Abstract:The interaction between gut microbiota and host metabolism regulates the physiological function of the host. Recent studies have shown that the microbiome-gut-brain axis exists in the host organism. The intestinal microflora can affect the central nervous system through various pathways, then regulates host behavior such as appetite. The dietary fiber is not easily digested and absorbed by the host, and used by gut microbial fermentation, resulting in a variety of metabolites. These metabolites serve as signaling molecules of the central nervous system and can modulate host appetite through different pathways. This article reviewed the effects of gut microbiota on the central nervous system and host appetite, as well as the mechanisms, aiming to highlight the role of gut microbiota on the host appetite.

Abstract:Mycobacterium ulcerans infection can cause chronic skin ulceration——Buruli ulcer, leading to tissue necrosis, apoptosis and immune suppression. The relatively painlessness of ulcers during the early stage of infection will delay the treatment for patients. Mycolactone is the cytotoxic basis of M. ulcerans, which targets both immune cells and some nonimmune cells. In this review, the chemical structure, cytotoxicity and molecular targets of mycolactone were summarized, as well as questions for mycolactone.

Abstract:Manganese oxides are highly reactive minerals and play an important role in elemental biogeochemical cycles in the natural environment. Bacteria can oxidize Mn(Ⅱ) to Mn(Ⅲ) and Mn(IV). More and more bacteria have been isolated from different environments. Furthermore, bacteria are key drivers of the global Mn cycle. Bacterial Mn(Ⅱ) oxidation is an enzymatic process. Enzymes responsible for bacterial Mn(Ⅱ) oxidation are multicopper oxidases and animal haem peroxidases (AHPs), but the latter is different from the former. In this mini-review, we focus on the advances in bacterial Mn(Ⅱ) oxidation by AHPs, manganese oxidizing bacteria, structure and characterization of AHPs, the genes of AHPs, and the process of oxidation. Challenges and future research needs are also discussed.

Abstract:Mitogen-activated protein kinase (MAPK) signaling pathway is one of the most important intracellular signaling pathways in which cells perceive exogenous stimuli and make an effective immune response. Recent studies have shown that the dysregulation of MAPK is highly associated with tuberculosis pathogenesis. The MAPK phosphatases (MKPs) are the prototypic phosphatase that could dephosphorylate the MAPKs, thus play an essential role in the regulation of cell stress, differentiation, proliferation and apoptosis by negatively regulating the activity of MAPKs. Among all the MKPs, MKP-1 has the strongest ability to dephosphorylate MAPKs. In this review, we summarize the role of MKP-1 in Mycobacterium tuberculosis infection and relevant research progress.

Abstract:[Objective] To study the effects of starane on maize soil bacterial diversity.[Methods] We analyzed composition and structure of bacterial communities in maize soils from the control and spraying herbicide of different periods by Illumina MiSeq high-throughput sequencing of the V4-V5 region of the 16S rRNA gene.[Results] We received a total of 260940 effective sequences, 167191 high-quality sequences and 12656 OTUs. The soil bacterial diversity and abundance decreased after 10 days of starane treatment, whereas the soil bacterial diversity and abundance increased after 60 days of starane treatment. Bacterial community composition analysis shows that the dominant phyla in five soil samples were Acidobacteria, Proteobacteria, Actinobacteria, Chloroflexi and Gemmatimonadete. After 10 days of starane treatment, the ratio of Acidobacteria increased in herbicide treated soil, whereas the ratio of Actinobacteria and Chloroflexi decreased. After 60 days of starane treatment, Proteobacteria decreased, Chloroflexi increased obviously.[Conclusion] Starane affected maize soil bacterial diversity and its impact varied with the application time.

Abstract:[Objective] To explore the effects of foot-and-mouth disease virus (FMDV) structural protein VP0 in type I IFN signaling.[Methods] A recombinant VP0 protein was constructed and expressed in eukaryotic cells followed by FMDV infection. The influence of VP0 protein during FMDV replication in BHK cells was determined by RT-PCR and the mRNA levels of IFN-β, RIG-I, ISG15, ISG20 and IRF3 was assessed. The dose-dependent effects of VP0 protein on Sendai virus (SeV)-triggered activation of IFN-β and NF-κB promoter as well as on RLRs-mediated activation of IFN-β promoter were examined by luciferase assay. Co-immunoprecipitation was done to detect the interaction of VP0 protein and the components of the RLRs signaling pathway.[Results] The recombinant VP0 protein was successfully expressed in HEK-293T cells and confirmed by Western blotting. At 4-6 h post-infection, VP0 protein significantly promoted the replication of FMDV in BHK cells (P<0.01 or P<0.05). VP0 protein clearly inhibited the gene expression level of IFN-β, RIG-I, ISG15, ISG20 and IRF3 (P<0.01 or P<0.05), respectively. In luciferase assays, FMDV VP0 protein distinctly inhibited SeV-triggered activation of the IFN-β and NF-κB promoters in a dose-dependent manner. The interferon regulatory factor 3 (IRF3)-activated IFN-β promoter and its upstream components, RIG-I, MDA5, VISA and TBK1 were also down-regulated in the presence of VP0 protein. The inhibition of IFN-β promoter induced by IRF7 was not observed. Furthermore, Co-immunoprecipitation showed that VP0 interacted with IRF3 protein in HEK-293T cells.[Conclusion] Our results indicated that VP0 may inhibit the activation of type I IFN signaling pathway via interaction with IRF3.

Abstract:[Objective] To clarify the role of nucleocapsid/polymerase proteins or helper proteins of human respiratory syncytial virus (RSV) in rescuing recombinant RSV, plasmids of RSV dicistronic minigenome encoding enhanced green fluorescent protein (EGFP) gene was constructed and rescued.[Methods] Based on the methods of gene synthesis and molecular cloning, RSV monocistronic minigenome plasmids, pUC57-RSV-EGFP encoding EGFP and pUC57-RSV-ORF1 encoding pseudo-virus protein, were constructed. Then, RSV dicistronic minigenome was further cloned through these two monocistronic minigenome plasmids and pBR322B vector, and identified by the analyses of restriction endonuclease and nucleotide acid sequence. Following co-transfecting helper plasmids to BHK-T7 cells together with RSV dicistronic minigenome plasmid, the function of helper proteins was evaluated based on the transcribed EGFP mRNA by real-time quantitative polymerase chain reaction (RT-qPCR) and the expressed EGFP by fluorescent microscope.[Results] The recombinant plasmid of RSV dicistronic minigenome encoding EGFP gene and driven by T7 promoter, pBR322B-RSVⅡ-EGFP, was constructed successfully. The differential expressions of EGFP both transcriptionally and translationally occurred following the dicistronic minigenome rescued by different combinations of the helper plasmids, which showed the helper proteins functioned differently in the replication of RSV.[Conclusion] The constructed RSV dicistronic minigenome containing EGFP gene is a useful tool for function analysis of the helper proteins, and the M2-1 protein, capable of elongating the transcription, is confirmed.

Abstract:[Objective] We improved the thermostability of lipase LipA from Burkholderia cecapia ZYB002 using protein engineering technology.[Methods] Lipase LipA mutant library was designed and screened using the following software, YASARA, FoldX, Rosetta, and Gromacs. We screened 27 thermostable lipase LipA mutants displaying the salt bridge effect among the resulting library of 341 variants, and then further screened using the site-directed mutagenesis technology.[Results] Three mutants LipA-Asn¹²⁵Asp, LipA-Asn¹²⁵Glu and LipA-Gln²⁶²Glu displayed improved thermostability. The T₅₀¹² value of LipA-Asn¹²⁵Asp, LipA-Asn¹²⁵Glu and LipA-Gln²⁶²Glu increased by 4.0℃, 5.5℃ and 4.4℃, respectively. The half-life of LipA-Asn¹²⁵Asp, LipA-Asn¹²⁵Glu and LipA-Gln²⁶²Glu at 55℃ increased by 2.23-fold, 3.8-fold and 2.6-fold, respectively.[Conclusion] It is feasible to screen thermostable mutant from the computationally designed library.

Abstract:[Objective] This work aims to compare the preventive and therapeutic effects of Bifidobacterium infantis 13.085 on suppression of shrimp tropomyosin (TM) sensitization in BALB/c mice. The balance of Treg/Th17 ratio and expression of Treg-and Th17-associated cytokines were also studied.[Methods] Shrimp tropomyosin was purified by ammonium sulfate and isoelectric precipitation. Four groups (Positive1, B. infantis therapy, Positive2 and B. infantis prevention) of BALB/c mice were immunized by intraperitoneal (IP) injection of purified TM together with incomplete Freund's adjuvant. Negative control animals received equal amounts of sterile PBS with Freund's adjuvant at each sensitization and challenge point. Allergic symptoms were evaluated by diarrhea, anaphylactic symptoms, HE staining analysis of lung tissue, changes of body weight and splenic viscera coefficient. The levels of specific IgE, IgG2a and histamine in sera were determined by ELISA. The amount of Treg and Th17 cell subsets were analyzed by flow cytometry. The mRNA expression levels of Treg-and Th17-associated cytokines and transcriptional factors were detected by quantitative PCR.[Results] The purity and yield of TM were 84.93% and 60.88%, respectively. In vivo study showed that B. infantis treatment ameliorated the development of diarrhea, anaphylactic symptoms and inflammation of lung tissue. The splenic viscera coefficient also decreased in B. infantis treatment groups compared to positive controls. At the end of experimental period (day 56), the levels of specific IgE and histamine (P<0.05) as well as IL-17A mRNA expression (P<0.01) significantly reduced, while the Treg/Th17 ratio remarkably increased (P<0.05) in B. infantis treatment groups compared to positive controls. The level of CD25 mRNA expression significantly enhanced (P<0.01) in B. infantis therapy group compared to Positive1. In addition, the levels of specific IgE and IL-17A mRNA expression in B. infantis therapy group were much lower than those in B. infantis prevention group (P<0.05), while the Treg/Th17 ratio and CD25 expression level were higher in B. infantis therapy group compared to B. infantis prevention group (P<0.05).[Conclusion] Therapeutic administration of B. infantis 13.085 can effectively alleviate TM-stimulated allergic inflammation and symptoms, which may attribute to the balance of Treg/Th17 ratio, increase of Treg-associated cytokines expression, inhibition of Treg-associated cytokines secretion, and thus leading to blockade of inflammatory antibodies and histamine release.

Abstract:[Objective] The effect of continuous low-expression region on validamycin A titer and biomass was evaluated through gene deletion experiments.[Methods] The deletion boundaries of large chromosome region were identified via transcriptome analysis. Meanwhile, deletion mutant was obtained via Cre-loxP system, titer of validamycin A was detected by HPLC and, biomass was represented by dry cell weight.[Results] The 1.2 Mb large region identified via transcriptome analysis was successfully deleted by Cre-loxP system. In comparison with parent strain TL01, deletion of 1.2 Mb region increased the biomass by 44% and showed no effect on validamycin A titer.[Conclusion] The strategy developed here can be applied to identify deletion boundaries of large chromosome region, and mutant of 1.2 Mb region deletion showed potential as host for heterologous expression of aminocyclitol biosynthetic gene clusters.

Abstract:[Objective] To study wolfberry rhizosphere fungal diversity in different regions, we attempted to explain the correlation between Ningxia wolfberry quality and soil microbial flora from the perspective of rhizosphere fungal community.[Methods] We used the miseq high-throughput sequencing method to analyze the Internally Transcribed Spacer (ITS) region in the rhizosphere soil samples of Lycium barbarum L. in four different regions of China. We analyzed Alpha diversity, Beta diversity and fungal community structure, and determined the main active ingredients of Lycium barbarum L. in the four regions, and analyzed the correlation between the quality of Lycium barbarum L. and soil physical and chemical factors and the diversity of rhizosphere fungi.[Results] Polysaccharide and betaine of Lycium barbarum L. in Zhongning were higher than Jinghe's and Xingren's, and Golmud's was the lowest. Species diversity and structure analysis of soil fungi in the rhizosphere of Lycium barbarum L. showed that species diversity and structure were similar in Jinghe, Xingren and Zhongning, Ascomycetes and Zygomycota accounted for about 80% of the total species, but Golmud's was different from other regions, ascomycetes accounted for 58% and Chytridiomycota and Neocallimastigomycota were much higher than the other three regions, nearly 25% of the proportion of total fungi. Beta diversity analysis showed that the similarity of fungal flora structure in the rhizosphere soil of the Lycium barbarum L. in four regions was Xingren, Jinghe, Zhongning and Golmud.[Conclusion] The research showed that the composition of fungal species in rhizosphere soil of Lycium barbarum L. was abundant and there were some differences in the population structure of soil fungi in the rhizosphere soil of different regions. Some active ingredients of Lycium barbarum L. had a certain correlation with the population structure of soil fungi in the rhizosphere. So, microorganism boost the growth and quality of Lycium barbarum L..

Abstract:[Objective] To study the effect of the heavy metal Cu on the bacterial composition and diversity in Allogynogenetic Carassius auratus intestinal.[Methods] Total DNA was extracted by DNA extraction kit. The 16S rRNA gene of intestinal microbial diversity was amplified by universal PCR primers. Then the clone library was built to analyze data.[Results] The main bacterial community in Allogynogenetic Carassius auratus intestinal contained Firmicutes, Proteobacteria and Bacteroidetes. At different concentrations of Cu, the contents of Firmicutes in Allogynogenetic Carassius auratus gut significantly decreased. The results of Rarefaction Curyiyuyiyuve, Venn and Diversity index showed that gut microbial diversity decreased obviously.[Conclusion] Cu caused the microbial diversity in Allogynogenetic Carassius auratus decreased. This result provides a basis for further studies on the health of Allogynogenetic Carassius auratus relevant to heavy metal Cu.

Abstract:[Objectives] To identify pH-signalling pathway (Pal)-related genes in mycoparasitic fungus Coniothyrium minitans and to understand the role of these genes in interaction between C. minitans and its host Sclerotinia sclerotiorum.[Methods] Six Pal-related homologues were obtained from the whole genome of C. minitans and designated as CmpalA, CmpalB, CmpalC, CmpalF, CmpalH and CmpalI. PEG-mediated protoplast transformation was used to create the deletion mutants of Pal-related genes. Five Pal-related genes were knocked out individually and the mutants designated as ΔCmpalA-33, ΔCmpalB-13, ΔCmpalC-5, ΔCmpalF-50 and ΔCmpalH-26. The biological characteristics, including colony morphology, mycoparasitism, oxalate degradation and antifungal activity,were compared between knock-out mutants and the wild-type strain.[Results] Compared to the wild type strain, five Pal-related genes-deletion mutants showed significantly reduced mycelia growth between pH 6 and 8. These results indicated that the disruption of these Pal-related genes increases sensitivity to neutral or alkaline pH. The sclerotia-infection assay showed that the parasitic activities of the five Pal-related genes-deletion mutants were dramatically reduced. qRT-PCR results showed that these Pal-related genes-deletion mutants suppressed expression levels of three mycoparasitism-associated genes Cmch1, Cmg1 and Cmsp1. Meanwhile, expression of CmpacC, the pH signaling pathway downstream gene, was also reduced in the Pal-related genes-deletion mutants. The oxalate degradation of the five Pal-related genes-deletion mutants at pH 6 were increased under pH 8, and the antifungal activity of those mutants were also increased at pH 8 comparison with the wild type.[Conclusion] Disruption of the Pal-related genes resulted in impaired C. minitans responses to ambient pH. The pH-signalling pathway (Pal) plays an important role in interaction between C. minitans and S. sclerotiorum, including mycoparasitism, oxalate degradation and antifungal activity in C. minitans against S. sclerotiorum.

Abstract:[Objective] The aim of this study is to gain the acclimated strain of Acidithiobacillus caldus SM-1 with high activity when growing at the temperatures below the optimum, and elaborate its genomic plasticity and adaptation at different temperatures.[Methods] Strains were incubated at 37℃, 40℃ and 45℃. We used 454 genome resequencing technology to find the single nucleotide mutant sites in genome. The genes including mutant sites were studied to understand their relationship with the temperature adaptation.[Results] By long-term breeding, we obtained strains with higher viability than unacclimated strain at temperatures (37℃), which is lower than initial optimum growth temperature (45℃). Resequencing results showed the genome of SM-1 with high plasticity. In the genome of different strains (incubated at 37℃, 40℃ and 45℃), 418, 384 and 347 single nucleotide mutation sites were accumulated, respectively. Among them, 20 mutant sites were commonly occurred in the three strains. The genes they affected are involved in heavy metals and toxic resistance system, DNA methylation and protein acylation, and nucleic acid metabolism. In comparison, the specific mutations of the strains, grown at the 37℃ and 40℃, were related to energy metabolism, signal transduction and DNA/RNA stability. Three genes contained mutant sites are common in L37 and M40, of which, Atc_1031 and Atc_1623 encode proteins related to transposon insertion, Atc_1130 encodes a hypothetical protein that is similar to the out membrane protein assembly factor B or the disulfide bond formation protein with 23% and 35% similarity. In addition, some single nucleotide mutations cause the amino acid changes of the related proteins during the adaption.[Conclusion] The genome of At. caldus SM-1 showed highly plasticity at different temperatures. The study provided a set of genomic data for understanding the temperature adaptability molecular mechanism of microorganisms. The study revealed that At. caldus SM-1 evolved to fit lower temperature through multiple pathways, not only by the general environment adaptation mechanism of microorganism, but also by the specific pathway of the strain.

Abstract:[Objective] The aim of this study was to screen the methylation tailoring gene-mcbD in marinacarboline biosynthesis pathway from the genomic sequence of Marinactinospora thermotolerans SCSIO 00652, and to characterize McbD both in vitro and in vivo.[Methods] Orf03255 (McbD) was selected by phylogenetic analysis of the methyltransferases from M. thermotolerans SCSIO 00652 with MfnG (D/L-Tyr methyltransferase) using Mega6. The mcbD gene was amplified from the M. thermotolerans SCSIO 00652 genomic DNA, cloned into pET28a(+) vector, and expressed in E. coli BL21 (DE3); the McbD protein was purified with Ni-NTA affinity chromatography. The McbD-mediated enzymatic reaction was performed with marinacarboline B as substrate and detected with HPLC. The ΔmcbD mutant was constructed with PCR-targeting genetic manipulation system. Difference in fermentation extracts between M. thermotolerans SCSIO 00652 wild-type and ΔmcbD was analyzed with HPLC.[Results] The N-His tagged McbD was successfully expressed in E. coli BL21 (DE3) in soluble form, and purified by Ni-NTA affinity chromatography; further biochemical reaction showed that McbD can catalyze the transformation of marinacarboline B to marinacarboline C with SAM. The ΔmcbD inactivated mutant completely abolished the production of marinacarboline C, and accumulated marinacarboline B.[Conclusion] McbD, responsible for the methylation tailoring of marinacarboline, is indispensable in marinacarboline biosynthetic pathway.

Abstract:[Objective] To explore the effect of methanogens on the carbon metabolism of anaerobic fungi.[Methods] End-metabolites of different carbon sources by two anaerobic fungi (Orpinomyces sp. and Neocallimastix sp.) with or without co-culture methanogens (Methanobrevibacter sp.) were compared after 96 h in vitro anaerobic batch fermentation.[Results] Co-culture F1 (Orpinomyces sp. +Methanobrevibacter sp.) greatly enhanced the production of methane, acetate and lactate after corn core and cassava fermentation compared to pure fungal culture F1^* (Orpinomyces sp.). In particular, lactate production by F1 reached (26.44±0.22) mmol/L when fermenting on cassava, 14 times more than those produced by F1^*. On the contrast, co-culture N3 (Neocallimastix sp. + Methanobrevibacter sp.) showed lower level of lactate production after corn core and cassava fermentation compared to the pure fungal culture N3^* (Neocallimastix sp.). In addition, lactate production varied greatly depending on the substrate amount and types of carbon. Lactate production by F1 showed an overall positive correlation with the amount of cassava, peaked (56.29±2.04) mmol/L when cassava amount was 2.0%. Besides, end-metabolites of five starch-rich materials fermented by F1 varied. In particular, there was a highly positive correlation (R₂=0.9554) between lactate yields and the proportions of amylopectin of substrates. Further fermentation on pure sugars by F1 was performed. Polysaccharide (maltodextrin) produced substantially greater amount of lactate than monosaccharide (glucose) and disaccharide (maltose).[Conclusion] Co-culture of anaerobic fungi with methanogens shifted the fungal carbon metabolism during fermentation, which depended on the carbon sources and fungal species.

Abstract:[Objective] Nicotinamide adenine dinucleotide (NAD⁺) plays a crucial role in controlling metabolism network. Improving its intracellular concentration or getting a high-NAD⁺-yield strain is of great significance for NAD⁺-dependent redox reaction rate.[Methods] First, we used endogenous regulation means to enhance the key genes of NAD⁺ synthesis pathway, such as over-expressing and co-expressing the key enzyme genes pncB, nadD and nadE. Second, we optimized NAD⁺ precursors supplement and fermentation conditions to increase NAD⁺ synthesis. Finally, we used Box-Bohnken method for optimal synthesis condition by 3 significant factors' interaction based on single-factor experiments and the response value of NAD⁺ content.[Results] According to different expression strategies, we constructed seven recombinant strains. Besides, the intracellular NAD⁺ content of the recombinant strain E. coli BL21/pET-21a-nadE-pncB was 405.2% higher than that of the original strain. Moreover, after optimization of induction conditions and NAD⁺ precursor concentration by Design Expert 8.0, NAD⁺ content reached 43.16 μmol/g DCW, 123.6% higher than that before optimization and 1029.8% higher than the original strain.[Conclusion] Co-expression the key enzyme genes pncB and nadE are essential to improve NAD⁺ synthesis. The recombinant strain with high NAD⁺ provides the feasibility to improve biocatalytic efficiency.

Abstract:[Objective] Based on the λ Red recombination system,a two-step PCR method was developed to delete small non-coding RNA (sRNA) and large chromosomal fragment in YYersinia pestis.[Methods] Two PCR procedures were done to amplify product formed of a kanamycin resistance gene flanked by long (600-1000 bp) homology extensions.The PCR fragment carrying a kanamycin resistance gene flanked by regions homologous to the target locus was electroporated into a recipient 201 strain of Yersinia pestis expressing the homologous recombination system encoded by plasmid pKD46,which promoted the replacement of the target gene with kanamycin resistant fragment.Finally,the recombinant clones were identified by PCR.[Results] The homologous extensions of 600-1000 bp were constructed by two PCR method,which increased the efficiency of homologous recombination,the sRNA RyhB1(108 bp) and RyhB2(106 bp) and the larger chromosome fragments 47-2 (10.4 kb),47-3(21.6 kb),47-3a (9.2 kb) and 47-3b (6.1 kb) were successfully deleted.[Conclusion] The two step PCR mutation technique was a simple and efficient method for the precise modification of sRNA and large fragment chromosome of Yersinia pestis.This method was suitable for gene knockout of whole genome of Yersinia pestis,which provided a powerful tool for gene expression and regulation,pathogenicity and virulence study of Yersinia pestis.