Abstract:Pasteurella multocida is an important gram-negative pathogenic bacterium that could infect wide ranges of animals. Humans could also be infected by P. multocida via animal bite or scratching. Current typing methods for P. multocida include serological typing methods and molecular typing methods. Of them, serological typing methods are based on immunological assays, which are too complicated for clinical bacteriological studies. However, the molecular methods including multiple PCRs and multilocus sequence typing (MLST) methods are more suitable for bacteriological studies of P. multocida in clinic, with their simple operation, high efficiency and accurate detection compared to the traditional serological typing methods, they are therefore widely used. In the current review, we briefly describe the molecular typing methods for P. multocida. Our aim is to provide a knowledge-foundation for clinical bacteriological investigation especially the molecular investigation for P. multocida.

Abstract:The progressive form of clinical Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by production losses, weight loss, chronic intractable diarrhea, and severe emaciation leading to death in cattle. Substantial economic losses to the animal husbandry are a result of infection. Cattles are usually infected in their youth through the oral route and will experience a long subclinical stage. At the early stage of infection, cellular immunity is the main immune response with bacterium excretion increased significantly after a subclinical period of 2 to 5 years. The majority of methods currently used to detect MAP are based on etiological detection, cellular and humoral immune response. Owing to the different mechanism of diagnostic methods varies a lot at a particular infection period. In this review, we illustrate the transmission route and the characteristic of immune responses of MAP, and also summarize the diagnostic methods of MAP.

Abstract:Antimicrobial peptides (AMPs) are bioactive short peptides produced in organisms. AMPs have important roles in resisting pathogen invasion. In recent years, several studies on intestinal microecology is hot and the influence of antimicrobial peptides on intestinal health is widely concerned. Relevant results have demonstrated that the expression level of AMPs can be used to assess the body's intestinal health, thereby an auxiliary method could be established through monitoring on the expression level of AMPs during disease prevention and treatment. In this paper, research advances in antimicrobial peptides in intestinal microflora structure and immune effect were summarized and analyzed to provide references for clinical diagnosis and treatment.

Abstract:[Objective] In order to demonstrate the ubiquitination regulation mechanism of histidine transporters. [Methods] By both ubiquitination sites prediction and site-directed mutagenesis, 3 potential ubiquitination sites, K30, K42 and K42 of Hip1p were mutated. These Hip1p mutants were cloned into the ubiquitination detection plasmid for measuring its ubiquitination level change. Effects of these mutants on both cell growth and histidine utilization were measured. [Results] By comparing the relative fluorescence of Hip1p and its mutants, ubiquitination sites mutation reduced the ubiquitination levels of Hip1p. Furthermore, double mutation of ubiquitination sites showed a synergy effect on reducing ubiquitination level. The ubiquitination sites mutants also influenced cell growth and enhance histidine utilization when histidine was used as the sole nitrogen source. [Conclusion] The ubiquitination levels could regulate the histidine metabolism and change the pattern for histidine metabolism, and provide clues for further investigation of regulation mechanisms involved in the amino acids transporter proteins.

Abstract:[Objective] This study was aimed to enhance the extracellular enzymes activities and soluble expression of CGTase from Geobacillus sp. B1 by directed evolution. [Methods] A library of CGTase mutants was constructed by introducing random mutagenesis using error-prone PCR to screen mutant enzymes with improved extracellular enzyme activities and soluble expression. After induction, expression and purification, the mutant enzyme was characterized. [Results] After screening, two optimum mutants ds-6 and ep-9 with extracellular alpha-cyclization activity are respectively 1.72 times and 2.18 times of the original enzyme. The sequence of ep-9 cgt gene showed that three nucleotides substitution, G2005A, A2037G and T2081G were observed, and two of them caused amino acid changes. According to the 3D structure of Geobacillus sp. B1 cyclodextrin glucosetransferase mimicked by SWISS-MODEL Repository, two amino acid mutations were in rotating angle between beta angle and the random coil. The wild-type CGTase or ep-9 genes was ligated with pET-28 (a)-OmpA vector, and expressed in E. coli BL21 (DE3). After induced by lactose, CGTases were purified and characterized. The results showed that the specific β-cyclization activity of the evolved CGTase was 1.31-fold than that of the wild-type CGTase, and the K_m decreased from 4.3 to 3.74 g/L. The pH stability of the evolved CGTase was better than wild-type CGTase. Site-directed mutagenesis demonstrated the key to improve the soluble expression level and extracellular enzyme activity was G2005A. [Conclusion] Directed evolution by error-prone PCR of Geobacillus sp. B1 CGTase gene is effective to improve extracellular enzyme activities and soluble expression, in particular mutation occurred in the G2005A.

Abstract:[Objective] Gene lef-11 is one of the high conservative late expression factors in Bombyx mori nucleopolyhedrovirus (BmNPV), and contributes to virus replication and proliferation. The effective RNA interference will increase the resistance of host cells to BmNPV. [Methods] Choosing two classical skeleton sh-RNA-loop and miRNA skeleton based on silkworm endogenous, we constructed targeted-lef-11 interference vectors: pIZ-shRNA1, pIZ-shRNA2 and pIZ-DsRed-amiR279 and optimized these vectors via different promoters. [Results] First, comparing the interference efficiency of shRNA-based and miRNA-based RNAi vectors on the same site, we found the interference efficiency of pIZ-DsRed-amiR279 on target gene was more than 90%, better than that of RNAi shRNA-based and could be applied for follow-up experiments. Second, we evaluated the antiviral effect of different interference vectors driven by A4, IE1, IE1-295, IE2, IE2-339, hr3A4 and hsp70 promoters and found that the most active promoter had less interference effect on the target gene and IE1 was the optimal screening seven promoters, which demonstrated that the accumulation of pre-miRNA was not contribute to interference. [Conclusion] The results showed that the interference effect on the target gene depended on kinds of factors, such as the interference skeleton, the activity of promoter and the function of target gene.

Abstract:[Objective] To study the role of capsule polysaccharide in pathogenesis of extraintesinal pathogenic Escherichia coli (ExPEC). [Methods] By using λ Red recombination system, we generated the capsular polysaccharide transport associated genes kpsE and kpsD double knockout mutants E058ΔkpsED and U17ΔkpsED. We then compared and analyzed the characteristics of the mutant strains and wild-type strains. [Results] The growth curves in Luria Bertani showed that the deletion of kpsED did not affect growth kinetics of the mutants. The abilities of resistance to serum and killing by chicken macrophages were significantly impaired. LD₅₀ results showed that the double mutants completely abolished the virulence, whereas the complementation strains restored the virulence to resemble that of wild-type strains, and the colonization and coinfection model demonstrated that the deletion of kpsED led to attenuation of virulence, because the double mutant showed significantly decreased colonization compared with the wild-type strains in all organs tested in chickens. [Conclusion] These results indicated that the virulence factors encoded by capsule genes were important for the pathogenesis of ExPEC.

Abstract:[Objective] We studied the diversity of endophytic bacterial communities in different species of halophytes growing in the same saline habitat, and analyzed the effect of rhizosphere soil physicochemical properties on endophytic bacterial communities. [Methods] PCR-based Roche FLX 454 pyrosequencing was applied to reveal the diversity of endophytic bacteria. [Results] Endophytic bacterial communities of the 16 species of halophytes mainly included 4 phyla, which were Proteobacteria, Tenericutes, Actinobacteria and Firmicutes. In terms of plant species classification, colonial differences existed among plant species at perspectives of composition of bacterial taxa; in the case of plant genus level, endophytic bacteria of different halophyte plant species but belonging to same plant genus exhibited similarity; as to plant family level, Actinobacteria and Proteobacteria comprised the main abundant phyla of the halophytes belonging to Chenopodiaceae; Proteobacteria comprised the main abundant phyla of the halophytes belonging to Zygophyllaceae; Tenericutes comprised the main abundant phyla of the halophytes belonging to Tamaricaceae; Proteobacteria, Fimicutes and Actinobacteria comprised the main abundant phyla of the halophytes belonging to Plumbaginaceae. The Cl^- in rhizosphere soil has significant effect on endophytic bacterial community structure. Moreover, there is a strong correlation between bacterial community and the combination of Cl^-, Mg²⁺ and total nitrogen. [Conclusion] Halophytes harbors diverse endophytic bacteria. In the same saline habitat, the distribution of endophytic bacteria showed host plant species-specific, and the Cl^- in rhizosphere soil was one of the factors determined the endophytic bacterial community.

Abstract:[Objective] To construct a Corynebacterium glutamicum strain system for L-theanine production by the secretion expression of γ-glutamyltranspeptidase. [Methods] Two genes ggt and △sp ggt (without signal peptide fragment) from Bacillus subtilis were cloned and expressed in C. glutamicum. Then the recombinant GGT was used for L-theanine production under the optimal conditions: GGT enzyme 0.9 U/mL, pH 10, 37℃, and 20 mmol/L L-glutamine and 60 mmol/L ethylamine were fed every two hours. Supplementation was ceased after 12 h to minimize the substrates residue in the final broth. [Results] Firstly, two different recombinant C. glutamicum strains C. glutamicum SYPA5-5/pXMJ19-△sp ggt (without signal peptide), C. glutamicum SYPA5-5/pXMJ19-ggt (with signal peptide) were successfully constructed. In comparison to C. glutamicum SYPA5-5/pXMJ19-△sp ggt, C. glutamicum SYPA5-5/pXMJ19-ggt showed the ability to secret GGT into the medium and 5-fold higher enzyme activity than that in the former strain. This finding suggested that the signal peptide of GGT was responsible for the secretion and could work in C.glutamicum strain system. Furthermore, the glucose concentration and the adding time of inducer IPTG on GGT production were optimized in shake-flask. The batch transformation conditions were also investigated. The optimal ratio of L-glutamine to ethylamine was 1:3, and optimal enzyme amount was 0.06 U/mL. The highest L-theanine production reached at 104.36 mmol/L at 12 h with the conversion rate of 86.9%. [Conclusion] This is the first time to use the C. glutamicum system for the efficient synthesis of L-theanine. Furthermore, the signal peptide of GGT is identified to function well in C. glutamicum, providing a possible strategy for constructing a secretion expression system in C. glutamicum.

Abstract:[Objective] To study the effect of peroxisome proliferations (PPs) on the development and pathogenicity of rice blast fungus Magnaporthe oryzae. [Methods] The peroxisomal proliferation and the expression of peroxisomal biogenesis related genes were detected in M. oryzae strain Guy11 under the induction of 6 PPs. Vegetative growth, conidial germination, appressoria formation and pathogenicity of the strain treated with PPs were compared with those of the control. [Results] Induced by 6 PPs, the quantity of peroxisome and the expression of PEX14, PEX8 and PEX11 were significantly increased. Vegetative growth, conidial germination, appressorial formation and pathogenicity were inhibited by the majority of the PPs. Of them, 2, 4-D and aspirin (ASA) exhibited higher inhibition rates than others. Further, the inhibition of 2, 4-D and Aspirin to the vegetative growth of Δpex5 and Δpex7 mutants of M. oryzae was found significantly increased than that of the wild type strain. [Conclusion] PPs could induce peroxisome proliferation in M. oryzae, inhibit the growth and development and reduce the pathogenicity of the fungus. This is the first investigation on the effects of PPs to filamentous fungi.

Abstract:[Objective] The effects of repeated freeze-thaw cycles(F-Tc) on cell shape of fermented Bacillus subtilis and the inhibitory activity to Alternaria alternata, the primary infection pathogen of jujube fruit shrunken disease, were determined using an endophytic B. subtilis strain St-zn-34 isolated from winter jujube. [Methods] The fermented broth was freeze-thawed after batch fermentation of the test strain. The population of living bacteria and bacterial endospores were determined by dilution methods of plate counting, and the inhibitory activity to A. alternata was tested by filter paper disks with filtrate of fermented broth. The shape of B. subtilis with different freeze-thaw cycles was observed under environment scanning electron microscope. [Results] Changes of pH, living bacteria, bacterial endospore counts in the fermentation broth and the inhibitory activity of filtrate at different time generally increased first and decreased afterwards. The inhibitory activity at 60 h was higher than other time points. The bacterial cells fermented for 60 h were treated with F-Tc, and the living bacteria count and inhibitory activity decreased gradually when cells of B. subtilis were freeze-thawed for 3 cycles, but it had no significant difference (P>0.05) after being freeze-thawed for more than 3 times. With the increase of freeze-thawing times, the bacteria cells became smaller than the normal cells, the surface twisted with one or more depressions, and the jelly flowed out under environment scanning electron microscope. The inhibitory activity of filtrate was determined and it showed a broad-spectrum inhibitive capability against twelve species of plant pathogens. The treatment of different temperature and proteinase to the filtrate showed that the temperature below 60℃ did not affect the inhibitive activity and had no significant difference compared with that of the control. However, the inhibition decreased with increasing of the temperature above 80℃ and was significantly lower than that of control. The inhibitory activity of filtrate to A. alternata decreased by treatment with proteinase K. [Conclusion] Repeated freeze-thaw cycles could affect the shape of B. subtilis and reduced the inhibitory activity of filtrate to A. alternata.

Abstract:[Objective] An endoglucanase gene (gluE1) was cloned from a thermalacidophilus (Alicyclobacillus tengchongensis CGMCC1504) isolated from a hot spring, and the sequence and biochemical characterization of enzyme were analyzed. [Methods] The full-length gluE1 was obtained based on genome sequencing, analysis of amino acid sequence of GluE1. gluE1 was ligated into pEASY-E2 vector and expressed in Escherichia coli BL21 (DE3) cells. GluE1 was purified to electrophoretic homogeneity by Ni²⁺-NTA metal chelating affinity chromatography, and then the enzyme characterizations were determined. [Results] The 1020 bp full-length gluE1 (50.5% GC content) encodes a 339 residues polypeptide (GluE1: 40.45 kDa). GluE1 showed the highest identity of 97% with endoglucanase in public databases, and <60% identities with other endoglucanase. GluE1 efficiently hydrolyzed CMC-Na, soluble starch and barley-β-glucan, which showed apparent optimal at pH 6.5 and 55℃. GluE1 was stable and active (>60%) at pH 5.0-10.0, and had a high stability at 37℃; and it exhibited K_m, V_max and k_cat values of 8.58 mg/mL, 416.67 U/mg and 280.90 s^-1 respectively. GluE1 was strongly inhibited by Ag⁺, Hg²⁺ and SDS, partial promoted by β-Mercaptoethanol, Pb²⁺, Mg²⁺, Ca²⁺ and Na⁺, 30% NaCl still retains more than 64% of the activity. The residual enzyme activity kept 93% after pre-incubation of the enzyme in 30% NaCl. [Conclusion] Endoglucanase gene gluE1 from Alicyclobacillus was first reported, and GluE1 showed a good pH stability and strong halo-tolerant property. GluE1 might have greater potential applications.

Abstract:[Objective] This study aimed to characterize microorganisms responsible for accumulation of ethyl carbamate precursor citrulline in fermented grains during Luzhou-flavor spirits fermentation process, to provide theoretical basis for clarifying mechanisms of ethyl carbamate formation. [Methods] High-throughput strain screening approach was used to isolate strains with high arginine utilization and high citrulline accumulation capability. Essential genes that comprise the arginine deiminase pathway of these isolates were verified and a modified Chinese liquor fermentation process was conducted by adding these strains. [Results] A total of 20 strains with high arginine utilizing ability were obtained. Among them, Lactococcus garvieae LD3, Bacillus amyloliquefaciens BG5, Pediococcus acidilactici PH7 and Staphylococcus pasteuri SH11 exhibited high efficacy to produce citrulline from arginine. These strains could also increase citrulline in fermented grains. [Conclusion] The accumulation of citrulline in fermented grains was confirmed to be corresponding to arginine utilization by Lactococcus garvieae, Bacillus amyloliquefaciens, Pediococcus acidilactici and Staphylococcus pasteuri through the arginine deiminase pathway.

Abstract:[Objective] To realize efficient biosynthesis of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol, we designed a co-expression system containing Candida parapsilosis CCTCC M203011 (S)-carbonyl reductase Ⅱ (SCRⅡ) and Bacillus sp. YX-1 glucose dehydrogenase (GDH) in Escherichia coli BL21(DE3), based on the optimal ratio between the specific activities of the two enzymes. [Methods] The enzymes SCRⅡ and GDH were purified from their corresponding recombinant E. coli strains. When the purified SCRⅡ and GDH were used for the reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol, the optimal ratio between their specific activities, the optimal temperature and pH were determined. Based on above results, a co-expression system E. coli BL21(DE3)/S-SD-AS-G harboring SCRⅡ and GDH was constructed. [Results] SCRⅡ and GDH exhibited specific activities of 1.3 U/mg and 13.5 U/mg. When the total enzyme activity was 1 U, the optimal ratio of their activities is between 1:1 and 5:1, and the optimal temperature and pH are 30℃ and 7.0, respectively. So we designed a co-expression system E. coli BL21/S-SD-AS-G, in which the ratio of the SCRⅡ and GDH genes is 1:1. The specific activities of SCRⅡ and GDH are 0.76 U/mg and 0.73 U/mg in the cell-free extracts of E. coli BL21(DE3)/S-SD-AS-G, respectively. The ratio between SCRⅡ and GDH activity is 1:1. Under the optimal conditions, the system showed excellent performance to produce (S)-1-phenyl-1,2-ethanediol with an optical purity and a yield both over 99% during the reduction of 2-hydroxyacetophenone. With respect to the recombinant E. coli BL21(DE3)/pET-SCRⅡ, the co-expression system obviously improved the yield of (S)-1-phenyl-1,2-ethanediol and reduced biotransformation time from 24 h to 13 h. [Conclusion] This work provides the research foundation on the construction of a co-expression system containing a target chiral catalyst and a cofactor-regeneration enzyme for efficient chiral biosynthesis based on the optimal ratio of SCRⅡ and GDH activities.

Abstract:[Objective] To analyze the effect of high 2-keto-L-gulonic acid (2-KLG) on important dehydrogenase, cofactor and transport proteins involved in 2-KLG synthesis. [Methods] First, the growth of Gluconobacter oxydans under high 2-KLG was observed. The real-time PCR was used to detect the expression of key sorbitol dehydrogenase gene sldAB, pyrroloquinoline quinone (PQQ) biosynthesis gene cluster pqqABCDE, and five genes encoding hypothetic PQQ transport proteins. [Results] According to results of the growth of G. oxydans under different 2-KLG concentration, 40, 80 and 120 g/L 2-KLG were decided to stimulate strains. Real-time PCR showed that PQQ synthesis genes pqqABCDE were not affected by high 2-KLG, but sorbitol dehydrogenase genes sldAB and part of genes encoding PQQ transport proteins were down-regulated under high 2-KLG stress. [Conclusion] The expression of sorbitol dehydrogenase genes was restrained by high 2-KLG, PQQ transport was probably inhibited, but PQQ synthesis was not affected.