Abstract:Abstract:We summarized proposals submitted and funded in the discipline of microbiology of the Department of Life Sciences of National Natural Science Foundation of China in 2016. The traits and concerns in different sub-disciplines as well as distinctive funding programs were addressed, and the prior funding fields were prospected. The information may provide references for researchers who apply funding at the discipline of microbiology.

Abstract:Abstract:Proteins are not only the main building blocks for the construction of tissue, but also crucial for metabolic activity in animals. The microbial community colonized in the gastrointestinal tract plays an important role in host nutrients metabolism, especially nitrogen nutrients. Bacteria in small intestine could metabolize parts of amino acids (AAs), which further affects the systemic AAs metabolism of host. Compared with that in small intestine, the density of bacteria is much higher and the retention time of chyme is much longer in large intestine. On the one hand, the metabolism and community structure of microflora could be affected by nitrogen nutrients entering the large intestine. Thus, the metabolism of nitrogen nutrients by large intestinal microflora can lead to the formation of several metabolites, which are generally presumed to be detrimental for the host health. This review summarized the effects of dietary protein on the community structure of large intestinal microflora, the microbial metabolites of AAs in large intestine and their impacts on intestinal physiology and host health.

Abstract:Abstract:Bacteria get a survival advantages in the complex environment through the chemotaxis system. Chemotaxis plays an important role in colonization and pathogenicity of bacteria, the legume–rhizobia symbiosis, and plant-microbe interactions. Phosphatase CheZ is an indispensable regulatory protein in the process of chemotactic signal adaptation. This review focused on the structure, mechanism of action, functional regulation, protein targeting and the status of the evolutionary of CheZ. This work can benefit the study of other bacterial chemotaxis systems.

Abstract:Abstract: Seeds with high oil contents are more susceptible to aflatoxin contamination after infected by Aspergillus species. However, in vitro studies showed that different types of fatty acids have striking difference on fungal growth, sporulation and aflatoxin biosynthesis in Aspergillus. Recent studies revealed that, although all fatty acids examined promote aflatoxin production, oxidized polyunsaturated fatty acids inhibit aflatoxin biosynthesis. The inhibiting effect is derived from oxylipins produced during autoxidation. In this article, we provide an overview for recent progress in fatty acids and oxylipins on fungal growth, sporulation and aflatoxin production in Aspergillus species.

Abstract:Abstract: [Objective] The objective of this research was to study plant cell wall degradation enzymes from Fusarium sp. Q7-31T. [Methods] Strain was cultured in liquid medium with 1% (W/V) peptone as nitrogen source, 0.5% (W/V) oat straw as carbon source, 120 r/min shaking at 20 °C for 3 days. The endoglucanase Egn21 was purified by using Sephacry S-100 chromatography and DEAE-sepharose ion-exchange column chromatography. Then the enzymatic properties and MADIL-TOF-TOF identification were analyzed. [Results] The molecular weight and isoelectric point (pI) of Egn21 was 44.25 kDa and 4.91, respectively. Egn21 had optimal activity with carboxymethyl cellulose at 40 °C and pH 6.0, stable at 45 °C and pH between 5.0 and 8.0, inhibited by Fe2+, Ca2+, K+, Na+, Mn2+ and inactivated by Hg2+, whereas Co2+, Zn2+ and Mg2+ had no effect. [Conclusion] The enzymatic properties and MADIL-TOF-TOF results suggested that Egn21 belongs to GH5 family.

Abstract:Abstract: [Objective] Hydrocarbon microseepage is a natural phenomenon that hydrocarbon gases of subsurface petroleum accumulations migrate upward by reservoir pressure. The detection of the activity and distribution of these highly specialized populations can be used to forecast the existence of oil and gas deposits. However, the hydrocarbon-oxidizing bacterial population are usually not predominant in soil samples above the typical onshore oil and gas reservoirs. It is hard to assess the abundance of hydrocarbon-oxidizing bacteria. [Methods] In this study, changes of microbial abundance and functional genes were studied. [Results] Under gaseous hydrocarbon condition, changes of methane and butane oxidizing bacteria were different. Furthermore, changes of functional genes indicated that genome analysis was more proper for microbial anomalies detection. [Conclusion] The profiling data of this study provide a comprehensive insight into gene expression profiles and lay the foundation for optimizing the microbial prospecting technology.

Abstract:Abstract: [Objective] We aimed at co-expressing heterologous D-hydantoinase and N-carbamoylase in Bacillus subtilis, and evaluating the feasibility of producing D-p-hydroxyphenylglycine by the recombinant B. subtilis whole-cell catalysis. [Methods] The Paco expression cassette was combined with the coding sequence of hyd or sd1 gene as an artificial gene to express D-hydantoinase. The PAE expression cassette was combined with the coding sequence of adc gene as an artificial gene to express N-carbamoylase. The D-hydantoinase and N-carbamoylase co-expression plasmids pHCS(sd1+adc) and pHCY(hyd+adc) were constructed, using plasmid pHP13 as carrier; the co-expression plasmids pUCS(sd1+adc) was constructed, using plasmid pUB110 as carrier. The additional copy of acoR and sigL gene was integrated at chromosome. The skf and sdp gene were knocked out in B. subtilis. All recombinant strains bearing co-expression plasmid were characterized by analyzing whole-cell catalysis activity. [Results] In the recombinant strains with plasmid pHCY and with pHCS, the whole-cell catalytic activity reached 0.21 U/mL and 0.31 U/mL, respectively. After the over-expression of acoR, sigL, and high-copy-number pUCS, the whole-cell catalytic activity reached 1.0 U/mL. [Conclusion] Overexpression of acoR, sigL and the deletion of skf, sdp genes had significant effects on the catalysis activity of recombinant whole-cell.

Abstract:Abstract: [Objective] Regulators of G-protein signaling (RGS) are negative regulatory factors of G protein and play important roles in growth development and pathogenicity of plant pathogen. However, biological functions of RGS in Colletotrichum gloeosporioides have not been studied so far. We cloned an RGS gene of CgRGS2 in C. gloeosporioides and analyzed its biological function. [Methods] Gene CgRGS2 was cloned using PCR and analyzed. The gene-knockout mutant of CgRGS2 was obtained by homologous recombination, and the complementary strain was also built based on the mutant. Biological function of CgRGS2 was determined through phenotypic analysis. [Results] CgRGS2 encoded a 574-amino acids protein, containing an RGS function domain in the N terminal. Comparing to the wild type, the knockout mutant of CgRGS2 had slow growth, thick aerial hyphae, reduced conidia with multi-end germination, sensitive to oxidative stress and SDS, decreased pathogenicity. [Conclusion] Protein CgRGS2 was involved in regulation of vegetative growth, conidium production and germination, oxidative stress response, cell wall integrity and pathogenicity of C. gloeosporioides.

Abstract:Abstract: [Objective] We studied the functions and characteristics of hfq gene in Mesorhizobium huakuii 7653R in adverse environment and symbiotic with its host plant. [Methods] The hfq mutant of 7653R was constructed via homologous recombination with small cloned fragments on suicide plasmids pK19mob to insert target gene. We applied 7653RΔhfq to characterize stress tolerance and symbiosis with host plant, in comparison with the complementary strains 7653R △hfq-C and the wild type. [Results] Mutant 7653RΔhfq presented lower growth rate, and higher mortality after heat shock-pretreated than that of the wild type, as well as the decreasing adaptability under the stress of 4.5% ethanol and 50 mmol H2O2. The defection of hfq affected the expression of some sRNAs in 7653R. Moreover, the mutant displayed significant reduced nodulation ability and nitrogenase activity compared with the wild type. [Conclusion] As a crucial post transcriptional regulatory factor, hfq plays an important role in Mesorhizobium Huakuii 7653R on both processes of stress resistance and symbiosis with the host plant Astragalus sinicus L.

Abstract:Abstract: [Objective] In this study, we constructed recombinant Kluyveromyces lactis strains to produce phospholipase C (PLC) of Bacillus cereus. The recombinant enzymes were purified and characterized. [Methods] We cloned the PLC encoding gene bcplc of Bacillus cereus. And the amplified fragments were inserted into pKLAC1 to obtain expression plasmids. K. lactis harboring the above plasmids was cultivated to express PLC that was purified by HisTrapTM affinity chromatography and characterized. [Results] PLC of B. cereus was cloned and expressed in K. lactis. The recombinant enzyme had shown activity of 19251 U/mg when using p-nitrophenyl phosphorycholine as substrate. Purified PLC exhibited optimum temperature at 80 °C and optimal pH at 9.0. The recombinant enzyme was stable below 40 °C and pH between 7.0 and 8.0. Cu2+ and Co2+ inhibited its activity whereas Zn2+, Mn2+, Ca2+ and Mg2+ stimulated its activity. [Conclusion] It is the first time to express and characterize the PLC gene in K. lactis. These research results provide reference for the study of recombinant PLC.

Abstract:Abstract: [Objective] To explore the genome sequence of Aureobasidium pullulans CCTCC M2012223, analyze the key genes related to the biosynthesis of important metabolites, and provide genetic background for metabolic engineering. [Methods] Complete genome of A. pullulans CCTCC M2012223 was sequenced by Illumina HiSeq high throughput sequencing platform. Then, fragment assembly, gene prediction, functional annotation, and GO/COG cluster were analyzed in comparison with those of other five A. pullulans varieties. [Results] The complete genome sequence of A. pullulans CCTCC M2012223 was 30756831 bp with an average GC content of 47.49%, and 9452 genes were successfully predicted. Genome-wide analysis showed that A. pullulans CCTCC M2012223 had the biggest genome assembly size. Protein sequences involved in the pullulan and polymalic acid pathway were highly conservative in all of six A. pullulans varieties. Although both A. pullulans CCTCC M2012223 and A. pullulans var. melanogenum have a close affinity, some point mutation and inserts were occurred in protein sequences involved in melanin biosynthesis. [Conclusion] Genome information of A. pullulans CCTCC M2012223 was annotated and genes involved in melanin, pullulan and polymalic acid pathway were compared, which would provide a theoretical basis for genetic modification of metabolic pathway in A. pullulans.

Abstract:Abstract: [Objective] The aim of this study was to identify the transport proteins that mediates the nuclear import of Newcastle disease virus (NDV) matrix (M) protein. [Methods] Chicken KPNA1 to KPNA6 gene and KPNB1 gene were cloned from DF-1 cells and then inserted into eukaryotic expression vectors. The constructed recombinant plasmids with a combination of grouping were transfected into HEK-293T cells to identify the transport proteins interacting with NDV M protein by co-immunoprecipitation (Co-IP) assay. Moreover, fluorescent co-localization assay was used to verify the transport proteins by co-expressing M and Ran protein mutant or M and its interactive protein deletant. [Results] The recombinant proteins could normally express in plasmid-transfected HEK-293T cells. Indirect immunofluorescence detection showed that the recombinant proteins except for Myc-KPNA2 displayed the same nuclear localization as NDV M protein. The results of Co-IP revealed that M protein could interact with KPNA1 and KPNB1. Further fluorescent co-localization indicated that co-expression of M and DN-KPNA1 did not change the nuclear localization of M, whereas co-expression of M and DN-KPNB1 or M and Ran-Q69L disrupted the nuclear localization of M, demonstrating that the nuclear import of M protein was dependent on KPNB1 and Ran protein. [Conclusion] KPNB1 and Ran protein jointly mediated the nuclear import of NDV M protein, showing that KPNB1 protein interacted with NDV M protein to form binary complex and then entered into the nucleus with the assistance of Ran protein.

Abstract:Abstract: [Objective] To study the regulation of sporulation controlled by two-component system (TCS) YvcPQ. [Methods] β-galactosidase experiment was used to verify the regulation of YvcP on kapD expression; bacterial one-hybrid assay, EMSA and RT-qPCR were applied to study the regulation of AbrB on yvcPQ expression; markerless gene deletion coupled with spore count was used to reveal the influence of yvcPQ and kapD expressions on sporulation. [Results] The transcriptional regulator AbrB up-regulated the expression of yvcPQ; YvcP promoted the expression of kapD to inhibit sporulation. [Conclusion] AbrB up-regulated the transcription of yvcPQ operon, then the increased YvcP strengthened the transcriptional acitivation of sporulation inhibitor gene kapD, and subsequently inhibited sporulation.

Abstract:Abstract: [Objective] To provide scientific data for studying the ecology of cyanophage, we studied the genetic diversity of psbA of cyanophage from paddy floodwater in northeast China and its phylogenetic positions. [Methods] Membrane separation and concentration of cyanophage, PCR-cloning-sequencing were applied to study the diversity of psbA of cyanophage from paddy floodwater in northeast China. [Results] In total 17 psbA sequences of cyanophage were obtained. Novel cyanophages were found by phylogenetic analysis. Compared to those of Japanese paddy floodwater, marine and lakes, psbA gene assemblage of paddy floodwater in northeast China was significantly different. [Conclusion] This is the first report to study genetic diversity of cyanophage from paddy floodwater in northeast China with a molecular marker of psbA by PCR-cloning-sequencing. The novel psbA assembly of cyanophage was found in paddy floodwater in northeast China.

Abstract:Abstract: [Objective] The complete genome of the extreme environmental resistant bacterium Deiococcus radiodurans R1 was analyzed by sequence comparative method and putative ferritin-like protein DRA0258 was screened. Molecular techniques were applied to validate and analyze its function. [Methods] We applied sequence alignment to analyze amino acid sequence of the hypothetical protein DRA0258 and detected its iron binding activity after purification. We used triple-fraction-ligation method to construct dra0258 null mutant and detected its survival rate under H2O2 treatment, catalase activity and total antioxidant capacity, using QRT-PCR to examine the relative transcriptional level change of the antioxidant relative enzymes and iron transport relative proteins. [Results] We confirmed DRA0258 obtained a certain iron binding activity. The survival rate assay with H2O2 treatment suggested that deletion of dra0258 reduced the cellular antioxidant activity of D. radiodurans. The attenuation of catalase activity, total antioxidant capacity as well as the reduction of relative transcriptional levels of antioxidant related genes verified that both the oxidative stress response systems and the iron regulation network were damaged. [Conclusion] This study verified DRA0258 is an iron-binding protein. Deletion of this gene would affect cellular iron transport system and reduce cellular antioxidant capability.