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He L.,State Key Laboratory of Agrobiotechnology | Zhou X.,State Key Laboratory of Agrobiotechnology | Yin X.,State Key Laboratory of Agrobiotechnology | Tian L.,State Key Laboratory of Agrobiotechnology | And 3 more authors.
DNA and Cell Biology | Year: 2015

Cholesterol is important for the growth and persistence of Mycobacterium tuberculosis in macrophages. The mce4 locus, which is conserved in both M. tuberculosis and Mycobacterium bovis, is thought to be responsible for cholesterol transport into the bacteria. However, the exact roles of specific genes within the sophisticated mce4 system remain poorly understood. In this study, Mce4A and Mce4E of M. bovis, two proteins that are encoded by the mce4 locus, were expressed in Mycobacterium smegmatis. The recombinant strain expressing the Mce4E protein (M. smeg::E) performed better than that expressing the Mce4A protein (M. smeg::A) in a minimal medium with and without glycerol or cholesterol, which may be the reason why M. smeg::E showed better survival in ANA-1 macrophages than did M. smeg::A. Cytokine expression profiles were similar in macrophages infected with either recombinant strain. We also investigated the role of CD36 in recognizing Mce4A and Mce4E proteins. However, CD36 did not appear to be specific for these proteins and showed little impact on the ultimate clearance of the recombinant strains. Reduced interleukin-1β, inducible nitric oxide synthase, and tumor necrosis factor-alpha mRNA expression at 6h postinfection in macrophages infected with M. smeg::E was observed using a CD36-specific monocular antibody to block the receptor, whereas no obvious changes in the expression of these cytokines were observed in cells infected with M. smeg::A with or without exposure to the CD36 antibody. Conclusively, the different performances of the recombinant strains suggest that the Mce4A and Mce4E proteins enhance mycobacterial adaptation to the harsh environment within macrophages after phagocytosis. © 2015, Mary Ann Liebert, Inc. Source


Chen K.,State Key Laboratory of Agrobiotechnology | Chen K.,Key Laboratory of Animal Epidemiology and Zoonosis | Chen K.,China Agricultural University | Luo Z.,State Key Laboratory of Agrobiotechnology | And 5 more authors.
Virology Journal | Year: 2011

Background: Chicken anemia virus (CAV) infection of newly hatched chickens induces generalized lymphoid atrophy and causes immunosuppressive. VP3, also known as Apoptin, is non-structural protein of CAV. Apoptin specifically induces apoptosis in transformed or tumor cells but not in normal cells. In particular, there are no known cellular homologues of Apoptin hindering genetic approaches to elucidate its cellular function. Although a number of Apoptin-interacting molecules have been identified, the molecular mechanism underlying Apoptin's action is still poorly understood. To learn more about the molecular mechanism of Apoptin's action, we searched for Apoptin associated proteins. Results: Using yeast two-hybrid and colony-life filter approaches we got five positive yeast clones. Through sequencing and BLASTed against NCBI, one of the clones was confirmed containing Gallus heat shock cognate protein 70 (Hsc70). Hsc70 gene was clone into pRK5-Flag plasmid, coimmunoprecipitation assay show both exogenous Hsc70 and endogenous Hsc70 can interact with Apoptin. Truncated Apoptin expression plasmids were made and coimmunoprecipitation were performed, the results show the binding domain of Apoptin with Hsc70 is located between amino acids 30-60. Truncated expression plasmids of Hsc70 were also constructed and coimmunoprecipitation were performed, the results show the peptide-binding and variable domains of Hsc70 are responsible for the binding to Apoptin. Confocal assays were performed and results show that under physiological condition Hsc70 is predominantly distributed in cytoplasm, whereas Hsc70 is translocated into the nuclei and colocalized with Apoptin in the presence of Apoptin in DF-1 cell. Functional studies show that Apoptin markedly down-regulate the mRNA level of RelA/p65 in DF-1 cell. To explore the effect of Hsc70 on Apoptin-mediated RelA/p65 gene expression, we have searched two Hsc70 RNAi sequences, and found that all of them dramatically inhibited the expression of endogenous Hsc70, with the #2 Hsc70 RNAi sequence being the most effective. Knockdown of Hsc70 show Apoptin-inhibited RelA/p65 expression was abolished. Our data demonstrate that Hsc70 is responsible for the down-regulation of Apoptin induced RelA/p65 gene expression. Conclusion: We identified Gallus Hsc70 as an Apoptin binding protein and showed the translocation of Hsc70 into the nuclei of DF-1 cells treated with Apoptin. Hsc70 regulates RelA/p65 gene expression induced by Apoptin. © 2011 Chen et al; licensee BioMed Central Ltd. Source


Jiao Y.S.,State Key Laboratory of Agrobiotechnology | Jiao Y.S.,China Agricultural University | Liu Y.H.,State Key Laboratory of Agrobiotechnology | Liu Y.H.,China Agricultural University | And 12 more authors.
Molecular Plant-Microbe Interactions | Year: 2015

In present study, we report our extensive survey on the diversity and biogeography of rhizobia associated with Sophora flavescens, a sophocarpidine (matrine)-containing medicinal legume. We additionally investigated the cross nodulation, infection pattern, light and electron microscopies of root nodule sections of S. flavescens infected by various rhizobia. Seventeen genospecies of rhizobia belonging to five genera with seven types of symbiotic nodC genes were found to nodulate S. flavescens in natural soils. In the cross-nodulation tests, most representative rhizobia in class a-Proteobacteria, whose host plants belong to different cross-nodulation groups, form effective indeterminate nodules, while representative rhizobia in class b-Proteobacteria form ineffective nodules on S. flavescens. Highly host-specific biovars of Rhizobium leguminosarum (bv. Trifolii and bv. viciae) and Rhizobium etli bv. phaseoli could establish symbioses with S. flavescens, providing further evidence that S. flavescens is an extremely promiscuous legume and it does not have strict selectivity on either the symbiotic genes or the species-determining housekeeping genes of rhizobia. Root-hair infection is found as the pattern that rhizobia have gained entry into the curled root hairs. Electron microscopies of ultra-Thin sections of S. flavescens root nodules formed by different rhizobia show that the bacteroids are regular or irregular rod shape and nonswollen types. Some bacteroids contain poly-b-hydroxybutyrate (PHB), while others do not, indicating the synthesis of PHB in bacteroids is rhizobia-dependent. The extremely promiscuous symbiosis between S. flavescens and different rhizobia provide us a basis for future studies aimed at understanding themolecular interactions of rhizobia and legumes. © 2015 The American Phytopathological Society. Source


Yan H.,State Key Laboratory of Agrobiotechnology | Yan H.,China Agricultural University | Ji Z.J.,State Key Laboratory of Agrobiotechnology | Ji Z.J.,China Agricultural University | And 10 more authors.
Systematic and Applied Microbiology | Year: 2016

With the increasing cultivation of medicinal legumes in agricultural fields, the rhizobia associated with these plants are facing new stresses, mainly from fertilization and irrigation. In this study, investigations on the nodulation of three cultivated medicinal legumes, Astragalus mongholicus, Astragalus membranaceus and Hedysarum polybotrys were performed. Bacterial isolates from root nodules of these legumes were subjected to genetic diversity and multilocus sequence analyses. In addition, the distribution of nodule bacteria related to soil factors and host plants was studied. A total 367 bacterial isolates were obtained and 13 genospecies were identified. The predominant microsymbionts were identified as Mesorhizobium septentrionale, Mesorhizobium temperatum, Mesorhizobium tianshanense, Mesorhizobium ciceri and Mesorhizobium muleiense. M. septentrionale was found in most root nodules especially from legumes grown in the barren soils (with low available nitrogen and low organic carbon contents), while M. temperatum was predominant in nodules where the plants were grown in the nitrogen-rich fields. A. mongholicus tended to be associated with M. septentrionale, M. temperatum and M. ciceri in different soils, while A. membranaceus and H. polybotrys tended to be associated with M. tianshanense and M. septentrionale, respectively. This study showed that soil fertility may be the main determinant for the distribution of rhizobia associated with these cultured legume plants. © 2016 Elsevier GmbH. Source


Jiao Y.S.,State Key Laboratory of Agrobiotechnology | Jiao Y.S.,China Agricultural University | Jiao Y.S.,MOA Key Laboratory of Soil Microbiology | Yan H.,State Key Laboratory of Agrobiotechnology | And 22 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2015

Five bacterial strains representing 45 isolates originated from root nodules of the medicinal legume Sophora flavescens were defined as two novel groups in the genus Rhizobium based on their phylogenetic relationships estimated from 16S rRNA genes and the housekeeping genes recA, glnII and atpD. These groups were distantly related to Rhizobium leguminosarum USDA 2370T (95.6% similarity for group I) and Rhizobium phaseoli ATCC 14482T (93.4% similarity for group II) in multilocus sequence analysis. In DNA–DNA hybridization experiments, the reference strains CCBAU 03386T (group I) and CCBAU 03470T (group II) showed levels of relatedness of 17.9–57.8 and 11.0–42.9%, respectively, with the type strains of related species. Both strains CCBAU 03386T and CCBAU 03470T contained ubiquinone 10 (Q-10) as the major respiratory quinone and possessed 16: 0, 18:0, 19:0 cyclo v8c, summed feature 8 and summed feature 2 as major fatty acids, but did not contain 20:3 v6,8,12c. Phenotypic features distinguishing both groups from all closely related species of the genus Rhizobium were found. Therefore, two novel species, Rhizobium sophorae sp. nov. for group I (type strain CCBAU 03386T5E5T5LMG 27901T5HAMBI 3615T) and Rhizobium sophoriradicis sp. nov. for group II (type strain CCBAU 03470T5C-5-1T5LMG 27898T5HAMBI 3510T), are proposed. Both groups were able to nodulate Phaseolus vulgaris and their hosts of origin (Sophora flavescens) effectively and their nodulation gene nodC was phylogenetically located in the symbiovar phaseoli. ©2015 IUMS. Source

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