Abstract:Nearly all bacteria require iron for growth and survival. In aerobic environment, ferric iron almost cannot be utilized directly by bacteria. But ferrous iron is mainly existing in the host gastrointestinal. Soluble ferrous iron [Fe(II)] is imported directly via membrane transporters into periplasmic, then ferrous iron is imported via ferrous iron transport systems into cytoplasm. Most of gram-negative bacteria uptake ferrous iron by Feo system. The Feo transport system in Escherichia coli consists of the feoA, feoB, and feoC genes. In addition to the Feo transport system, the Yfe transport system and the Efe transport system and the Sit transport system are involved in the transport of ferrous iron. In this review, we described the component and functional mechanism of Feo system in gram-negative bacteria, to provide reference for studying other bacteria ferrous iron transport mechanism.

Abstract:Recent studies have found that the matrix protein of paramyxoviruses is a multifunctional viral protein. In addition to inhibiting the transcription and translation of cell genes, regulating the replication and transcription of viral genome and recruiting cellular proteins to facilitate viral assembly and budding, the matrix protein can enhance the replication of paramyxoviruses through its ubiquitination and phosphorylation. However, as a member of paramyxoviruses, the matrix protein of Newcastle disease virus (NDV) is only demonstrated to participate in viral assembly and budding. Moreover, the functions of matrix protein identified in other paramyxoviruses still remain unknown in NDV. This review compares the functions of matrix protein between NDV and other paramyxoviruses, and focuses on the relationship of matrix protein to the virulence, replication and pathogenicity of NDV. Meanwhile, challenges and research prospects of NDV matrix protein are also discussed.

Abstract:[Objective] Based on different potato virus Y isolates gene sequencing, we studied the diversity of potato virus Y strains, to provide information for molecular detection, prevention and control of the virus. [Methods] P1 gene of 15 samples of potato virus Y of Heilongjiang Province was cloned and then the sequences of genes were analyzed by using phylogenetic tree. [Results] Samples were divided into two groups. According to a comparative analysis, 10 samples have highly conservative and homologous genes. They are the dominant population in the research area and have certain genetic distance to other domestic samples and foreign samples. In another group, 5 samples differ significantly with local dominant population in term of P1 gene. These 5 samples also have some differences and their P1 genes are close to those of other domestic samples and foreign samples. By comparing PVY strain data provided by uploaded sequences in GenBank, it found that P1 gene of test samples is similar with PVY^NTN-NW strains. These 15 samples as well as other domestic samples are evolved from PVY^N strains. [Conclusions] The P1 gene analysis demonstrated that PVY is influenced by environment significantly and PVY of 10 samples in Heilongjiang develops local characteristics in the long-term evolution. The later 5 samples reflect that most PVY in China may be introduced by foreign cultivars. At the same time, PVY spreads through regional resource exchange and tuber transportation in China.

Abstract:[Objective] We isolated bacterial strains with chitin-degrading activity from the digesta of large yellow croakers (Pseudosciaena crocea) fed with chitin-enriched trash fish, and characterized potential chitinases thereof. [Methods] Chitin-degrading strains were screened with colloidal chitin agar from the digesta of P. crocea fed with trash fish. The chitinase gene (chi-X) was cloned and expressed in Escherichia coli, and the enzymatic properties of the chitinase (CHI-X) were characterized. [Results] A Citrobacter freundii strain with chitin-degrading activity was isolated. The chitinase gene encodes a protein containing 493 amino acid residues, with a proposed glycoside hydrolase family-18 catalytic domain. CHI-X could hydrolyze colloidal chitin. The optimal pH for CHI-X was 4.0 at optimal temperature (60 ℃). CHI-X was active over a broad pH range, with around 90% of the activity maintained after incubation at pH between 3.0 and 11 for 1 h. The enzymatic activity of CHI-X was stimulated by Mn²⁺, Li⁺, and K⁺, but inhibited by Ag⁺. The enzyme was stable after treatment by proteases and grouper intestinal juice. CHI-X hydrolyzes colloidal chitin into GlcNAc and (GlcNAc)₂. Furthermore, an synergic effect was observed between CHIX and ChiB565 (a chitinase from Aeromonas veronii B565) on colloidal chitin. [Conclusion] CHI-X from intestinal bacterium may be potentially used as feed additive enzyme for warm water marine fish.

Abstract:[Objective] To simplify the PNA-FISH (Peptide nucleic acid-fluorescence in situ hybridization) test, molecular beacon based PNA probe combined with fluorescence scanning detection technology was applied to replace the original microscope observation to detect Listeria monocytogenes. [Methods] The 5′ end and 3′ end of the L. monocytogenes specific PNA probes were labeled with the fluorescent group and the quenching group respectively, to form a molecular beacon based PNA probe. [Results] When PNA probe used for fluorescence scanning and N1 treatment as the control, the false positive rate was 11.4%, and the false negative rate was 0; when N2 treatment as the control, the false positive rate decreased to 4.3%, but the false negative rate rose to 18.6%. When beacon based PNA probe used for fluorescence scanning, taken N1 treatment as blank control, the false positive rate was 8.6%, and the false negative rate was 1.4%; taken N2 treatment as blank control, the false positive rate was 5.7%, and the false negative rate was 1.4%. Compared with PNA probe, molecular beacon based PNA probe can effectively reduce false positives and false negatives. The success rates of hybridization of the two PNA probes were 83.3% and 95.2% respectively; and the rates of the two beacon based PNA probes were 91.7% and 90.5% respectively, which indicated that labeling the both ends of the PNA probe dose not decrease the hybridization rate with the target bacteria. [Conclusions] The combination of liquid phase PNA-FISH and fluorescence scanning method, can significantly improve the detection efficiency.

Abstract:[Objective] The aim of this study is to investigate the effects of AMP metabolism on the physiological function of Torulopsis glabrata. [Methods] Strain cgade12Δade13Δ was constructed by deleting cgade12 and cgade13 with homologous recombination, and was used to study the effects of AMP metabolism on carbon metabolism by comparing the ATP levels, enzymes activity and inter-metabolite concentrations of carbon metabolism to that of ATCC55. And the effects of AMP on metabolisms on organic acid tolerance were studied by compared the cell growth and intracellular environment of cgade12Δade13Δ to that of ATCC55 under organic acid stress. [Results] The ATP levels of mutant cgade12Δade13Δ was decreased by 12.50% when compared with that of strain ATCC55. The enzymes activity of citrate synthetase, malate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase was increased by 31.26%, 19.45%, 28.96%, 18.36% and the intracellular citric acid, α-ketoglutarate, malic acid, succinic acid contents were increased by 44.11%, 73.60%, 50.00%, 65.68%, respectively, compared with the corresponding value of strain ATCC55. However, the intracellular concentration of pyruvic acid in mutant cgade12Δade13Δ was decreased by 20.00% which led to a 73.11% reduction of pyruvic production in fermentation broth. Compared with strain ATCC55, the cell concentrations of cgade12Δade13Δ were increased by 8.71%, 11.21% and 12.71% grown in YNB with 0.4% pyruvic, 0.6% malic acid and 0.2% acetic acid, respectively. Grown in YNB with 0.2% acetic acid the H⁺-ATPase activity, cell membrane integrity, cell membrane electric potential of mutant cgade12Δade13Δ was increased by 7.04%, 8.71%, 25.14% than that of strain ATCC55, respectively, while the ROS concentration was decreased by 19.51%. [Conclusion] The deletion of genes cgade12 and cgade13 resulted in a reduction in ATP level but led to an increase in activity of TCA cycle and organic acid tolerance.

Abstract:[Objective] Microbes-induced mineralization is one of the hottest issues in the field of geomicrobiology. Strain DHS C013^T isolated from the surfaces of rocks in the Karst region was used to investigate microbial influence on the formation of carbonate and its morphology in the metallogenic system consisting NaHCO₃ and Ca(NO₃)₂·4H₂O. [Methods] Strain DHS C013^T was inoculated into malt extract-glucose-yeast extract peptone (MGYP) liquid medium. After cultivation we put the fermented solution, supernatant, hypha pellets, sterile MGYP liquid medium and ultrapure water into the metallogenic system separately. Scanning electronic microscope was applied to observe the crystals at the bottom of the petri dishes. [Results] In the metallogenic system with ultrapure water, only standard calcite of rhombohedron was found. However, special morphology of CaCO₃, such as dumbbelllike, spherulite and scaly cylindrical shapes, were found in the metallogenic system with actinomycetes, hyphae fragment and their cell metabolism products. These calcium carbonates of special morphology might be resulted from their nucleation on smaller hypha pellets, hyphae fragment or extracellular secretion. [Conclusion] Actinomycetes can induce the formation of CaCO₃, and the mycelium and metabolites have important effects on regulating and influencing CaCO₃ morphology. Our data provide new evidence for further understanding of the biological mineralization mediated by actinomycete and its metabolic products.

Abstract:[Objective] To study the role of Snf1/AMPK kinase in cell wall integrity in Saccharomyces cerevisiae JY102. [Methods] SNF1 deletion mutant of S. cerevisiae was created by homologous recombination. Congo red and Calcofluor white were applied to evaluate the cell wall integrity for the mutant strain. qRT-PCR was used to analyze the transcription of cell wall-related genes. [Results] The SNF1 disruption mutant was severely sensitive to Congo red and Calcofluor white, manifesting the impairment of cell wall integrity. The mutant exhibited apparently growth defect at high temperature. The results of qRT-PCR revealed down-regulated transcription of several genes involved in β-glucan biosynthesis. [Conclusion] The deletion of Snf1 impairs the cell wall integrity by reducing the transcription of β-glucan-related genes, suggesting a new role of Snf1 in the activation of cell wall synthesis.

Abstract:[Objective] To develop a new method for efficient expression and rapid preparation of biologically active anthrax edema factor (EF). [Methods] EF was fused with GST and expressed in the host E. coli BL21-CodonPlus (DE3)-RIL by IPTG induction. The crud protein was extracted by permeabilization, and then EF was purified by onestep affinity chromatography. cAMP assay, Native-PAGE and competitive inhibition analysis were carried out to evaluate EF's biological activity. [Results] EF was expressed in soluble form and then purified to 96% purity by single-step. The recombinant EF was able to bind furin-nicked protective antigen (PA) to form edema toxin, which could elevate the intracellular cAMP level of CHO-K1 cells dramatically. [Conclusion] This work provides a timesaving method for purification of EF with high purity and good biological activity, which might be valuable for anthrax-related study.

Abstract:[Objective] The aim of this study is to explore the changes of sensitivity to tetracyclines and the resistance mechanism of Aeromonas hydrophila (Ah) after in vitro induction by exposure Ah to the increasing concentration of Doxycycline (DO). [Methods] The sensitive Ah isolates were first screened from clinical isolates and then incubated on TSA solid medium containing DO of 1/4×MIC value. The resistant strains were obtained through continuous subculture by increasing the concentration of DO in the medium for geometric series. The minimal inhibitory concentrations (MICs) of induced strains to DO and 16 other non-selected antimicrobials were determined. Meanwhile, MICs were also determined after adding efflux pump inhibitor 1-methyl-2-pyrrolidinone (NMP) to the medium. The relationship between sensitivity changes and efflux function were analyzed. Then five genes of tet from induced strains were amplified and sequenced. [Results] The MICs of induced strains to DO increased significantly after induction, whereas the MICs of strains to those non-selected tetracyclines also increased. The MICs of induced strains to fluoroquinolone increased much more than that of control. The induced strains exhibited a little higher sensitivity to aminoglycoside and rifampicin. However, the MICs of all induced strains to DO decreased after adding NMP to the medium. The detection of tet genes indicated that tetA and tetE were positive in No. 7 after induction and the tetC gene was positive in No. 2 before and after induction. The tetE gene was detected in strains No. 1, 3, 4, 5, 6 and 7 no matter whether it was induced or not. [Conclusion] This study suggested that tetE gene may be the predominant gene mediating tetracyclines-resistance of Ah, which would provide theoretical basis to clarify the resistance mechanism of Ah to tetracyclines.

Abstract:[Objective] Biopesticides are safe and environment friendly. We evaluated the biocontrol effect of Pythium oligandrum broth (POB) and its toxicity to animals and plant growth. [Methods] Animal, antagonist, pot, and field experiments with mice, Mycosphaerella melonis, and cucumber seedlings were carried out to study animal toxicity, control of gummy stem blight, plant growth, fruit yield and quality with POB produced from self-isolated P. oligandrum CQ2010. [Results] Mouse showed normal weight, appearances, performances and no pathogenic changes in organs and tissues with a large amount of POB supplied by lavage. The inhibition rate of POB against M. melonis was 51.95%, similar to thiophanate methy (800 times dilution) but much higher than chlorothalonil (200 times dilution). Malondialdehyde concentration was reduced whereas activities of peroxidase and superoxide dismutase were stimulated in seedling leaves irrespective of POB supplied before and after pathogenic inoculation. POB also decreased the pathogenic incidence and disease index with relative control efficacy from 54.8% to 64.1%. Thus, POB could alleviate cell membrane damage caused by pathogenic microbes, stimulate physiological reactions related to disease defense, and increase disease-resistant abilities of plants. Moreover, POB increased chlorophyll content, root activity, and uptake of nitrogen, phosphorus and potassium, resulting in growth acceleration, fruit yield increment, and quality improvement. [Conclusion] POB is safe to animals and could control gummy stem blight of cucumber seedlings, promote plant growth, increase fruit yield, and improve the qualities.

Abstract:[Objective] In order to improve biofuel production by Desmodesmus insignis, we studied the effect of different nitrogen sources and concentrations on the growth, total lipids, carbohydrate and starch accumulation of Desmodesmus insignis. [Methods] D. insignis was cultivated in basic general-11 medium containing 5 different initial nitrogen concentrations (3, 6, 9, 12 and 18 mmol/L) supplied in the form of sodium nitrate, ammonium bicarbonate and urea. Biomass was determined by dry weight, total lipids by gravimeter, and carbohydrates and starch by phenolsulfuric acid method. [Results] NaNO₃, NH₄HCO₃ and CO(NH₂)₂ were all suitable for the growth of D. Insignis. When 3 mmol/L NaNO₃ was used, the peak lipid content reached to 32.61% (d.w). The maximum content and productivity of carbohydrate and starch were 56.54% (d·w), 55.33% (d·w) and 0.24 g/(L·d), 0.23 g/(L·d) respectively, when 18 mmol/L NH₄HCO₃ was used. Urea could also obtain relative high content of biomass, total lipids, carbohydrates and starch. [Conclusion] Integrating these results with production costs, we could suggest ammonium bicarbonate and urea as nitrogen source at the concentration of 18 mmol/L.

Abstract:[Objective] This study is aimed to establish an in vitro pull down method combining Avi-tag technology to screen and indentify the CodY targets in the whole genomes of Bacillus thuringiensis strain YBT-1520. The method offers a potential biocatalyst forscreening target sequences of transcription factors in vitro. [Methods] CodY proteins were labeled with biotin using Avi-tag technology. B. thuringiensis strain YBT-1520 genomes were enzymatically digested to 100-400 bp DNA fragments that had adapters with known sequences on both ends. Then, the target genes of CodY protein were screened by streptavidin biotin resin. [Results] The 46 targets of CodY were mainly involved in amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, transport, regulation, DNA replication repair, and two component regulatory systems. [Conclusion] Compared with chromatin immune co-precipitation technology in vivo, this pull down method is simple, cheap, fast and specific to biotin streptavidin system.

Abstract:[Objective] In order to obtain piericidin intermediates of low toxicity by metabolic engineering, we studied the function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1. [Methods] The methyltransferase pieB2 gene disrupted Streptomyces piomogeues var. Hangzhouwanensis was constructedby double crossover recombination. The methyltransferase gene pieB2 was also PCR amplified and cloned into the plasmid pET28a for overexpressing N-(His)₆-tag PieB2 in E. coli BL21(DE3)/pLysE. The recombinant PieB2 was purified by affinity chromatography via AKTA FPLC system. The PieB2 catalyzed reactions were performed using SAM and demethyl-piericidinas substrates. [Results] The disruption mutant LQ-9 produced demethyl-piericidininstead of piereicidin A1, which was restored by in trans complementation of the pieB2 gene. The N-(His)6-tag PieB2 was expressed in E. coli in soluble form and was successfully purified via Ni²⁺ mediated affinity chromatography. In vitro biochemical experiments showed that PieB2 could convert demethyl-piericidininto piereicidin A1 in the presence of SAM. The demethyl-piericidin intermediat showed an attractive biological activities as well as piericidin A1. [Conclusion] We confirmed that PieB2 is function as a SAM dependent methyltransferase in the biosynthetic gene cluster of piericidin A1.

Abstract:[Objective] The fluorescent protein and gD envelope protein of equine herpes virus type 1 (EHV-1) were used to study the impact of tags on gD protein subcellular localization in BHK-21 cells. [Methods] With the EHV-1 genome as a template, the gD complete gene was amplified by PCR technique. The product of PCR was cloned to pAcGFP1-C1 and pDsRed2-N1 plasmids. The recombinant plasmids were designated as pAc-GFP-gD (GFP-gD) and pDs-gD-Red (gD-Red). The GFP gene was inserted into the posterior position of gD gene signal peptide sequence. The modified gD gene signal peptide sequence was cloned to pVAX-1 plasmid, so that pVAX-S-GFP-gD' (S-GFPgD') recombinant plasmid was constructed. Meanwhile, the flag tag was added to N-terminal of gD sequence and they were cloned to pVAX-1 expression vector for constructing pVAX-Flag-gD recombinant plasmid. The BHK-21 cells were transfected with the 4 different recombinant plasmids and the subcellular localizations of fusion proteins were determined by lasar confocal scan microscopy. [Results] Four eukaryotic expression vectors were constructed successfully. In BHK-21 cells, the vast majority of gD envelope proteins was localized in Golgi, and a small amount of gD was localized in the nucleus. [Conclusion] Our finding reveals that the fluorescent protein of different insertion sites has no significant effects on the subcellular localization of gD, and provides a useful reference for other researchers.

Abstract:[Objective] We studied the effects of a plant fermentation extract on destroying biofilms of P. aeruginosa, to provide basic information for treatment of P. aeruginosa related infection diseases. [Methods] Strains of P. aeruginosa in clinical specimens were isolated by streaking plate method and identified by PCR and sequencing. Virulence factors were examined using reporter strains, and biofilms were measured by test tube method and a confocal laser scanning microscopy. [Results] A total of 16 strains of P. aeruginosa were isolated from clinical specimens from a local hospital, among them PA007 strain showed a maximum response when treated with plant fermentation extract. It shows that 1% plant fermentation extract significantly reduced the production of biofilm, pyocyanine and 3-oxo-C12-HSL (P<0.05). Besides, 1% plant fermentation extract also deceased the bioactivity of LasA protease and survival rate of persisters (P<0.05). The qRT-PCR result indicated that the expressions of lasI and pqsA genes were also markedly inhibited at the presence of 1% plant fermentation extract (P<0.05). [Conclusion] The studied plant fermentation extract has anti-infection effect against some P. aeruginosa strains, suggesting a great potential to work as natural antibiotics.