the contrary, overexpression of HSP27 not only reduced EMCV-induced autophagy and inhibited EIF2S1-ATF4 signaling pathway, but also up-regulated the expression of the proteins in MyD88/NF-κB signaling pathway and promoted the nuclear translocation of p65 and the transcription of inflammation-related cytokines.[Conclusion] HSP27 can not only negatively regulate EMCV-induced autophagy by inhibiting the EIF2S1-ATF4 signaling pathway, but also positively regulate the EMCV-triggered MyD88/NF-κB signaling pathway, playing multiple regulatory roles in EMCV infection.

Abstract:Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only licensed vaccine for prevention of tuberculosis, whereas the genomic changes lead to differences in the protective effect and safety among strains.[Objective] To study the correlation between sigH-rshA overexpression and pathogenicity of BCG strains in DU2 groups Ⅲ and Ⅳ.[Methods] The recombinant BCG China and BCG Japan strains overexpressing sigH-rshA were established by molecular cloning, and the gene expression was verified by RT-PCR and Western blotting. We used macrophages of mouse origin and server combined immune-deficiency (SCID) mouse infection model to assess the pathogenicity of the recombinant BCG. We employed enzyme-linked immunosorbent assay (ELISA) to assess the secretion of tumor necrosis factor-α (TNF-α).[Results] The overexpression of SigH-RshA protein significantly promoted the intracellular proliferation of BCG and stimulated macrophages to produce more TNF-α. The results of the experiment with SCID mice showed that overexpression of sigH-rshA increased the virulence of recombinant BCG strains to immunodeficient mice.[Conclusion] The overexpression of sigH-rshA can enhance the pathogenicity of BCG strains to macrophages and mice.

Abstract:The intestinal microorganisms in Hexagrammos otakii at different growth stages are analyzed. [Objective] We investigated the changes of intestinal microorganisms in H. otakii and the relationship with the nutrition via metagenomics. [Methods] HiSeq sequencing of the intestinal microbial samples from larvae, juveniles, and young fishes was carried out to analyze the changes in community composition, compare the diversity of microbial community, and explore the interactions between microorganisms and functional relationship in the growth. [Results] The abundance of the dominant Proteobacteria in the intestine of fish decreased, while that of the dominant Firmcutes increased. The dominant genus was Vibrio at larval stage (37.8%), Photobacterium at juvenile stage (77.8%), and Lactococcus at young fish stage (42.5%), suggesting the significant variation of community composition in the growth period. Moreover, the microbial diversity was high at the larval stage and the richness was high at the juvenile stage. Species difference was correlated with the growth. Specifically, at the young fish stage, the differential taxa were Firmcutes, Bacilli, Streptococcaceae, Lactobacillales, Lactococcus and Lactococcus lactis. As for the juvenile stage, the biomarkers were Photobacterium and Photobacterium toroni. At the larval stage, the biomarkers were Phaeobacter inhibens, Colwellia aestuarii, C. polaris, etc. The microbes were mainly involved in the Kyoto encyclopedia of genes and genomes (KEGG) pathway of metabolism and the abundance of this function rose with the growth. To be specific, the intestinal microbiota played an important role in carbohydrate metabolism, amino acid metabolism, nucleoside acid metabolism, energy metabolism, co-factor and vitamin metabolism. Similar results were also presented in terms of protein annotation, which showed that carbohydrate metabolism (3 350) and amino acid metabolism (2 424) dominated the metabolic pathways. The analysis of functional differences demonstrated that microbial functions gradually adapted to the needs of the fish body and environment during the growth. The differential functions were energy metabolism and glucose degradation at the juvenile stage, and the cell growth, death, and apoptosis at the larval stage (mainly in photosynthesis). As for the young fish stage, the major differential function was the biosynthesis of secondary metabolites, followed by carbohydrate metabolism such as glycolysis and gluconeogenesis. [Conclusion] The intestinal microbial structure of H. otakii was significantly different among the larvae, juveniles and young fishes. The functions of the intestinal microbiota changed with the growth and development of the fish. The nutrients for the growth of the fish can be determined based on the differential species and differential functions. This study is expected to improve the breeding efficiency of this fish and lay a theoretical basis for ecological breeding.

Abstract:[Objective] To screen out the lactic acid bacteria that can inhibit the biofilm formation of Listeria monocytogenes (LM), identify the active component of the bacteria, and analyze the function of the active component. [Methods] We employed crystal violet staining to screen out the lactic acid bacteria of which the extract could inhibit the biofilm formation of L. monocytogenes. We analyzed the potential active component that inhibited biofilm formation by acid neutralization, protease treatment, and heat treatment and hypothesized that the main active component was extracellular crude polysaccharide (ECP). We then employed ethanol precipitation to extract the ECP from the lactic acid bacteria and analyzed the inhibitory activity of ECP on the biofilm formation and growth of L. monocytogenes. The morphology and structure of the biofilm treated with ECP were characterized by laser confocal scanning microscopy and scanning electron microscopy. [Results] The inhibition rate of Limosilactobacillus fermentum CSC-19 fermentation supernatant on the biofilm formation of L. monocytogenes 1516-2LM was 81.7%. After heat treatment and protease treatment, the activity of the fermentation supernatant did not significantly change (P>0.05), which indicated that the active component in the fermentation supernatant may be ECP. ECP inhibited the biofilm formation of L. monocytogenes in a concentration-dependent manner, while it did not inhibit the growth of L. monocytogenes. The results of confocal laser scanning microscopy and scanning electron microscopy showed that ECP destroyed the three-dimensional and honeycomb-structured biofilm and left only a few scattered adherent cells on the surface of the glass slide. [Conclusion] The ECP of L. fermentum CSC-19 inhibits the biofilm formation of L. monocytogenes, which is expected to be applied to the prevention and control of food contamination.

Abstract:Polyglutamic acid, as a natural multifunctional polymer, has become a research hotspot in recent years. Microbial fermentation is currently an effective way to produce polyglutamic acid which is difficult to be synthesized by chemical methods. [Objective] To explore the roles of degS, degQ, degU, swrA, rocA, and putM genes in the polyglutamic acid synthesis of Bacillus subtilis and realize the regulation of the synthesis pathway through molecular modification. [Methods] The genetically engineered B. subtilis strains were constructed by knocking out degS, degQ, and degU or overexpressing swrA, rocA, and putM, respectively. The key nodes in the synthesis pathway were analyzed based on the content change of extracellular polyglutamic acid secreted by the engineered strains. [Results] In shake flask culture, the extracellular polyglutamic acid contents of the recombinant strains B. subtilis 168-swrA, 168-rocA, and 168-putM were 1.28, 1.47, and 1.37 times that of the original strain, respectively; the extracellular polyglutamic acid contents of B. subtilis 168-△degS, 168-△degQ, and 168-△degU were 1.01, 0.98 and 0.94 times that of the original strain, respectively. In static culture, B. subtilis 168-△degU could not form an intact biofilm, and the biofilm formation of B. subtilis 168-△degS, 168-△degQ, 168-swrA, 168-rocA, and 168-putM was 1.48, 1.31, 1.77, 2.59, and 2.16 times that of the original strain, respectively. The extracellular protein content was positively correlated with the biofilm formation. [Conclusion] The deletion of degS, degQ, and degU did not significantly affect the synthesis of polyglutamic acid, while the overexpression of swrA, rocA, and putM significantly improved the ability of B. subtilis to synthesize polyglutamic acid.

Abstract:[Objective] Streptococcus thermophilus IMAU20246 is a strain with good fermentation performance and high production of exopolysaccharides (EPS), while its gene cluster and pathway for EPS synthesis remain unclear. Therefore, whole-genome sequencing and bioinformatics tools can be employed to identify the gene cluster and decipher the mechanism of EPS synthesis. [Methods] The whole genome of S. thermophilus IMAU20246 was sequenced and bioinformatic analysis was performed to analyze the EPS biosynthesis-related gene clusters and pathway. Further, quantitative real-time PCR (qRT-PCR) was carried out to determine the expression levels of the EPS gene cluster at different time points. [Results] An EPS gene cluster of 18.1 kb was identified in the genome of S. thermophilus IMAU20246, including 15 genes. The strain synthesized 7 sugar nucleotides including UDP-glucose, dTDP-glucose, dTDP-rhamnose, UDP-galactose, UDP-galactofuranose, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine by transporting glucose, mannose, fructose and galactose, lactose, trehalose, cellobiose, and sucrose, respectively. The results of qRT-PCR showed that the genes in the EPS gene cluster were expressed during the cell growth. In particular, the glycosyltransferase genes epsE, epsF, epsH, and epsJ reached the highest expression levels at the time point of 6 h, when the EPS yield peaked. [Conclusion] In this study, the gene cluster and pathway for EPS synthesis in S. thermophilus IMAU20246 were analyzed, which will provide a theoretical basis for further utilization of the strain.

Abstract:[Objective] To study the secondary metabolites of the Aspergillus jensenii LW128 isolated from the deep-sea sediment of northwest Pacific and the antibacterial activity.[Methods] Based on silica gel column chromatography, Sephadex LH-20 column chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC), secondary metabolites were isolated from the crude extract of the fermentation broth of strain LW128 and purified. Their structures were elucidated on the basis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data, and by comparison with the data of literature. The antibacterial activity of secondary metabolites was evaluated by broth microdilution. [Results] Ten known compounds, diorcinol D (1), diorcinol K (2), diorcinol I (3), (+)-(7S)-7-O-methylsydonic acid (4), (+)-sydonic acid (5), pseudaboydin B (6), anthraquinone aversin (7), 6,8-di-O-methylnidurufin (8), 5-methoxysterigmatocystin (9), and sterigmatocystin (10), were isolated and identified. Compounds 1−3 showed inhibitory effect on the growth of several pathogens. [Conclusion] Aspergillus jensenii LW128 can be a potential resource for antibacterial drugs.

Abstract:[Objective] Metabolites from members of Lentzea (Actinobacteria) have a wide range of bioactivities, thus showing potential application value in biopharmaceutical field. This study aims to establish a method to identify members of Lentzea based on ribosomal protein biomarkers and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). [Methods] We searched public genome database to obtain the sequences of 15 ribosomal proteins from the type strains of Lentzea and calculated the theoretical molecular weights. The molecular weight comparison was performed to investigate the matching of 15 ribosomal proteins between the type strains of Lentzea and that between the type strains of Lentzea and its closely related genera (Actinosynnema and Saccharothrix). Identification criteria for Lentzea at the species and genus level were proposed based on the number of ribosomal protein matches. Subsequently, the identification criteria were tested with target and non-target organisms and improved. [Results] MALDI-TOF MS peaks from an unknown organism were compared with the weight list of 15 ribosomal protein biomarkers from each Lentzea type strain and the number of peaks that matched each strain’s biomarkers was obtained. Based on the maximum of matches and peak intensity mode, the organism can be identified at the genus or species level. [Conclusion] This study established a method for identifying Lentzea members based on 15 ribosomal protein biomarkers and MALDI-TOF MS, which can provide a reference for the rapid identification of other Actinobacteria groups.