Abstract:Stenotrophomonas maltophilia is a gram-negative bacilli, widely distributed in the natural environments. As a novel, conditional pathogenic bacterium associated with high mortality, S. maltophilia could infect human or other organisms. In recent years, more and more studies have shown that extracellular proteases from bacteria are the key proteins which could lead to the incidence of hosts. Therefore, to explore the compositions and functions of extracellular proteases from S. maltophilia will not only help elucidate the pathogenic mechanisms, but also provide information on using them as the targets during the clinical treatment in the near future. This paper summarizes the characterizations, functions and applications of the extracellular proteases from S. maltophilia.

Abstract:Infectious bursal disease virus (IBDV) is an important representative of Birnaviridae, which causes infectious bursal disease (IBD), one important immuno-suppressive and fatal disease threatening the poultry husbandry. The naturally occurring reassortants of IBDV induced new risks to disease prevention and control. Here, we reviewed the main types of the genome segments reassortants and intragenic recombination, the inherent mechanism and the biological significances were analyzed, which would give us new insights into the virus genetic evolution research and the disease control strategy.

Abstract:Brucellosis, caused by Brucella species, is a worldwide zoonosis. As facultative intracellular pathogens, Brucella possess non-classical virulence factor, but its virulence is very powerful and can elicit chronic infections of both animals and humans. Evasion of host anti-infectious immunity is a prerequisite for chronic infections, this ability appears increasingly crucial for Brucella virulence. As successful pathogens, Brucella can escape or suppress innate immunity and modulate adaptive immunity to establish long lasting infections in host cells. In this review, we address the molecular mechanisms of Brucella to evade anti-infectious immunity. This will shed new insights on Brucella virulence and will, potentially, open new prophylactic avenues.

Abstract:[Objective] To find an efficient and fast method for microbial immobilization, we compared simultaneity culture method and adsorption method on morphology and intrastructure of combined mycelial pellets, as well as their o-chlorophenol biodegradation efficiency. [Methods] The o-chlorophenol degrading photosynthetic bacterium PSB-1D was immobilized onto mycelial pellets formed by Phanerochaete chrysosporium DH-1 to form combined mycelial pellets. The morphology and intrastructure of pellets formed by two immobilization methods were observed by optical microscope and scanning electron microscope. Then, their differences were analyzed. Using the sterile medium as control, o-chlorophenol removal efficiency of free photosynthetic bacteria, single mycelial pellets and combined mycelial pellets formed by two methods were studied. [Results] Photosynthetic bacteria were largely concentrated in the core region of pellets formed by simultaneity culture method and grew in clusters on each mycelium and their intersections. As compared with simultaneity culture method, photosynthetic bacteria mainly grew in the transition region of pellets formed by adsorption method. With the same inoculation amount of spores and photosynthetic bacteria, the simultaneity culture method could immobilize more bacteria with little time. Moreover, average diameter, dry weight and dry wet ratio of pellets formed by simultaneity culture method were bigger than that by adsorption method, and their desorption amount were less. The o-chlorophenol degradation followed a first-order kinetics model. The combined mycelial pellets formed by simultaneity culture method could degrade above 89% of o-chlorophenol in medium with an initial concentration of 50 mg/L after incubation for 7 days. And the half-life periods (t_1/2) were shortened to 2.8 days. [Conclusion] The study provides the theoretical foundation for the practical application of the new biomass carrier to organic wastewater treatment.

Abstract:[Objective] This study is aimed to screen and identify a bacterium with the ability to degrade lignocellulose, to perform its genomic analysis, and to determine its related enzymatic activities. [Methods] Using a bleaching/dyeing method with three kinds of lignin analogues (Azure-B; Phenol red; Guaiacol), we separated and screened a bacterium strain, with a strong ability to degrade lignocellulose, from soil enriched by decaying wood and leaves. We identified the species of this bacterium according to its 16S rRNA gene and core gene sequence analysis. In order to understand the trend of enzymatic activities within a certain period, we used ultraviolet spectrophotometry on manganese peroxidase (MnP), laccase (Lac), carboxymethyl cellulose (CMCase) and filter paper (FPA). The whole genome was sequenced by Illumina MiSeq and 454 GS Junior platforms. The protein sequences were annotated from the whole genome and compared with COG and KEGG databases through BLASTp to determine several potential lignocellulose-degrading enzymes and pathways. Some of the annotated genes were further verified by realtime RT-PCR. [Results] We obtained strain S12 which was identified as Raoultella ornithinolytica. The bacterium grew to stationary phase after being incubated in CMC-Na liquid medium for 28 h, at which its cellulose degradation related enzymatic activities reached to peak values. Bioinformatic analysis results showed that strain S12 has some significant genes that encode enzymes working in the lignin degradation pathway, such as peroxidase, Fe-Mn superoxide dismutase, catechol 1,2-dioxygenase, protocatechuate 3, 4-dioxygenase, etc. The expression levels of these genes were higher when strain S12 was grown in a medium with lignin as the unique carbon source than in a medium with glucose as the unique carbon source. Also, strain S12 has a complete cellulose degradation and ethanol generation pathway. [Conclusion] Raoultella ornithinolytica S12 has the ability to degrade lignocellulose effectively, which is significant in promoting the development of the lignocellulose application industry.

Abstract:[Objective] To investigate the production of adenosine modified with N⁶-(Δ²-isopentenyl) and 2-thiomethyl groups from marine-derived Streptomyces xinghaiensis NRRL B24674^T. [Methods] Bioinformatics analysis was carried out to search the genome sequence of S. xinghaiensis NRRL B24674^T and the secondary metabolites were purified by silica gel column chromatography, gel chromatography and high-performance liquid chromatography, and the chemical structure was elucidated by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). [Results] Two proteins involved in such a biosynthetic pathway were found in the genome of S. xinghaiensis NRRL B24674^T; 2-methylthio-N⁶-(4-hydroxyisopentenyl)-adenosine has been purified from the liquid culture of S. xinghaiensis NRRL B24674^T, and its chemical structure was elucidated by analysis of high-resolution mass spectrometry (HR-MS) and NMR data. [Conclusion] Such an adenine modification process was present in S. xinghaiensis NRRL B24674^T, and it is the first time to report this kind of adenine modification from actinomycetes Streptomyces. Bioinformatics analysis implies that Streptomyces can also have this kind of RNA or adenine modification.

Abstract:[Objective] We aimed to express and characterize biochemical properties of Chi92, a chitinase from Aeromonas veronii B565, and study its potential application as aquafeed supplement. [Methods] The chitinase gene chi92 was cloned from A. veronii strain B565 and expressed in Pichia pastoris GS115. The recombinant chitinase (Chi92) was purified and characterized. Chi92 was supplemented in diets containing P. pastoris powder and fed to zebrafish for 14 days. By comparing with the control group, effect of Chi92 supplementation on growth, feed utilization, microvilli morphology, and disease resistance was investigated. [Results] The complete gene sequence encoded a polypeptide with 864 amino acids. Chi92 exhibited optimal activity at pH 6.0 and 40℃, and was resistant to proteases and not substantially inhibited by metal ions. Chi92 had high chitinase activity (69.4 U/mL). The specific activity was 809.2 U/mg and 235.6 U/mg on colloidal chitin and β-1,3-1,4-glucan, respectively. Thin-layer chromatography and electrospray ionization-coupled mass spectrometry revealed that N-diacetylglucosamine was the dominant product of Chi92 when colloidal chitin was used as substrate. Moreover, Chi92 showed advantages over other chitinases for degradation of yeast cell wall. Supplementation of Chi92 in diet containing yeast product significantly improved the intestine microvilli length and density of zebrafish after two weeks of feeding. Marginally improved growth performance, feed utilization, as well as disease resistance were also observed in the Chi92 supplement group. [Conclusion] The pH stability, resistance against metal ions/chemical reagents/proteases, and high yeast cell wall degradation activity of Chi92 suggest its potential use as feed additive enzyme for warm water aquaculture.

Abstract:[Objective] The aim of this study was to express Mycobacterium tuberculosis Ag85A protein and to evaluate its immunogenicity in mice. [Methods] The cold expressed system and chaperone competent cells BL21 were combined to express Mycobacterium tuberculosis Ag85A protein, then the protein was purified with affinity chromatography and identified by Western Blot analysis. [Results] The immunogenicity of the purified Ag85A protein was evaluated in C57BL/6 mice. Results show that high level of specific IgG was elicited in the serum, and the splenocytes and lymph node cells of immunized mice could produce more Th1 cytokines, such as IFN-γ and TNF-α, after stimulated with specific antigen. [Conclusions] Ag85A protein can induce strong specific humoral and Th1 type cellular immune responses, providing an important biological material for further research and application.

Abstract:[Objective] The performances of Lactobacillus acidophilus NCFM, Lactobacillus plantarum121 and Lactobacillus pentosus ML32 in removing benzo(a)pyrene [B(a)P] in simulated starch conditions were studied. To provide a novel way to reduce the potential risk of B(a)P to human, several factors which affect the binding of the 3 Lactobacillus strains to the chemical toxin were investigated in starch system. [Methods] The percentage of B(a)P bound by Lactobacillus strains was determined with HPLC after incubation at 37℃ for 4 h. The B(a)P-removing capabilities of Lactobacillus strains in various simulated starch systems were evaluated, including the concentrations, types and gelatinization of starch, incubation time, pH and starch-hydrolyzed products. [Results] The B(a)P-binding percentages of the Lactobacillus strains increased as starch concentrations were elevated from 2% to 10%. Starch types, either from corn, potato or sweet potato, had little effects on the ability of these bacterial cells to bind B(a)P. The gelatinization of starch favored Lactobacillus strains to bind more B(a)P. The percentage of these bacterial cells to bind B(a)P grew fast at 37℃ for the first 4 h-incubation time, and then slowly increased. The three Lactobacillus strains bound more B(a)P when starches were acidified to pH of 3 to 4 or alkalified to pH of 8 to 9. The viable cells of three Lactobacillus strains removed more B(a)P via their binding than their dead cells did. Moreover, both glucose and maltose, the end products after starch is hydrolyzed, improved significantly the performance of the 3 bacterial strains to remove B(a)P. [Conclusion] All of the three Lactobacillus strains perform good ability to bind B(a)P in the presences of starch. Their B(a)P-removing ability would be improved with an increased starch concentration, and the gelatinization of starch as well as the supplements of glucose and maltose. Thus, the selected Lactobacillus strains in our current work should be promising as a biological agent to reduce the occurrence of B(a)P in starch-based food products.

Abstract:[Objective] To provide scientific data for studying the ecology of cyanophages isolated from Daqing wetland by analyzing their genetic diversity and phylogenetic positions. [Methods] Liquid enrichment culture and double-layer plate methods were used to isolate cultivable cyanophages by using host cyanobacteria Anabaena PCC7120, and the DNA of cultivable cyanophages was extracted. The biomarker genes of g20 encoding capsid assembly protein and pol encoding DNA polymerase in podoviruses were PCR amplified. The PCR products were cloned and sequenced. The sequences were constructed with references sequences into the phylogenetic trees to clarify the phylogenetic positions of cultivable cyanophage. [Results] One g20 sequence and four pol sequences were obtained. Phylogenetic analysis showed that the g20 sequence belongs to the cultivable cyanophage group (Cluster δ). Three pol sequences were closely related to cyanophage groups PG-Pol-I and PG-Pol-II that were observed in an alkaline paddy floodwater in Da'an, Jilin province, China; another pol sequence formed a unique clade. [Conclusion] The g20 gene from cultivable cyanophages infecting Anabaena PCC7120 belongs to the Cluster δ, and the pol genes are closely related to those of paddy floodwater in Da'an, China.

Abstract:[Objective] The impact of inoculation with the biocontrol agent Bacillus subtilis on bacterial communities in rhizospheric soil of Nicotiana tabacum was assessed by using 454 pyrosequencing technology. The control effect of Tpb55 on tobacco black shank was also studied. [Methods] Two treatments were done as follows: irrigating root with Bacillus subtilis strain Tpb55 inoculants (10⁸ CFU/mL) and the control. Soil samples from tobacco rhizosphere were collected at 0d, 10d and 22 d after the treatment. Genomic DNA of soil samples was extracted and amplified for the 16S rDNA V1-V3 tags, and then the tags were sequenced by 454 sequencing. Qiime was used to analyze soil bacterial diversities. [Results] A total of 41207 high quality sequences were obtained from all samples, which were classified into 25 phyla. The dominant bacteriophyta were Actinobacteria, Proteobacteria and Acidobacteria in all samples. The content of Actinobacteria was decreased gradually in the development of disease, whereas Proteobacteria showed an opposite tendency. Acidobacteria revealed a marked increase andexceeded control in content after inoculation with Tpb55. The control showed a significant decline in Bacillaceae, as well as Oxalobacteraceae which was known as an indicator for bacterial diversity. However, Bacillaceae showed an increasing tendency and Oxalobacteraceae was relatively constant in Tpb55 treatment. The Chao 1, ACE and Shannon index of treatment showed a constant improvement of variety and richness. In 10 d and 22 d after Tpb55 inoculation, the number of sequences with high homology of V1-V3 regions of Tpb55 16S rDNA was 31 and 45, respectively. The disease index of tobacco black shank in inoculated tobacco (5.29) was significantly lower than the control (38.52). [Conclusion] Tpb55 could improve the diversity of soil bacterial community and ecosystem stability, which presented a possible reason for its biocontrol efficacy on tobacco black shank.

Abstract:[Objective] To identify and characterize an electrogenic bacterium SE6 isolated form forest soil. [Methods] Pure culture of the strain was obtained by anaerobic incubation. It was identified based on morphology, physiology and biochemistry, and 16S rRNA gene sequencing analysis. The strain was inoculated in a dual chamber microbial fuel cell with LB medium as anolyte and potassium ferricyanide as catholyte, to characterize its electrogenic ability. Electrochemical impedance spectroscopy was conducted to analyze internal resistances of the MFCs. Extracellular electron transfer mechanism of the strain was explored by cyclic voltammetry. Biofilm on the anode surface was observed using scanning electron microscope. [Results] The 16S rRNA gene sequence of strain SE6 was 100% phylogenetically related to Clostridium sporogenes. Their morphological, physiological and biochemical characteristics were identical. The maximum power density of the MFCs inoculated with SE6 was 44.42 mW/m². The anodic resistance, cathodic resistance and ohmic resistance were (1488±193) Ω/cm², (0.92±0.01) Ω/cm² and (20.69±1.76) Ω/cm², respectively. Cyclic voltammograms indicated the existence of an electrochemically active substance, of which the peak currents were linearly correlated with the scanning rates. The 1 μm-rodshaped bacteria densely attaching to the anode surface were observed in scanning electron micrographs. [Conclusion] A novel electrogenic strain of C. sporogenes was isolated from forest soil, which transfers electrons extracellularly to electrode with high resistance.

Abstract:[Objective] Food phytochemicals as biofilm inhibitor of pathogens have been highlighted. Our study aimed to investigate the effects of resveratrol on biofilm formation of an aquatic pathogen Vibrio parahaemolyticus, and to elucidate the important regulatory genes. [Methods] In the subinhibitory concentrations, the inhibition of resveratrol aganist biofilm and exopolysaccharides of V. parahaemolyticus was detected, and the differentially expressed genes were analyzed based on RNA-Seq. Four genes involved in biofilm formation was validated by qRT-PCR. [Results] The minimum inhibitory concentration of resveratrol against V. parahaemolyticus was 20 μg/mL. Resveratrol at the subinhibitory concentration of 5 μg/mL and 10 μg/mL significantly decreased the biofilm development and exopolysaccharides production in V. parahaemolyticus (P < 0.05). Scanning electron microscopy micrographs showed a significant reduction of adherence and extracellular polymeric substances. RNA-Seq analysis revealed that 22.6% up-regulated and 77.4% down-regulated gene (P < 0.05) after treatment by 10 μg/mL resveratrol among 106 differential gene expressions. These differential genes in V. parahaemolyticu focused on 7 metabolic pathways, and 14 genes involved in biofilm formation were down-regulated by resveratrol, such as outer membrane protein (W, YedS, OmpK), quorum sensing (LuxS), flagellin (FlaA), fimbrial assembly protein (PilQ), hemolysin secreted protein. qRT-PCR confirmed that the expressions of luxS, trh, tlh and flaA, was significantly repressed in the presence of resveratrol, which was consistent with transcriptomics data. [Conclusion] Inhibitory activity of resveratrol on biofilm formation was assicated with multiple genes and diverse cellular processes in V. parahaemolyticus. These findings suggest that resveratrol would disturb various metabolic pathways, particularly quorum sensing system, adhesion process and membrane proteins secretion, resulting in inhibition of attachment and biofilm development. The present work provided valuable information to explore molecular mechanism of resveratrol as an novel anti-biofilm compound.

Abstract:[Objective] To study the immunological cross-reactivity and cross-protection characteristics of OmpU in Vibrio species. [Methods] The ompU genes from 10 Vibrio strains were cloned, sequenced, followed by bioinformatics analysis. Western blot and whole-cell ELISA assay were used respectively to determine immunological cross-reaction feature and subcellular location of OmpU with rabbit serum against recombinant OmpU from V. parahaemolyticus ATCC17802, V. alginolyticus ATCC33787, V. vulnificus ATCC27562, V. mimicus ATCC33653 and V. cholera Vb0. Finally, the cross-protective property of recombinant OmpU (V. cholera-derived) was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in mice. [Results] The similarities of OmpU proteins of Vibrio ranged from 73.0 to 100% intra-species, and from 58.6 to 89.0% inter-species. Furthermore, homologous epitopes were found in OmpU and shared by different species of Vibrios. Western blot of rabbit serum against recombinant OmpU showed cross-recognition intra- and inter-species. Bands were observed ranging from 35 to 40 kDa. Whole-cell ELISA assay further confirmed that the antiserum of recombinant OmpU from V. parahaemolyticus ATCC17802, V. vulnifgicus ATCC27562 and V. mimicus ATCC33653 recognized the tested Vibrio species, implying that epitopes of OmpU were located on the cell surface. Recorded relative percent survival of the vaccinated group varied from 43.0 to 100%, showing that mice were protected from Vibrio infection after immunization with OmpU protein. [Conclusion] OmpU was a conserved antigen among tested Vibrio species and might be a universal vaccine candidate for the prevention of Vibriosis.

Abstract:[Objective] We attempted to obtain a fungus producing thermotolerant dextranase by screening samples from soil. [Methods] The fungus producing thermotolerant dextranase was isolated and screened by auxotrophic medium, combined with Pour Plate method and Flat Transparent Circle method. The strain was identified by its colony, cell morphology and cultural characteristics, as well as ITS rDNA sequence analysis. The dextranase produced by the strain was characterized. [Results] We obtained the strain DG001 producing thermotolerant dextranase, which was identified as Paecilomyces lilacinus. The optimum catalytic conditions for the dextranase were 55℃, pH 5.0, and the optimum substrate concentration was 5% dextran T70. The dextranase was stable below 60℃ and between pH 4.0 and 7.0. Urea, Mn²⁺ and Mg²⁺ could increase enzyme activity, and the low concentration of Mn²⁺ and Urea could increase enzyme activity to 116.91% and 110.14% respectively, whereas Cu²⁺ had a strong inhibitory effect on the dextranase. The dextranase, identified as endo-dextranase, hydrolyzed dextran T2000 with main products as isomalt and isomaltotriose. The enzyme-substrate affinity increased with the increasing substrate molecular weight. [Conclusion] Strain DG001 producing thermotolerant dextranase was obtained through successful screening, bearing a high activity in a wide temperature range and a good thermal stability. This enzyme shows a promising prospect of application in sugar industry and in the preparation of different molecular weight dextran.

Abstract:[Objective] wzz is involved in the synthesis of O antigen and plays a role in virulence in many gram-negative bacteria. However, the function of wzzE in avian pathogenic Escherichia coli (APEC) is unclear. The aim of this study is to elucidate the role of wzzE in the synthesis of LPS and virulence. [Methods] Mutant strain with wzzE deletation was constructed. Biological characteristics of the wild and mutant strains, such as LD₅₀, adherence and invasion to DF-1 cells, phenotype of LPS and endotoxin titer, were detected. [Results] There was no significant difference in growth, adherence and invasion to DF-1 cells, serotype and the endotoxin titer between the wide strain and the mutant. However, compared with the wild-type strain, the virulence of DE17ΔwzzE had dropped by 10 times. [Conclusion] wzzE is not involved in the synthesis of LPS in DE17, but participates in the pathogenic process of APEC. However, the mechanism of wzzE in APEC virulence needs to be studied in the future.