Abstract:Phage therapy has become an important choice to control multi-drug-resistant bacteria. As viruses containing both proteins and nucleic acids, phages can induce the generation of specific neutralizing antibodies in the host. This article reviews the phage-specific neutralizing antibodies during phage therapy, particularly the law of neutralizing antibody generation, the influence of the antibodies on phages' antibacterial efficacy, and the countermeasures against the influence. In summary, phage particles do induce the generation of specific neutralizing antibodies in the body, and the level of antibodies is correlated with the usage, type, dosage, and structural proteins of phages, the immunity of the host, and infected sites, treatment duration, etc. The generation time and yield are different among different types of antibodies. They all can neutralize phages and reduce the antibacterial efficacy. Therefore, in phage therapy, measures for controlling the neutralizing antibodies or strategies for different statuses/types of infection should be taken to reduce the generation of neutralizing antibodies and achieve the best therapeutic efficacy.

Abstract:CRISPR/Cas system is distributed in most bacteria, with immune defense mechanisms and showing polymorphisms among different species. Campylobacter jejuni is an important foodborne pathogen worldwide, and the disease caused by infection with it is also a typical self-limiting disease. Its pathogenic mechanism is complex, which has not been clearly clarified, and together with the polymorphisms of CRISPR/Cas system in C.jejuni, there exist many limitations in analyzing the relationship between CRISPR/Cas system and C.jejuni. In this paper, the structure, mechanism and technical application of CRISPR/Cas system in C.jejuni were reviewed, which provided new ideas for exploring the pathogenic mechanism of C.jejuni.

Abstract:Thymine glycol (Tg) is one of the common oxidative DNA damage bases. It can stall DNA polymerase and RNA polymerase that perform DNA replication and transcription, thus leading to the termination of the corresponding biological processes and further causing cell death. Therefore, Tg in DNA needs to be repaired. Endonuclease Ⅲ (EndoⅢ) is a bifunctional DNA glycosylase capable of excising Tg from DNA, thus initiating a base excision repair pathway for restoring Tg to a normal T base. The genomes of bacteria, archaea, and eukaryotes possess the gene encoding EndoⅢ. The available studies mainly focus on the EndoⅢ in bacteria and eukaryotes while rarely concern archaeal EndoⅢ. We reviewed the research progress on the EndoⅢ in hyperthermophilic archaea and proposed the future research directions in this field.

Abstract:Breast milk is the ideal food for infants. As the third most abundant solid ingredients in breast milk, human milk oligosaccharides (HMOs) have an important impact on the growth, development and health of infants. A few HMOs have been used in infant formulas. Among them, 2'-fucosyllactose (2'-FL) has the highest content and comprises 30% of the total HMOs in secretor milk. With high nutritional and medical values, 2'-FL is regarded as a new type of food nutrition fortifier, and it boasts multiple functions such as growth promotion, cognition benefits, immune enhancement, anti-allergic activity, anti-viral activity, and intestinal flora regulation. However, originating from breast milk, 2'-FL is unlikely to be obtained by separation. Thus, it is urgent to synthesize 2'-FL in artificial manners, which include chemical and enzymatic synthesis and whole-cell biosynthesis. Cost-effective and easy to scale up, whole-cell biosynthesis of 2'-FL has received worldwide attention. At present many foreign multinational companies have begun the industrial production and commercial application of 2'-FL, whereas the relevant work in China remains in research. Therefore, it is of great significance for China to master the synthesis method of 2'-FL. This paper aimed to review the functional characteristics of 2'-FL and systematically describe the key technologies and the latest progress in whole-cell biosynthesis. The strategies of rate-limiting steps to increase production were also discussed. We sought to provide a biological basis for the synthesis and commercial production of 2'-FL. Further, the paper facilitated the development of infant formulas close to human milk.

Abstract:Bacteriocins are a class of ribosomally synthesized antimicrobial peptides produced by bacteria, which endow bacteriocinogenic strains with unique survival advantages. Different from linear bacteriocins, circular bacteriocins have unique N-to-C terminal covalent linkage, and thus they have strong heat tolerance, adaptability to a wide range of pH, as well as certain resistance to protease. Therefore, they show great potential in food antisepsis and antagonization of resistance bacteria. Circular bacteriocins show higher similarity in tertiary structure than in primary structure, laying a basis for the classification. The biosynthesis mechanism of circular bacteriocins is still unclear, whereas the cyclization mechanism is attracting the interest of scholars, given that it can provide scaffolds for the synthesis and the modification of other types of peptide. The antibacterial mechanism of circular bacteriocins is mainly associated with perforation on the cell membrane and the consequential outflow of intracellular substances. With the antibacterial activity similar to or different from antibiotics, they can be potential candidates against the resistance pathogens. In this paper, research on circular bacteriocins was summarized, and the structure-activity relationship, biosynthesis pathway, the mode of action, as well as the application potential of them were highlighted, respectively.

Abstract:[Objective] Anthracnose, a major disease of tea-oil tree (Camellia oleifera), is mainly caused by Colletotrichum fructicola. In this study, we investigated the biological function of the small-molecule GTPase Rab7 of C. fructicola, aiming to provide a theoretical basis for the prevention and control of anthracnose. [Methods] The CfRAB7 gene knockout vector was constructed based on the principle of homologous recombination. After PEG-mediated protoplast transformation, resistance screening, and verification by PCR and electrophoresis, the mutant strain ∆Cfrab7 and the complementary strain ∆Cfrab7/CfRAB7 were obtained. The growth, sporulation, appressorium formation, stress response, and other biological characteristics of ∆Cfrab7 were explored. [Results] On the PDA and MM plates, ∆Cfrab7 showcased significantly decreased colony diameter, spore production, and appressorium formation. ∆Cfrab7 failed to penetrate cellophane. The oxidative stress (H₂O₂) had higher inhibition rate on the growth of ∆Cfrab7 than on that of WT and ∆Cfrab7/CfRAB7. ∆Cfrab7 did not cause disease spot on the leaves of Ca.oleifera. Furthermore, CfRab7 was required for homotypic vacuole fusion, which was essential for pathogen invasion. [Conclusion] Our findings reveal that CfRAB7 gene plays a vital role in the growth, sporulation, appressorium formation, oxidative stress response, homotypic vacuole fusion, and pathogenicity of C. fructicola.

Abstract:[Objective] To investigate the mechanism of honokiol at different concentration in inhibiting Escherichia coli 10389 biofilm (BF) formation. [Methods] We employed triphenyltetrazolium chloride (TTC) method to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of honokiol for the test strain and tetrazolium salt (XTT) reduction assay to investigate the influence of honokiol concentration on BF formation of the test strain and the influence over time. Through qRT-PCR, we examined the effect of honokiol concentration on the expression of genes related to BF formation and quorum sensing of the test strain. We detected the effect of honokiol at sub-MIC on the expression of furanosyl borate diester (AI-2) in E.coli 10389 and its regulated downstream genes associated with BF formation by bioluminescence and qRT-PCR. [Results] Honokiol inhibited the BF formation of E.coli 10389, but the mechanism was different for different concentration of honokiol. Among them, honokiol at MIC significantly increased the mRNA expression of toxin gene hha and bacterial acid regulator ariR involved in the BF formation of E.coli 10389 and significantly decreased the mRNA expression of toxin overexpression-modulating gene ybaJ compared with the control group. Honokiol at sub-MIC can suppress AI-2 secretion by E.coli 10389 and decrease the mRNA expression of its regulated downstream genes related to BF formation. Compared with the control, 16 mg/mL honokiol reduced the mRNA expression of colanic acid gene mqsR, mucoid gene mcbR, and flagellum formation-related genes csrA, flhD, flhC, and flic by 65.21%, 55.01%, 73.16%, 62.01%, 60.30%, and 59.71%, respectively. [Conclusion] Honokiol suppresses the BF formation of E.coli 10389, but the mechanism is different for honokiol of different concentration. Among them, honokiol at MIC mainly inhibits BF formation by affecting the expression of related genes, and honokiol at sub-MIC by suppressing the expression of the AI-2 synthase luxs gene of Luxs/AI-2 system, decreasing AI-2 secretion, and further influencing the synthesis of capsular polysaccharide, mucoid, and flagellin.

Abstract:As the first metabolic enzyme in the histidine metabolic pathway, histidine aminonia-lyase (HutH) controls the metabolism of histidine in bacteria. HutH is highly conserved in most bacteria and participates in the balance of bacterial energy metabolism.[Objective] To investigate the effects of HutH on the biological characteristics and pathogenicity of Vibrio parahaemolyticus. [Methods] The hutH gene-deleted mutant ΔhutH and the complementary strain CΔhutH were constructed by homologous recombination. The effects of HutH on the growth characteristics, histidine utilization ability, histidine metabolism related gene expression level, motility, biofilm, environmental tolerance, cytotoxicity and mouse toxicity of V.parahaemolyticus were researched. [Results] Compared with wild strains, the deletion of hutH gene did not affect the growth characteristics, acid and alkali tolerance, salt tolerance and swarming ability of V.parahaemolyticus. But the growth of ΔhutH was significantly inhibited in M9-limited medium with histidine as the only carbon source. In addition, we confirmed that the deletion of hutH significantly decreased the transcription level of histidine metabolism related genes in hut operon and increased the expression level of the VP0889 gene. The deletion of hutH resulted in the decrease in biofilm formation ability, swimming ability, toxicity to HeLa cells and mortality to institution of cancer research female mice. [Conclusion] This study shows that the deletion of hutH affects the ability of V.parahaemolyticus to metabolize histidine, and it is the first time to confirm that HutH plays an important role in the biofilm formation, motility and toxicity toward mice of V.parahaemolyticus, which provides ideas for bacterial prevention and control by regulating histidine metabolism.

Abstract:[Objective] Listeria monocytogenes, the important foodborne pathogen, uses opuCA gene to defend against osmotic stress. In this study, we explored the roles of opuCA gene in growth, pathopoiesis, oxidative stress resistance, and osmotic stress resistance of L.monocytogenes, hoping to lay a basis for clarifying the mechanism of OpuCA-mediated bacterial adaptation and infection. [Methods] We constructed the opuCA deletion mutant (ΔopuCA) and complementary mutant (CΔopuCA) and then compared the growth, oxidative stress tolerance, osmotic stress tolerance, and cellular adhesion and invasion of the wild strain and the mutants by molecular biology technology, infection biology technology, and laser scanning confocal microscopy. [Results] opuCA deletion had no influence on bacterial growth but decreased the osmotic stress tolerance. Additionally, ΔopuCA showed low tolerance to Cu²⁺and Cd²⁺compared with the wild strain, but the tolerance to thiol-specific oxidant diamide demonstrated no obvious variation. Furthermore, loss of opuCA significantly reduced bacterial adhesion and invasion to Caco-2 cells, and actin aggregation, thereby influencing the intercellular migration of bacteria. [Conclusion] opuCA plays a critical role in bacterial osmotic stress tolerance, oxidative stress resistance, and intracellular infection. This study expands our understanding of the OpuCA-mediated in vitro adaption and pathopoiesis of L.monocytogenes, which lays a basis for the prevention and control of the foodborne diseases caused by L.monocytogenes.

Abstract:[Objective] β-glucosidase, also known as β-D-glucosidase hydrolase, is a key rate-limiting enzyme for cellulose degradation, which is a cellulase. The β-glucosidases from thermophilic archaea have been verified to have tolerance to acid and high temperature, which have become one of the research hotspots of thermostable enzymes. We studied the prokaryotic expression and enzymatic properties of a GH3 family glucosidase from Thermofilum adornatum, which has not been reported, aiming to mine superior β-glucosidase. [Methods] We obtained the GH3 amino acid sequence of T.adornatum from NCBI database and constructed the recombinant plasmid pET-30a(+)-TaBgl3. The recombinant protein TaBgl3 was expressed in Escherichia coli BL21(DE3) competent cells under induction. The properties of the enzyme were studied after purification with magnetic beads. [Results] TaBgl3 had a molecular weight of 77.0 kDa and the best performance at pH 5.0 and 80℃. The treatment at 70℃ for 1-4 h improved the enzyme activity, and that at the optimum temperature of 80℃ for 2 h showed the most significant effect, which increased the enzyme activity by more than 40%. The enzyme still had the relative activity of above 60% after being treated 37℃ and pH 5.0-8.0 for 1 h. When the substrate was 4-p-nitrophenyl-β-D-glucopyranoside (pNPG), the enzyme had the specific activity of 144.23 U/mg, the K_m value of 1.81 mmol/L, the maximum reaction rate of 268.10 μmol/(mg·min), and the catalytic efficiency of 115.47/s. Cu²⁺, Li⁺ and EDTA at the final concentration of 5 mmol/L all increased the enzyme activity, which had the maximum increase of 39%. Fe³⁺ (5 mmol/L) and 5% β-mercaptoethanol had inhibitory effect on the enzyme activity. In addition, SDS, ethanol and glucose greatly inhibited the enzyme activity. [Conclusion] TaBgl3 is an acidic thermostable enzyme with good thermal stability and thermal activation characteristics, which can shed light on the future theoretical research and industrial production.

Abstract:[Objective] The Pamir Plateau is characterized by a cold and oligotrophic environment exposed to strong radiation, in which cold-adapted microorganisms are abundant. Using a cultivation-dependent approach, we aimed to reveal the diversity of cold-adapted bacteria and archaea present in the Pamir Plateau. [Methods] Soil samples were collected from a gradient of altitudes in the Pamir Plateau, from which aerobic bacteria and archaea were isolated by different cultivation methods. For the isolation of cold-adapted bacteria, two bacterial culture media were used and the incubation was only conducted at 4℃. For cultivation of archaeal ones, two archaeal culture media were employed, and cultivation was at two temperatures, i.e., 4℃ and 15℃. Sampling was conducted at four altitudes:1 000-2 000 m, 2 000-3 000 m, 3 000-4 000 m and 4 000-5 000 m. All microbial isolates were classified based on their 16S rRNA gene sequences. [Results] A total of 419 strains of aerobic prokaryotes were isolated from the soil samples, which belonged to 118 different species, including 115 bacterial species and 3 archaeal species. These microorganisms fell into 49 genera, 28 families, 18 orders, 8 classes, 5 phyla, and 2 domains. Gammaproteobacteria was the predominant order of cold-adapted bacteria and Actinomycetes constituted the most diverse one. Gammaproteobacteria dominated at altitude of 1 000-4 000 m and Actinobacteria prevailed at altitude of 4 000-5 000 m (the highest altitude). The archaeal isolates belonged to haloarchaea of Natrinema and Haloterrigena, and they were obtained from the samples at altitude of 1 000-2 000 m and at the cultivation temperature of 15℃. Furthermore, among the four culture media used in this work, Gammaproteobacteria isolates were mainly obtained with the two bacterial media whereas the haloarchaea only grew on the archaeal media. Additionally, 23 strains represented new taxa among all isolates, and they belonged to Actinobacteria, Proteobacteria, Bacteroidetes, Firmicutes and Euryarchaeota.[Conclusion] A number of cold-adapted bacteria and three archaeal species were isolated as pure cultures from the Pamir Plateau soil samples. These microorganisms exhibited an altitude-dependent distribution. Furthermore, we found that the isolation experiment was benefitted from utilization of different culture conditions, which was a strategy to be exploited in future work. Together, the present study has yielded the first insights into the composition and diversity of cold-adapted bacteria and archaea in the Pamir Plateau. The obtained microorganisms provide unique resources for exploration of their ecological functions and biotechnological applications.

Abstract:[Objective] Microorganisms with potential growth-promoting effects on plants were isolated from the rhizosphere of Nitraria tangutorum Bobr. in Lanzhou City, which laid a foundation for research and development of related preparations.[Methods] With the plate streak method, 6 bacterial strains were isolated and purified from the rhizosphere of N.tangutorum, and the morphological characteristics of the strains were observed. The physiological and biochemical properties were examined based on Gram staining and other tests. The effect of each strain on quinoa growth was determined. The 16S rRNA gene of a strain with excellent growth-promoting effect was sequenced and the circular genome map for the strain was plotted.[Results] According to the biochemical identification results, the 6 strains belonged to Acinetobacter, Agrobacterium, Paenibacillus, Bacillus, Sphingomonas, and Pseudomonas, respectively. Further identification by 16S rRNA gene sequencing suggested that BC4, with strong growth-promoting activity, belonged to Enterobacter.[Conclusion] BC4 shows strong growth-promoting activity, which lays a theoretical basis for development and utilization of bacterial resources of N.tangutorum in Lanzhou.

Abstract:Marine epiphytic actinomycetes are potential sources of natural bioactive products with unique structures. [Objective] To isolate and identify the secondary metabolites of Streptomyces ardesiacus SCSIO LO23 which was derived from marine Onchidium sp. (Mollusca), and analyze their biosynthetic gene clusters and biosynthetic pathways. [Methods] A variety of media were used to optimize the fermentation of the strain. Further large-scale fermentation and extraction were performed to yield the germicidins. The structures of the germicidins were characterized by spectroscopy and single-crystal X-Ray diffraction. The whole genome of the strain was sequenced by Illumina HiSeq. antiSMASH and BLAST were combined for gene function annotation and biosynthetic pathway analysis of germicidins in the strain. The biosynthetic gene was further verified by heterologous expression. [Results] Six germicidins were isolated and identified from the fermentation products of AM3 medium, which were germicidin A (1), germicidin B (2), germicidin D (3), germicidin H (4), isogermicidin A (5), and isogermicidin B (6). Compounds 2 and 6 showed excitatory effect on zebrafish neurobehavior at the concentration of 30 μmol/L. The biosynthetic gene was verified by bioinformatic analysis and heterologous expression, and the biosynthetic pathways of compounds 1-6 were deduced. [Conclusion] The structures, bioactivity, and the biosynthetic genes of germicidins from S.ardesiacus SCSIO LO23 are clarified, which lays a solid foundation for the study of this type of compounds.

Abstract:[Objective] To analyze the distribution, genomic characteristics, and genetic evolution of the prophages found in Clostridium perfringens. [Methods] Phage search tool enhanced release (PHASTER) was used to predict the prophages carried by C.perfringens. The prophages were classified into groups based on ANI value. Comprehensive antibiotic research database (CARD), ResFinder 4.1, virulence factors database (VFDB), and BacMet (antibacterial biocide & metal resistance genes database) were first employed to predict various genes related to antibiotic resistance, virulence, antibacterial biocide and metal resistance that can be encoded by the prophages, respectively. CRISPRCasFinder was then applied to predict the CRISPR-Cas system in C.perfringens, and MEGA 7.0 to analyze the genetic evolution of the prophages. [Results] Each C.perfringens genome was found to carry an average of 2.67 prophages and the size of prophage showed a bimodal distribution, accounting for 2.23% of C.perfringens genome on average. The prophages were not found to carry antibiotic resistance genes, but possessed genes of virulence factors such as alpha toxin, sialidase, and hemolysin, and genes related to the metal ion metabolism. The prophages clustered into 3 groups, and most of the prophages in Group 1 were intact and only existed in C.perfringens type A. C.perfringens with a complete CRISPR-Cas system carried a few prophages, but the number of spacers in CRISPR-Cas system was in negative correlation with the number of the prophages. The prophages showed far genetic distance from C.perfringens phages, and only structural proteins and some enzyme genes have been found to display high homology. [Conclusion] C.perfringens carries prophages, but its CRISPR-Cas system has little influence on the number of prophages. The virulence genes and metal resistance genes carried by the prophages can effectively enhance both the pathogenicity and adaptability of C.perfringens. However, their potential functions and impact on the genetic evolution of C.perfringens need to be further analyzed.

Abstract:Potato common scab (CS) caused by pathogenic Streptomycetes has occurred widely in China and led to increasing damage to the quality and commercial value of potato. Since the pathogen is soil-borne and seed-borne, antagonistic microorganisms are regarded as an important method for the prevention and control of this disease. [Objective] We aim to screen out the antagonistic bacterial strains from the soil in the field with serious CS, reveal the mechanism of antagonism against Streptomyces scabies, and evaluate the environmental adaptability of the targeted strains. This study can provide a theoretical basis for developing applicable microbial agents. [Methods] The strains with antagonistic effects were screened out via the plate confrontation method and pot experiment and then identified based on morphological, physiological, biochemical, and molecular characteristics. Their metabolites with antifungal functions were detected via high performance liquid chromatography coupled with mass spectrometry.[Results] Three Gram-positive strains with antagonistic effects were identified as Bacillus amyloliquefaciens and designated HZ11-4, HS-12, and HZ13-1, which showed the inhibition zone diameters of 34, 29 and 30 mm to S.scabies and the control effects of 68.57%, 57.15%, and 65.96%, respectively. The genes coding for synthetases of surfactin, iturin, and fengycin were amplified from these strains, and existence of the lipopeptide antibiotics was detected by high performance liquid chromatography. S.scabies could be inhibited only by surfactin which was not the main active compound of the strains. The three strains exerted inhibitory effects on a variety of pathogens such as Alternaria solani, Fusarium oxysporum, Rhizoctonia solani, and Verticillium dahliae. They could live in a wide pH range of 5-9, tolerate 1%-7% NaCl and high temperature of 100℃, promote potato growth, and were insensitive to fluopicolide + propamocarb, flusilazole, pyraclostrobin, kasugamycin, zhongshengmycin and thiophanate-methyl. [Conclusion] Owing to the good environmental adaptability and broad-spectrum resistance, B.amyloliquefaciens HZ11-4, HS-12, and HZ13-1 can be used to develop compound microbial agents against potato soil-borne diseases. This study verifies for the first time that surfactin, iturin A, and fengycin are not the main active substances to inhibit S.scabies.

Abstract:Objective] Pichia pastoris (syn. Komagataella phaffii) has been extensively used as a versatile cell factory for the production of industrial enzymes and chemicals. However, well-tuned co-expression of multiple genes is a common challenge for P.pastoris in metabolic engineering and synthetic biology. Therefore, in this work, we constructed a set of terminators and paired them with varied promoters to tune the protein levels in P.pastoris.[Methods] We constructed the P.pastoris strains expressing reporter genes (egfp and lacZ) under the control of truncated constitutive 3-phosphoglycerate kinase (PGK1) promoters, and then measured the transcript levels of reporter genes, yEGFP fluorescence intensity and β-galactosidase activity of these strains. Next, we created a total of 27 promoter-terminator pairs to regulate the transcription of egfp, and used 6 promoter- terminator pairs to alter the secretory expression of β-fructofuranosidase (β-Ffase). [Results] The promoter activities of the truncated P_PGK1 variants (P_PP, P_PE, P_PG and P_PD) relative to that of the native P_PGK1 ranged from 70% to 190%. Furthermore, when paired with the weak promoter P_PG, moderate promoter P_PE, and strong promoter P_PD, the terminators had the tuning ranges of 4, 7 and 10 folds (comparing between the strongest and weakest terminator), respectively. Finally, we demonstrated the utility of the promoter-terminator pairs for tuning the expression of the industrial enzyme β-Ffase, which showed an overall tuning range of 6 folds. [Conclusion] The promoter-terminator pairs constructed not only provide valuable information for understanding the modulatory roles of terminator regions in gene expression but also serve as a useful toolbox enabling the metabolic engineering of P.pastoris and the application of P.pastoris in synthetic biology.

Abstract:[Objective] To investigate secondary metabolites of the mangrove-derived fungus Aspergillus sp. WHUF0343 from the root soil of an unidentified mangrove plant sample collected in Yalong Bay, Sanya, China, and their activity. [Methods] The column chromatography on silica gel and Sephadex LH-20, and semi-preparative high-performance liquid chromatography (HPLC) were used to isolate and purify the secondary metabolites from the fermented extract of Aspergillus sp. WHUF0343. Their structures were elucidated by modern spectroscopy methods such as nuclear magnetic resonance (NMR) and mass spectrometry (MS) combined with relevant literature data. Their antibacterial and cytotoxic activities were evaluated by broth microdilution assay and MTS assay, respectively. [Results] Ten compounds were isolated, which were respectively identified as isoechinulin A (1), neoechinulin A (2), neoechinulin E (3), preechinulin (4), neoechinulin D (5), variecolorin J (6), dehydroechinulin (7), questinol (8), emodin (9) and catenarin (10). Compounds 2, 9 and 10 exhibited moderate inhibitory activities against Helicobacter pylori, Staphylococcus aureus and their drug-resistant strains. Compound 1 demonstrated cytotoxic activities against malignant melanoma cell line B16, human hepatocellular carcinoma cell line HepG2 and human breast cancer cell MCF-7. [Conclusion] Aspergillus sp. WHUF0343 has potential research value in microbial drugs.

Abstract:[Objective] Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) technique provides mass spectral fingerprints of characteristic proteins for microbial identification. This study aims to characterize the ribosomal protein biomarkers from Actinobacteria via genomics and MALDI-TOF MS techniques. [Methods] Actinobacteria representatives were chosen from MALDI-TOF MS spectral library. By searching against genome database, we acquired the ribosomal protein sequences from the target or reference strains of target species and calculated the theoretical molecular masses. The mass peaks in the MALDI-TOF mass spectra of target strains were annotated with the calculated molecular masses of ribosomal proteins. [Results] Mass peaks annotated in the spectra of 142 strains of 114 species, 53 genera, 24 families from 8 orders were assigned to 31 ribosomal proteins. The number of annotated ribosomal proteins varied significantly among strains. The number of mass peak annotations also varied considerably among different subunit proteins. A total of 15 ribosomal proteins were annotated in over half of the spectra, and the ribosomal protein with the most mass peak annotations was L36. [Conclusion] This study identified 15 common ribosomal protein mass peaks in MALDI-TOF mass spectra of Actinobacteria. The results can support the establishment of a method for the identification of Actinobacteria by matching characteristic mass peaks of ribosomal protein biomarkers in MALDI-TOF mass spectra.

Abstract:[Objective] The aim of this study is to screen an ideal adjuvant for an inactivated porcine deltacoronavirus (PDCoV) vaccine to induce mucosal immunity and reduce the side effect of the vaccine. We used different mucosal adjuvants to prepare the inactivated PDCoV vaccines. We then used mouse model to evaluate the humoral, cellular and mucosal immune responses induced by the inactivated vaccines via different immunization routes. [Methods] The adjuvants IMS1313 and GEL02 were respectively combined with polyactin A (PA), CpG ODN2395, and monophosphoryl lipid A (MPLA) to prepare the inactivated PDCoV vaccines, which were then used to immunize BALB/c mice intranasally. The inactivated PDCoV vaccine prepared with ISA201 adjuvant was used to immunize BALB/c mice subcutaneously. The inactivated PDCoV vaccine without adjuvant was used as a control to immunize BALB/c mice intranasally. The mice were immunized once again at the same doses in the same ways 14 days post the primary immunization. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the expression levels of IgG, IgG1, IgG2a, IL-4, and IFN-γ in serum and bronchial lavage fluid (BALF) samples, as well as the levels of sIgA in feces and BALF samples. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the proliferation of spleen lymphocytes. We observed and recorded the clinical manifestations of immunized mice. Meanwhile, we observed the pathological changes of major organs and tissues of immunized mice via hematoxylin-eosin (HE) staining to evaluate the safety of the vaccines. [Results] The ISA201 group had high expression levels of antibodies (IgG and IgG1) and IL-4 in BALF and serum and low expression levels of IgG2a, IFN-γ, and sIgA in feces. GEL02, GEL02+2395, GEL02+PA, and GEL02+MPLA groups showed higher expression levels of antibodies (IgG, IgG1, and IgG2a), IL-4, and IFN-γ in BALF and serum and higher expression level of sIgA in feces than IMS1313, IMS1313+2395, IMS1313+PA, and IMS1313+MPLA groups. In particularly, the expression levels of IgG2a in BALF and serum samples and IFN-γ and sIgA in BALF and feces samples of GEL02+2395 group were significantly higher than those of other groups. The vaccines prepared with the adjuvant combinations GEL02+2395 and IMS1313+2395 promoted T lymphocyte proliferation. No obvious pathological changes were observed in the main organs and tissues of mice. The mice immunized with the vaccines prepared with the adjuvants GEL02 and GEL02+2395 had the mildest adverse reactions. [Conclusion] The inactivated PDCoV vaccine prepared with GEL02 and CpG ODN2395 can not only enhance the humoral immunity but also improve the cellular immunity and mucosal immunity in mice, which provides basic information for the research and development of novel mucosal adjuvants for PDCoV vaccines.

Abstract:Tellurite as a strong antimicrobial agent is highly toxic to a variety of microorganisms, while its toxicity mechanism remains indistinct. [Objective] The main goal of this work is to uncover the global changes of cell metabolism under tellurite stress and reveal the toxicity mechanism of tellurite. [Methods] The transcriptomes of Escherichia coli MG1655 exposed to tellurite stress and under normal conditions were compared to reveal the differentially transcribed genes. [Results] After being treated with 10 µg/mL tellurite for 1 h, the cells exhibited an obvious adaptive response with many metabolic processes influenced. The transcription levels of the genes involved in ribosome metabolism and flagellar assembly changed significantly, implying the two pathways were affected by tellurite. The genes encoding the transcriptional factors and small RNAs and those functioning in the cell motility, metal ion metabolism, and membrane function also showed varied transcription levels, which might participate in the metabolism regulation and damage repair to resist the toxicity of tellurite. [Conclusion] This work can facilitate the study of the toxicity mechanism and promote the clinical application of tellurite.

Abstract:The sulfur cycle mediated by microbes in sediments plays an important role in the decomposition of organic matters and nutrient cycling. However, little is known about the microbial diversity and potential regulatory mechanisms involved in sulfate reduction and sulfur oxidation in aquaculture ecosystems. [Objective] To explore the vertical distribution pattern of sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) and their environmental driving factors. [Methods] In this study, the abundance, diversity and community composition of bacteria, SRB and SOB in surface (0-1 cm), middle (10-11 cm) and bottom (20-21 cm) sediments originated from a freshwater prawn (Macrobrachium rosenbergii) aquaculture pond were investigated by high-throughput sequencing and real-time PCR (qPCR). [Results] The gene copy numbers of bacteria (16S rRNA), SRB (dsrB) and SOB (soxB) showed a trend of dramatic decline from the surface to the middle layer (analysis of variance, P<0.05), but the difference between the middle and bottom layers was not significant (P>0.05). The α-diversity analysis showed that species richness and evenness of three microbial populations both gradually decreased with depth, implying that the microbial sulfur cycling processes mainly occur on the surface layer. The γ-, δ- and β-proteobacteria were the dominant taxa of bacteria, SRB and SOB, respectively. Specifically, SRB was dominated by Desulfobacca and Desulfosarcina; the former has the lowest proportion in the surface layer, while the latter was the opposite. Thiobacillus, as the major genus of bacteria and SOB, was more abundant in the middle layer. Redundancy analysis and Mantel test revealed that the key environmental factors driving the variation of bacterial communities were NO₃^-, SO₄^2-, total organic carbon (TOC) and total organic nitrogen (TON), while the SRB community variation was mainly affected by arsenic (As), TON, NO₃^- and lead (Pb), and SOB responded to the changes in total carbon (TC), NO₂^-, NH₄⁺ and TON. [Conclusion] The abundance, diversity and community structure of bacteria, SRB and SOB in aquaculture pond sediment exhibited distinct vertical distributions, which could be driven by multiple environmental factors.

Abstract:[Objective] To screen out the bacterial strains that can control the Fusarium root rot and promote the growth of Capsicum annuum L. and clarify their disease-controlling and growth-promoting effects. [Methods] The soil samples were collected from the rhizosphere of healthy C.annuum plants, and then plate confrontation method was employed to screen the biocontrol bacteria with Fusarium solani and F.oxysporum as the indicator fungi. After that, selective media were used to screen the growth-promoting bacteria with inorganic phosphorus-solubilizing, organic phosphorus-solubilizing, nitrogen-fixing, or potassium-solubilizing activities. Further, the disease-controlling and growth-promoting effects of the selected strains were determined qualitatively and quantitatively. The amount of phosphorus solubilized, nitrogen fixed, and potassium solubilized were respectively determined by molybdenum-antimony anti-colorimetric method, Kjeldahl method, and flame atomic absorption spectrometry. We then determined the 16S rDNA sequences of the strains with excellent characteristics and prepared the bacterial inoculants with different combination formula. Finally, pot experiments were carried out to measure the disease-controlling and growth-promoting effects of the inoculants. [Results] We screened out 323 strains with excellent functions, including 78 antagonistic strains, 87 organic phosphorus-solubilizing strains, 107 inorganic phosphorus-solubilizing strains, 128 nitrogen-fixing strains, and 123 potassium-solubilizing strains. Some strains had multiple functions, and 6 combinations with excellent characteristics were obtained, which involved 8 strains. Strains XP271 and XP181 were identified as Bacillus subtilis, XP125 as B.tequilensis, XP236 as B.halotolerans, XP79 as B.megaterium, XP171 as B.circulans, XP248 as Cellulosimicrobium funkei, and XP167 as Pseudomonas synxantha. The inoculants were prepared according to two combination formula, which demonstrated the best controlling effect of 88.52% on Fusarium root rot and increased the plant height, branch number, and biomass of C.annuum by about 10 cm, 2 branches, and 5-21 g. Moreover, after the inoculation, the content of available nitrogen, available phosphorus and available potassium in the rhizosphere soil, as well as the activities of soil enzymes such as urease, sucrase, and alkaline phosphatase increased, while the activity of soil catalase decreased. Additionally, the microbial biomass carbon, nitrogen-fixing genes, and nitrogen-fixing microorganisms in the soil increased significantly. [Conclusion] The rhizosphere soil of C.annuum harbors rich bacteria with excellent disease-controlling and growth-promoting effects, which can be prepared into inoculants for the control of Fusarium root rot and the growth-promoting on C.annuum.

Abstract:[Objective] This study aims to determine and characterize the pathogen of the diseased Trionyx sinensis in a farm in Xiantao, Hubei Province. [Methods] The suspicious pathogenic bacteria were isolated from the diseased T.sinensis and identified based on the morphological, physiological, and biochemical characteristics and the phylogenetic tree constructed with the 16S rRNA gene sequence. The artificial infection tests, drug sensitivity tests, multilocus sequence typing (MLST), eBURST analysis, and whole-genome sequencing were then carried out for the isolates. [Results] Three predominant strains, HX8, FG10, and GC20, were isolated from the diseased T.sinensis and all identified as Pseudomonas aeruginosa. Artificial infection tests confirmed that the isolates were the pathogen causing the disease of T.sinensis. Drug sensitivity tests demonstrated that the three isolates were sensitive to eight antibiotics (such as enrofloxacin and norfloxacin) and resistant to six antibiotics (such as florfenicol, doxycycline, and sulfagan). According to the results of MLST, the three isolates all belonged to sequence type 252 (ST252). The further eBURST analysis showed that ST252 formed a clonal complex CC252, of which ST252 was the founder ST. The whole genome of strain FG10 had a size of 5.65 Mb, the average G+C content of 65.3%, and 5 956 coding sequences. The whole genome has been deposited at GenBank under the accession number JAJGXC000000000. In comparison with the virulence factor database (VFDB), 873 virulence-related genes were predicted, which were mainly associated with adhesion, secretion systems, and toxin. In comparison with the comprehensive antibiotic resistance database (CARD), some drug (such as fluoroquinolone, carbapenem, and peptide) resistance related-genes were predicted.[Conclusion] We isolated and identified the pathogens of the diseased T.sinensis and preliminarily analyzed the prevalence and predicted the genes related to the virulence and drug resistance of P.aeruginosa, which provided a basis for the prevention and control of infections caused by P.aeruginosa in aquaculture.

Abstract:[Objective] Breast milk-derived Bifidobacterium animalis subsp. lactis Probio-M8 has excellent probiotic properties. We analyzed the genetic characteristics of Probio-M8 at the genome-wide level and compared the genomes between Probio-M8 and other B.animalis subsp. lactis strains with probiotic efficacy. [Methods] We constructed the core gene set and pan gene set based on the genome data of 21 strains and 1 model strain DSM10140^T of B.animalis subsp. lactis in the NCBI database. Further, we constructed a phylogenetic relationship of this population and analyzed the genetic characteristics and functional genes of Probio-M8. [Results] The pan gene set of the 22 strains contained 1 618 genes, including 1 514 (93.57%) core genes, which indicated the core gene set was highly conserved. A phylogenetic tree was constructed based on the 1 514 core genes, which showed that AD011 was in a single branch, while Probio-M8 and the other strains were in the same clade with the type strain DSM10140^T. Probio-M8 had short genetic distance with V9, BB-12, Bi-07, and HN019. The genomes of Probio-M8, V9, BB-12, Bi-07, and HN019 carried three inherent resistance genes dfrA43, tetW, and rpoB with the same copy number among strains and did not contain virulence genes, which indicated the virulence and resistance genes were conserved. Furthermore, Probio-M8, V9, BB-12, and HN019 all had 47 carbohydrate metabolism-related genes. Bi-07 lacked GH49 gene and one GH13 gene copy, while Probio-M8 had an extra copy of GH49 gene, which indicated that the 5 probiotic strains had similar genes for carbohydrate metabolism. [Conclusion] The core gene set is highly conserved among the 22 strains of B.animalis subsp. lactis. The strains Probio-M8, V9, BB-12, Bi-07, and HN019 have similar genetic characteristics. This article supplies data support for the follow-up studies of Probio-M8.

Abstract:Raw milk usually needs to be refrigerated at 4 ℃ before being put into production. In this process, microbial contamination will cause deterioration of the refrigerated milk.[Objective] To investigate the dynamic changes of differential proteins expressed by microorganisms during raw milk refrigeration and thereby to lay a theoretical foundation for raw milk refrigeration [Methods] Label-free technology was used to study the species origins and functions of microbial proteins during the 6-day cold storage of raw milk at 4 ℃ and the pathways involved, screen differential proteins, and explore the pathways of the main differential proteins to explore their changing laws.[Results] A total of 341 microbial proteins were identified during refrigeration, of which 60.12% were detected after 3 days of freezing. The analysis of the cluster of orthologous groups (COG) of proteins and kyoto encyclopedia of genes and genomes (KEGG) suggested that the functions of the proteins and the pathways involved changed with time. The number of proteins involved in glycolysis/gluconeogenesis, ATP-binding cassette (ABC) transporter, amino sugar and nucleotide sugar metabolism increased significantly after 4 days of cold storage. The number of differential proteins at adjacent time points gradually increased over time, and different pathways were enriched for them. [Conclusion] The pathways involved in the proteins produced by microorganisms in raw milk during refrigeration and the functions are complex, with the most obvious changes on the 4th day, which may be a key time point for quality control of raw milk.

Abstract:[Background] Poly-γ-glutamic acids (γ-PGAs) with different molecular weights are of high application value in agriculture, cosmetics, and medicine, and biosynthesis of γ-PGA with specific molecular weights has attracted the interest of scholars. [Objective] To synthesize γ-PGAs of different molecular weights in a γ-PGA producer, Bacillus subtilis KH2. [Methods] Three γ-PGA hydrolases of B.subtilis PgdS, B.subtilis YwtE, and B.licheniformis SGH, were heterologously expressed and their hydrolytic capacities were investigated. γ-PGAs with different molecular weights were yielded in B.subtilis KH2 through the modification of enzymatic degradation conditions. [Results] All the three hydrolases can cleave high-molecular-weight γ-PGA into low-molecular-weight γ-PGA, particularly PgdS. The molecular weight of γ-PGA decreased from 1 600 kDa to 180 kDa with the addition of PgdS. Finally, the addition amount and time of PgdS were optimized to yield γ-PGA with the molecular weight of 210-600 kDa in KH2. [Conclusion] γ-PGAs with diverse molecular weights were synthesized in B.subtilis KH2 through the controllable treatment with hydrolase. This study provided a promising approach for the sustainable production of γ-PGA with different molecular weights in a wide range under mild synthesis conditions.

Abstract:Autoinducer-2 (AI-2), a quorum sensing signal molecule ubiquitous in bacteria, influences a variety of bacterial physiological processes including biofilm formation and motility. However, the role of AI-2 in regulation of these phenotypes and the underlying mechanism have not been reported for Plesiomonas shigelloides. [Objective] To reveal the mechanism through which AI-2 regulates biofilm formation and motility of P.shigelloides by regulating the intracellular cyclodiguanosine monophosphate (c-di-GMP) level, and provide a new idea for the prophylaxis and treatment of P.shigelloides infections. [Methods] Firstly, we constructed the luxS gene knockout strain (ΔluxS) by homologous recombination method. Soft agar plate assays and crystal violet staining assays were employed to compare the swimming motility and biofilm formation between ΔluxS and the wild type. Then, we identified the potential receptor protein DosC (SAMEA2665130_2180) of AI-2 by sequence alignment.Vibrio harveyi MM32 bioluminescence and isothermal titration calorimetry (ITC) were employed to test the binding affinity of the ligand-binding domain of DosC (DosC-LBD) to AI-2. Afterwards, we studied the effect of AI-2 on DosC activity by in vitro enzyme activity assay and intracellular c-di-GMP quantitative assay. Finally, we constructed the dosC knockout strain (ΔdosC) according to the above method and compared its swimming motility and biofilm formation with those of the wild type. [Results] AI-2 bound to DosC-LBD with high affinity. Compared with the wild type, ΔluxS and ΔdosC showed significantly weakened swimming motility and biofilm formation and dramatically increased intracellular c-di-GMP level. HPLC demonstrated that AI-2 enhanced the phosphodiesterase (PDE) activity of DosC. [Conclusion] The c-di-GMP-metabolizing enzyme DosC enhances its PDE activity in response to AI-2, thus affecting the intracellular c-di-GMP level and regulating the biofilm formation and swimming motility of P.shigelloides.

Abstract:[Objective] To develop monoclonal antibodies (MAbs) against chicken Toll-like receptor 21 (chTLR21) and determine the expression of chTLR21 in chicken tissues challenged by avian pathogenic Escherichia coli (APEC) or highly pathogenic avian influenza virus (HPAIV). [Methods] The 6-week-old BALB/c mice were immunized with the synthetic polypeptide chTLR21 (203-225 aa) conjugated with keyhole limpet hemocyanin (KLH). The chTLR21 (203-225 aa) conjugated with bovine serum albumin (BSA) was employed as the coating protein in indirect enzyme-linked immunosorbent assay (ELISA). The positive hybridoma cells were screened by indirect ELISA. The MAbs selected by ELISA were detected by indirect immunofluorescence assay (IFA) and then used to trace the localization of chTLR21 in chicken macrophages (HD11) and determine the expression of chTLR21 in tissues of the chickens infected with APEC O1 serotype E516 strain and HPAIV H5N6 strain. [Results] An indirect ELISA method was established, with the optimal antigen coating concentration of 2.5 μg/mL and serum dilution of 1:6 400. Four positive hybridoma cell strains were obtained and named 1G3, 2C10, 3B6, and 4F11, which belonged to the IgG2b, IgG1, IgG2b, and IgG2a subclasses, respectively. The MAb 3B6 had a robust fluorescence reactivity. The results of IFA demonstrated that chTLR21 was localized in the cytoplasm of HD11 cells. Compared with that in mock birds, the expression of chTLR21 was up-regulated in liver, spleen, lung, kidney, and bursa of Fabricius while down-regulated in pancreas of the 35-day-old specific pathogen-free (SPF) chickens infected with APEC E516 strain while exhibited no changes in the tissues of the chickens challenged with HPAIV H5N6 strain. [Conclusion] We developed the MAbs against chTLR21, among which the MAb 3B6 can be used to probe the localization and expression of chTLR21 in both chickens and macrophages, providing a tool for studying the effect of chTLR21 on the pathogenesis of bacterial or viral infections in chicken.

Abstract:[Objective] Cycas sp. is a rare and endangered tree species and can live stably in dry and hot valleys for a long time, which is closely related to the microorganisms in coralloid roots. The paper explored the differences in the species and community composition of endophytic microorganisms in coralloid roots of different species of Cycas under the same habitat. [Methods] Five Cycas coralloid roots of the same genus and different species in the Panzhihua park in Sichuan Province were molecularly identified by metagenomic sequencing technology, and the differences in microbial types, functional genes and metabolic pathways among Cycas plants were analyzed.[Results] The dominant groups of endophytic microorganisms in the samples were basically the same at the phylum level but with different relative abundance. Basidiomycota, Ascomycota, Glomeromycota and Cryptomycota were the dominant groups in the fungal kingdom, and in the bacterial kingdom Cyanobacteria, Proteobacteria, Firmicutes, Spirochaetes and Actinobacteria were dominant. There were some differences in the relative abundance of microbial communities of different species of Cycas in the fungal and the bacterial kingdoms. The relative abundance of Cyanobacteria in Cycas pectinate, C.panzhihuaensis, C.rumphii and C.guizhouens was much higher than that in C.balansae, while the relative abundance of Actinomycetes and Glomeromycota in C.balansae were much higher than that in C.pectinate, C.panzhihuaensis, C.rumphii and C.guizhouens. Through KEGG analysis, it was found that the differentially expressed gene functions of microorganisms among different species of Cycas were mainly related to environmental adaptation and energy metabolism pathways. There were abundant functional genes in carbohydrate metabolism, amino acid metabolism, folding, sorting, and degradation and signal transduction. [Conclusion] The dominant groups of endophytic microorganisms in the coralloid roots of different species of Cycas planted in the same place were basically the same at the phylum level, but the relative abundance were different. Cyanobacteria and Actinomycetes in the coralloid roots participated in the synthesis and metabolism of nitrogen and carbohydrates, which may be an important factor for Cycas to adapt to the barren environment of dry and hot valleys.

Abstract:[Objective] To explore the effect of sRNA Mpr5 of Mycobacterium tuberculosis (M.tb) on stress resistance of Mycobacterium and host cell physiology. [Methods] The recombinant Mycolicibacterium smegmatis (M.smeg) strain (M3-Atc) overexpressing the sRNA Mpr5 from M.tb was developed. The wild-type M.smeg (T1-Atc) which was transformed with an empty plasmid (pSI) was used as control. The in vitro growth status and colony morphology of these two strains were observed. The resistance of the recombinant strain to hypoxia, starvation, and 0.02% sodium dodecyl sulfate was investigated. M3-Atc was used to infect the human non-small-cell lung cancer A549 cell line and the proliferation in the cells was detected with the spread plate method. Meanwhile, the physiological structure changes of the cells were observed based on immunofluorescence staining.[Results] The in vitro growth and colony morphology of M3-Atc were similar to those of the wild type. The anti-surfactant ability of M3-Atc was significantly improved at 4 h (P<0.05). In the case of starvation, colony number of M3-Atc was smaller than that of the wild type at the early stage (2-12 h) (P<0.05). In the instance of hypoxia, colony number of M3-Atc was larger than that of the wild type in 0-3 days (P<0.05), and the growth rate was lower than that of the wild type after 3 days. Mpr5 overexpression failed to affect the infection to epithelial cells and the cytotoxicity, but reduced the early intracellular survival rate and proliferation.[Conclusion] Overexpressing Mpr5 influences the response of Mycobacterium to hypoxia and starvation and changes its ability to infect epithelial cells, which may finally affect the pathogenicity.