Abstract:Metapneumovirus, including human metapneumovirus and avian metapneumovirus, is an important pathogen in human and avian infections. The attachment protein is one type of glycoprotein on the surface of metapneumovirus particles, which belongs to type Ⅱ transmembrane protein. Compared with other paramyxoviruses, the length of the amino acid sequence, the homology and the biological functions (such as the attachment of the virus, the mediation of cell fusion, the influence of viral replication in cell culture and in vivo, and the immuno-protective effect) of the G proteins of metapneumovirus are different. Meanwhile, the G protein of metapneumovirus plays an important role in immunosuppression and immune evasion. At present, few related research has been carried out in China. The aim of this paper is to review and discuss the latest research and progress in the study of the attachment proteins of metapneumoviruses. The future research direction is prospected.

Abstract:Two microbiotas were identified in shrimp and other crustaceans, gut and hemolymph microbiaotas. The microbe species and number of gut microbiota are much more than that in hemolymph microbiota. They all contain probiotic and pathogenic bacteria in the two microbiotas. The gut and hemolymph microbiaotas have important functions in host metabolism, nutrition and organic development and immune responses. Different regulation mechanisms were identified in shrimp for homeostasis of gut and hemolymph microbiaotas. The reactive oxygen species produced by dual oxidases is mainly involved in homeostatic regulation of gut microbiota; and antimicrobial peptides regulated by a C-type lectin and prophenoloxidase activating system are related to homeostatic regulation of hemolymph microbiota. The studies of composition, functions and homeostatic regulation of microbiotas in shrimp are beneficial for healthy aquaculture of shrimp and other economic crustaceans.

Abstract:[Objective] Our previous study found that a Tn5 transposon inserted mutant in pilT gene of Xanthomonas oryzae pv. oryzicola (Xoc) RS105 strain (the causal agent of bacterial leaf streak) significantly reduced bacterial virulence in susceptible rice plants, and the ability to trigger hypersensitive response (HR) on non-host tobacco. [Methods] To reveal the roles of pilT gene in bacterial pathogenicity in rice, we constructed a deletion mutant of pilT gene in Xoc RS105 named RΔpilT. Then we tested phenotypes of the mutant compared to the wild-type RS105 in bacterial virulence, motility and biofilm formation. [Results] The pilT mutant RΔpilT significantly reduced bacterial virulence in rice and the ability to trigger HR on tobacco. In addition, compared to RS105 strain, RΔpilT mutant displayed severely weakened motility on semisolid medium, and increased the production of biofilm. Full virulence, bacterial motility and biofilm formation was restored to the mutant when Xoc pilT was expressed in trans. Through qRT PCR analysis, we found that pilT gene was regulated by clp, rpfG, pilA and pilC. [Conclusion] These results indicate that pilT gene is essential for full bacterial virulence in rice possibly due to the association with the reduced motility and increased biofilm production by the pilT mutation, implying that the importance of further investigation on the function of PilT in composition of type IV pili in Xoc.

Abstract:[Objective] This study aims to study metabolic characteristics of methionine by caecal and colonic bacteria compared with ileal bacteria from pigs using in vitro fermentation. [Methods] We took ileal, caecal and colonic chyme from four Duroc×Landrace×Yorkshire hybridization pigs as inoculum, and fermented in vitro separately. Control group 1 was without adding the chyme and Control group 2 was without adding methionine. [Results] Methionine disappeared 21.9% in the cecum group, significantly higher than that in the ileum group (16.3%) and colon group (16.7%) (P<0.05). The cecum group had the highest amount of short chain fatty acids (P<0.05), with the highest decrease of pH value. Moreover, the cecum group had the highest concentration of microbial crude protein, compared with the other two groups (P<0.05). Nevertheless, gas production and ammonia nitrogen (NH₃N) concentration in the ileum group were significantly higher than those in the cecum and colon group (P<0.05). At the phylum level, total bacteria and Firmicutes population were similar among all groups (P>0.05), and the counts of Bacteroidetes was highest in the cecum group. At the genus level, Escherichia coli counts in the cecum and colon group were significant lower than that in the ileum group (P<0.05), whereas the counts of Clostridium leptum and Clostridium cluster XIVa in the cecum and colon group were both higher than those in the ileum group. Bifidobacterium counts were similar among all groups (P>0.05). [Conclusion] In vitro fermentation using methionine as a substrate, the utilization rate of methionine by caecal bacteria is higher than that by ileal bacteria from pigs, accomplished with altering other fermentation parameters and producing microbial protein. Compared with in vitro fermentation of ileal bacteria, the counts of Clostridium leptum and Clostridium cluster XIVa are higher, whereas the Escherichia coli counts is lower after fermentation of the large intestinal bacteria.

Abstract:[Objective] The biosynthesis ability of secondary metabolites in the lovastatin-producing strain Aspergillus terreus HZ01 was analyzed, for strain improvement and genome mining of secondary metabolites through genetic engineering. [Methods] The transcriptome of Aspergillus terreus HZ01 under the condition of lovastatin fermentation was analyzed. Meanwhile, the main by-products were separated and identified by chromatographic separation and spectroscopy. [Results] The biosynthesis of lovastatin was very active, and most of other PKS and NRPS genes are silent besides of 4 PKS, 6 NRPS and 1 PKS-NRPS. In addition, 10 major by-products were isolated and identified from the cultures. [Conclusion] Aspergillus terreus HZ01 is an excellent lovastatin producing strain, which will also be a valuable chassis cell for the microbial cell factory construction and biosynthesis pathway identification.

Abstract:[Objective] In order to investigate how NDM-1 affect the metabolism of A. baumannii, we analyzed the differentially expressed proteins (DEPs) between clinically isolated multidrug-resistant A. baumannii MDR-ZJ06 (bla_NDM-1^-) and ABC3229 (bla_NDM-1⁺) which have a close genetic relationship. [Methods] We used 2-DE coupled with MALDI-TOF MS/MS to analyze the differentially expressed proteins (DEPs) between A. baumannii MDR-ZJ06 and ABC3229. Based on GO annotations, we analyze DEPs by KEGG, Function classification, enrichment analysis and protein-protein interaction network. [Results] We found that there were 51 DEPs, including 11 up-regulated proteins and 41 down-regulated proteins in ABC3229. These proteins were predominantly involved in reducing carbohydrate metabolism, amino acid metabolism, fatty acid metabolism, cell wall synthesis, and increasing of iron transport system formed. [Conclusion] These results uncover the effect of NDM-1 on bacteria may be resistant to antibiotics by slowing down the metabolism of bacteria and increasing the uptake of iron, thereby the bacteria will systemic resistance antibiotics.

Abstract:[Objective] We isolated and purified a nitrogen-fixing bacterium AZ16 and a phosphate-solubilizing bacterium XT37 from the sediment of Halophila ovalis in Xisha, China. Furthermore, we optimized their culture conditions and explored their potential for solid microbial agents. [Methods] The strains were identified by morphological, biochemical and molecular characteristics. The nitrogenase and phosphate solubilizing activities were measured by acetylene reduction method and molybdenum antimony anti-coloring method. The fermentation conditions were optimized by single factor method and response surface method. Safety of the strains was identified by hemolysis test and acute toxicity experiment. [Results] Strain AZ16 is a gram-negative bacterium, belonging to Sagittula stellate, producing yellow, round, sticky colonies on the selective nitrogen fixation medium. The maximum nitrogenase activity was 34.63 nmol C₂H₂/(mL·h). The optimum growth conditions were as follows: salt content 2.5%, pH 7.5, fermentation temperature 33 inocula concentration 5.0%. Strain XT37 showed closest resemblance with Bacillus sp., gram-positive bacteria, developed yellow, round, plicated in the selective nitrogen fixation medium. And the phytase activity was 239.5 μg/L. The optimum conditions were as follows: salt content 2.5%, pH 6.7, fermentation temperature 28 inocula concentration 5.0%. Hemolysis tests and acute toxicity experiments proved that the two strains were in the actual non-toxic level. [Conclusion] Nitrogen-fixing bacterium AZ16 and phosphate-solubilizing bacterium XT37 can resist high salt concentration and can also survive under alkaline condition, hence these strains have the potential for multi-functional mixed microbial agents.

Abstract:[Objective] Elimination of 3-phenoxybenzoic acid (3-PBA) is the key to solve the pyrethroid pesticides contamination environment. The aim of this study was to isolate efficient 3-PBA-degrading strains from the plant rhizosphere contaminated by pyrethroid pesticides. [Methods] 3-PBA-degrading strains was screened by using enrichment domestication, isolating and purification methods with 3-PBA used as the sole carbon source. Then the effcient 3-PBA-degrading strain was identified by morphological, physio-biochemical tests and 16S rRNA sequence analysis, and its growth and degradation kinetics and degradation characteristics were studied. Moreover, the degradation conditions were optimized by the Box-Behnken response surface analysis. [Results] A novel 3-PBA-degrading strain BPBA031 isolated from the soybean rhizosphere in northern Sichuan Province was classified into Enterobacter ludwigii, which could be resistant to high concentration of 3-PBA (1600 mg/L). The growth of strain BPBA031 and 3-PBA degradation respectively followed the logistic growth kinetic model (μ_m=0.09149 h^-1, X_m=1.1145) and first-order degradation kinetic model (k=0.02085, t_1/2=33.24 h). The optimum conditions for 3-PBA degradation were culture temprature of 34-37 , 3-PBA concentration of 30.0 mg/L and initial pH 7.58 after 48 h of incubation. [Conclusion] Enterobacter ludwigii BPBA031, a highly effcient 3-PBA-degrading strain, can be used as a potential strain resource for bioremediation of environment polluted by 3-PBA or pyrethroid pesticides.

Abstract:[Objective] To obtain a new aspartate kinase (AK) with higher activity and better properties of highly produced aspartic acid family from Corynebacterium pekinense by spatial structure transformation, and attenuate or release the feedback inhibition of AK. [Methods] The Gln(Q)316 of AK was mutated by site-directed mutagenesis, and mutant strain with the highest activity was selected by high throughput screening. The mutant was expressed in Escherichia coli BL21. AK of the wild type and mutant strain was purified by Ni²⁺-NTA column and then the characterized. [Results] The mutant Q316P was constructed, and successfully expressed in E. coli BL21. Compared with the wild type, V_max of Q316P was increased by 8.53 times, and the n values was decreased from 2.15 to 1.29. The optimum temperature was increased from 25 ℃ to 30 ℃, the optimum pH was decreased from 8.0 to 7.5 and the half-time period was increased from 3.8 h to 5.0 h. The inhibition of the substrate inhibitor threonine was not only relieved but also threonine had an active effect on the activity of Q316P. Q316P had better resistance to methanol and K⁺. [Conclusion] Our findings provided a reference for the construction of high-yield aspartic acid family engineering strain.

Abstract:[Objective] The aim of this study was to characterize Vibro harveyi acyl-ACP synthetase (AasS) with free fatty acids of different chain length and other carboxylic acids as substrates in vitro. [Methods] We analyzed the catalytic activity of AasS in vitro qualitatively and quantitatively by Urea-PAGE and UV spectrophotometry, respectively. [Results] AasS could catalyze the synthesis of fatty acyl-ACPs using straight chain free fatty acids with different chain length as substrates, and the enzymatic activity was much higher than C6-C12 fatty acids. In the case of 3-hydroxyl fatty acids, AasS showed higher activities to C8-C14 chain length as substrates. Furthermore, AasS could utilize such as 20 amino acids, benzoic acid and salicylic acid as substrates, and all formed corresponding acyl-ACPs. [Conclusion] Vibro harveyi acyl-ACP synthetase (AasS) catalyzed different substrates with different activities, and this finding could provide a reference for analyzing the metabolism of amino acids.

Abstract:[Objective] To isolate high effective phosphate-solubilizing microbial strains from growing sunflower in saline-alkali soils for bio-fertilizer production. [Methods] Phosphate-solubilizing fungus M1 was isolated by petri-dish methods and identified using ITS rDNA sequence homology analysis. The phosphate-solubilizing capacity of M1 was measured by broth medium and soil pot experiment. The effect of isolate M1 on plant growth promotion was studied in field experiment. Organic acids and phytohormone produced by isolate M1 were analyzed by means of LC-MS. [Results] Isolate M1 was identified as Aspergillus japonicus and had a strong ability to dissolve insoluble phosphates. The rate of Ca₃(PO₄)₂ dissolved by M1 was 63.30% and the concentration of available phosphorus was 1020.89 mg/L at 6 d shaking incubation. Soluble phosphorus in AlPO₄ liquid culture by M1was lower than in Ca₃(PO₄)₂ added medium, the content concentration of available phosphorus was 995.69 mg/L and the solubilized rate of AlPO₄was 48.59%.The greatest concentration of available phosphorus dissolved by isolate M1 from Jinning rock phosphate was 363.64 mg/L after 6 d shaking incubation. Isolate M1 combining salt resistance properties as well, the greatest content of NaCl was 10%. In greenhouse pot experiment, isolate M1 had a significant growth-promoting effect on corn in four kinds of soils (paddy soil, viscous fluvo-aquic soil, salinized fluvo-aquic soil and calcareous fluvo-aqui soil) under three kinds of insoluble phosphates treatment such as Ca₃(PO₄)₂, AlPO₄ and Kaiyang rock phosphate with 4 replicates per treatment. Compared with control (CK), inoculation with M1 increased the fresh weight of corn biomass by 2.14%-90.91% and dry weight of corn biomass by 22.15%-268.28%, and soil available phosphorus content increased 21.81-24.24 mg/kg. The adaptability of isolate M1 with four kinds of soils is greater than strain DSM 821. Field experiment showed that isolate M1 had a greater effect on enhancement of peanut yield, the yield was average 4.46 t/hm² and increased 22.19% being greater over control. 7 organic acids and 2 phytohormones were analyzed in liquid culture of Ca₃(PO₄)₂, AlPO₄ and Kaiyang rock phosphate. Out of seven acids, significant increases in the concentration of oxalic acid (616.16 mg/L) and citric acid (413.69 mg/L) were recorded in 3 liquid cultures by isolate M1, respectively. The concentration of indole acetic acid (IAA) was 15.45-77.58 mg/L, zeatin was 0.06-0.11 mg/L. [Conclusion] One new phosphate- solubilizing isolate M1 was obtained and identified as Aspergillus japonicus. Isolate M1 could solubilize insoluble phosphates in petri dishes, broth medium as well as pot experiments and increased soil available phosphate and corn biomass significantly in pot condition. So Aspergillus japonicus M1 strain have a better potential for bio-fertilizer production in the future.

Abstract:[Objective] To investigate the phenotypes of Azorhizobium caulinodans ORS57 mutant strains that lacked the flagellar motor proteins FliN and FliM respectively, and the functional mechanism of these proteins. [Methods] The gene knock-out mutant strains (ΔfliN, ΔfliM) were constructed by homologous recombination and triparental conjugation. The phenotypes including cell growth, chemotaxis, exopolysaccharide production, biofilm formation and cell flocculation were investigated. [Results] Mutants ΔfliN and ΔfliM did not shown any difference in cell growth compared with the wild-type strain. However, both mutants lost their flagella when observed under electron microscope. In addition, ΔfliN and ΔfliM were impaired in chemotaxis, exopolysaccharide production, and biofilm formation. To the contrary, the flocculation ratio of both mutants was higher than that of the wild-type strain within the same amount of time. [Conclusion] The flagellar genes fliN and fliM could regulate the flagellum formation, cell motility, exopolysaccharide production, biofilm formation and cell flocculation of Azorhizobium caulinodans ORS571.

Abstract:[Objective] To study the division of Rhodococcus sp. R04 cells and the effect of biphenyl on cellular morphology and cell division. [Methods] Using fluorescence microscopy, scanning electron microscopy and transmission electron microscopy, we analyzed the division of R. R04 cells cultured under different conditions. [Results] Rhodococcus sp. R04 exhibited asymmetric and symmetric division, and the proportion of the two types of division was about 7:3. The culture conditions did not affect the proportion of asymmetric division cells. The septum was located on the center to the poles, with some clustering at 30% and 50% of the cell length. Rhodococcus sp. R04 degraded biphenyl, but its division would be inhibited by biphenyl. Microscopic observation showed that Rhodococcus sp. R04 cells formed filaments and exhibited abnormal division in early exponential phase under biphenyl culture conditions. As the culture time was prolonged, the cells finally restored normal division. [Conclusion] Biphenyl/polychlorinated biphenyl has a strong effect on the reproduction and growth of Rhodococcus sp. R04 cell, but it does not affect the mode of cell division.

Abstract:[Objective] We cloned and expressed the agnE gene involved in nicotine-degrading in Agrobacterium tumefaciens and characterized the AgnE protein. [Methods] The agnE gene fragment (1029 bp) was amplified with PCR. Recombinant plasmid pET28a-agnE was constructed and transformed into Escherichia coli BL21(DE3) for heterologous expression, and the expressed protein was detected with SDS-PAGE. The enzyme activity of AgnE was determined spectrophotometrically by monitoring the decrease of 2,5-dihydroxy pyridine at 320 nm. The effect of temperature, pH and metal ion on enzyme activity was investigated. [Results] The agnE gene was cloned and the recombinant plasmid pET28a-agnE was expressed. The molecular weight of the recombinant protein was 42.0 kDa, and the protein was expressed in the form of inclusion bodies in cells. The AgnE protein had high enzyme activity at pH 8.0 and and 20 ℃. In addition, Fe²⁺ showed significant promoting effect on enzyme activity. [Conclusion] The activity of AgnE protein with 2,5-dihydroxy pyridine dioxygenase was clarified.

Abstract:[Objective] The physiological function of regulatory protein RHOGL007659 in Rhodococcus sp. R04 and the metabolic properties of the mutant strain were investigated to explore its regulation mechanism of biphenyl degradation. [Methods] The RHOGL007659 gene was knocked out by homologous recombination, and the growth of the wild-type strain and deficient mutant strain in different carbon sources were compared. The transformation of biphenyl by the wild-type strain and the deficient mutant strain was detected by HPLC. Total RNAs were extracted from the wild-type strain and deficient mutant strain, and quantitative RT-PCR was carried out to analyze the transcriptional variation of bphA, bphB, bphC and bphD between the wild-type strain and the deficient mutant strain. BphB and BphD were expressed in Escherichia coli BL 21(DE3) and polyclonal antibodies were prepared after purification. BphB and BphD expression levels in the wild strain and deficient mutant strains were detected by Western blot. [Results] The deficient mutant strain R04Δ7659 of RHOGL007659 gene was obtained. The biomass of the deficient mutant strain significantly reduced in biphenyl culture, and almost did not grow. HPLC analysis showed that the deletion of RHOGL007659 gene resulted in inability to transform biphenyl by Rhodococcus sp. R04. Q-RT PCR experiment disclosed that the key genes of biphenyl degradation were down regulated in different degrees under biphenyl culture condition in the RHOGL007659 deficient mutant strain, the same results were obtained by Western blot. [Conclusion] RHOGL007659 is a regulatory protein of the upper biphenyl degradation gene in Rhodococcus sp. R04, and functions as a positive regulatory factor of the metabolism of biphenyl by Rhodococcus sp. R04.

Abstract:[Objective] Bacterial wilt is a soil-borne disease caused by Ralstonia solanacearum. Extracellular polysaccharides are one of the key pathogenic factors of R. solanacearum. The physiological functions of extracellular polysaccharides on the pathogenesis of bacterial wilt were studied by constructing a mutant with synthesis of extracellular polysaccharides deficiency. [Methods] First, we cloned the homologous arm of epsD from the genome of R. solanacearum FJAT-91, then inserted into suicide plasmid pK18mobsacB. Second, the Gm gene was inserted into homologous arm to obtain recombinant plasmid pK18-epsD. Third, the recombinant plasmid was transformed into R. solanacearum FJAT-91 competent cells. The epsD gene deletion mutant was constructed by homologous recombination. Finally, we detected the differences of the biological characteristics of mutant strains and wild-type strains. [Results] Compared with the wild-type strains, the mutant strains showed different features. The mutant showed: grew slowly and decreased the yield of extracellular polysaccharides; significantly reduced the abilities of swimming motility and swarming motility; significantly decreased the colonization ability in roots and stems of tomato; had the Attenuation Index (AI) with 0.905 and the avirulence to tomato which indicated a nonpathogenic strain. [Conclusion] These results suggested that extracellular polysaccharides played the key role in pathogenicity of R. solanacearum. This research provides excellent materials and research foundation for the development of plant vaccines.

Abstract:[Objective] Sequencing and analysis of Phomopsis sp. XP-8 genome are beneficial to reveal the potential metabolic pathways of this strain and the key genes related to the biosynthesis of pinoresinol and its glycoside derivatives and other secondary metabolites. [Methods] We sequenced Phomopsis sp. XP-8 genome by the Illumina HiSeq 2500 high throughput sequencing platform. Then gene prediction and functional annotation were analyzed using different softwares. [Results] The final assembled genome size was approximately 55.2 Mb with an overall GC content of 53.5%. Further annotation analyses predicted 17094 protein-coding genes and 310 non-coding RNA genes. A large set of candidate genes involved in the production of pinoresinol, its glycoside derivatives and other secondary metabolites were identified. Orthology and phylogenetic analysis revealed that Phomopsis sp. XP-8 and 5 Ascomycota share 12635 orthologous genes and 5626 gene families. [Conclusion] Phomopsis sp. XP-8 possessed genomic basis for production of diverse secondary metabolites, including pinoresinol and its glycoside derivatives. This study provides basis for the further metabolic engineering of pinoresinol and its glucoside production.