Abstract:Zinc is an indispensable trace element and important for both bacteria and eukaryotes. Zn homeostasis is established by Zn²⁺ transport and regulation system. Hosts have developed mechanisms for Zn restricting or toxicosis in response to infections. In order to grow and multiply in the infected host, bacteria have progressed strict zinc transportation and regulation system, such as ZnuABC. Zinc is critically involved in a plethora of metabolic and virulence pathways and is paramount important in infection. Therefore, there could be possibilities to use zinc transporters as a very promising target for the development of novel antimicrobial strategies.

Abstract:Haustoria, one of the fundamental characteristics of obligate parasite, is a micro-branch produced by biotrophic fungi and oomycetes, which is composed of haustorial body, extrahaustorial matrix and extrahaustorial membrane. It is an abnormal structure that can invade the host cell interaction with the plant. Haustoria is not only the key factor of biotrophic fungi carrying on the living specimen nutrition way but also represents significant roles in the nutrition biosynthesis and inhibiting the defense reaction of host. The dee_per understanding of haustoria will favor us acquaint obligate parasite enormously, so that we can control the corresponding diseases better. This paper summarized the function on nutrition and pathogenicity of haustoria, and the problems and the research trend in this area were discussed.

Abstract:Replant disease devastates crop growth, and its disease etiology has not been fully elucidated. The latest research revealed its close association with the changes of rhizosphere microecology, especially the changes in rhizosphere microbial community structure. With the advance of modern high-throughput DNA sequencing technology, the relationship between rhizosphere microbiome and replant diseases has become a popular research focus. This review comprehensively analyzes the currently available literatures regarding to the relationship between rhizosphere microbiome, crop growth and replant diseases. In summary, a new perspective for the disease etiology and possible prevention regimen has been arisen from better understanding the association between replant disease and the dynamic changes of rhizosphere soil microbial population.

Abstract:[Objective] Using Aspergillus niger as host to express β-mannanases from Stachybotrys chartarum.[Methods] Through sequence analysis of Stachybotrys chartarum genome, two β-mannanase genes (s16942 and s331) were identified. The primers were designed based on the DNA sequence and the β-mannanase genes (s16942 and s331) were obtained, and then inserted to the vector pGm. The expression plasmids were transferred into Aspergillus niger. β-mannanase producing strains (G1-pGm-s16942 and G1-pGm-s331) were isolated after screening several transformants using amdS selection plates and confirmed by PCR fragment sequencing.[Results] The molecular weight of the enzymes from G1-pGm-s16942 and G1-pGm-s331 were about 48 kDa and 60 kDa respectively by SDS-PAGE gel analysis, and the recombinant proteins did not present in the negative control. Assays of enzymatic property using the crude enzyme preparations indicated that the enzyme from G1-pGm-s16942 exhibited maximum activity (521 U/mL) under the optimum conditions (pH 7 and 60℃); the enzyme from G1-pGm-s331 exhibited maximum activity (84 U/mL) under the optimum conditions (pH 7 and 50℃).[Conclusion] This was the first study of the heterologous expression of the β-mannanase genes from Stachybotrys chartarum in Aspergillus niger host and the β-mannanase genes could be expressed successfully with high activities and protein titers.

Abstract:[Objective] Azorhizobium caulinodans ORS571 can fix nitrogen not only as a free-living organism and an associative-symbiotic bacterium by colonizing the root surface of non-leguminous plants, but also as a symbiotic bacterium by interacting with leguminous plant Sesbania rostrata. Due to its ability to grow and fix nitrogen under three conditions, A. caulinodans uses sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioral responses. Chemotaxis appears crucial for the growth of A. caulinodansin complicated environment and the construction of associative relationship with the plant. However, little is known about the chemotactic pathway of A. caulinodans. Thus, our study aimed to compare the chemotaxis-like genes of A. caulinodans with those of well-studied species.[Methods] NCBI protein BLAST was used for searching sequence similarity with default parameter values against the genomes of A. caulinodans. HMMER3, based on Pfam database, was used for comparative analyses of methyl-accepting chemotaxis protein (MCP).[Results] There was a major chemotaxis cluster in A. caulinodans and the CheR methylated MCPs independently of pentapeptide motif. There were 43 MCP homologs containing diverse signal-sensing architectures in A. caulinodans. In addition, cytoplasmic domains of these MCPs were all composed of 38 heptad repeats.[Conclusion] Despite the extremely high homology presented between the chemotactic system of A. caulinodans and those of well-studied species, A. caulinodans shows its own unique characteristics. The classification of these chemotactic pathways by comparative genomics enables us to better understand how A. caulinodansresponds to changes in environment via exquisite signal transductions in chemotaxis system.

Abstract:[Objective] To study the inhibition of methicillin-resistant Staphylococcus aureas (MRSA) biofilm by honokiol.[Methods] We used triphenyl tetrazolium chloride method to evaluate the inhibition of biofilm formation and mature by honokiol. We used congo red agar and spectrophotometer to detect the influence of honokiol on polysaccharide intercellular adhesion formation and extracellular DNA release. RT-PCR analysis was used to determine the effect of honokiol on expression of icaA, cidA and agrA.[Results] Honokiol showed strong antimicrobial activity both on biofilm formation and mature biofilm of MRSA 41573. Minimum inhibitory concentration was 10 μg/mL for biofilm formation and 50 μg/mL for mature biofilm. Minimum bactericidal concentration was 20 μg/mL for biofilm formation and 100 μg/mL for mature biofilm. Honokiol showed synergy effect with vancomycin and it significantly increased the sensitivity of mature biofilm to vancomycin. Polysaccharide intercellular adhesion formation and extracellular DNA release were effectively inhibited by honokiol. Extracellular DNA release decreased by 28.3% when honokiol at 1/8 MIC. After incubated with 1/2 MIC of honokiol for 16 h, the relative expression of icaA, cidA and agrA of MRSA41573 was reduced by 59.1%, 56% and 72.3%, respectively.[Conclusion] Honokiol can significantly inhibit biofilm formation of MRSA41573 and its mechanism is mainly the inhibited expression of icaA and cidA to influence the synthesis of polysaccharide intercellular adhesion and extracellular DNA. Moreover, it also affect biofilm formation by QS system.

Abstract:[Objective] Nonribosomal peptide synthetase (NRPS) plays an important role in plant-pathogenic fungi interaction. In this study, we analyzed the role of VmNRPS12 during Valsa mali (V. mali) infection and pathogenecity, which may shed new insights into the pathogenesis of V. mali.[Methods] Based on the genome of V. mali, we obtained one NPRS, designated as VmNRPS12. The expression profile of VmNRPS12 was analyzed by qRT-PCR. Through Double-joint PCR and PEG-mediated protoplast transformation, VmNRPS12-knockout mutants were generated. PCR and Southern blot hybridization were applied to verify the positive mutant. Then genetic complementation was performed by transforming mutant protoplast with VmNRPS12 original gene. Finally, vegetative growth, conidiation and pathogenicity were examined.[Results] qRT-PCR analyses showed that VmNRPS12 was upregulated during the early infection stages of V. mali, and expressed remarkably at 48 hours post inoculation (138.6-fold). Compared with the wild type 03-8, VmNRPS12-knockout mutant showed no obvious change in vegetative growth and conidiation on PDA medium. Notably, the VmNRPS12-knockout mutant exhibited significantly reduced virulence on apple twigs. Furthermore, complementation of VmNRPS12 restored the pathogenecity of the VmNRPS12-knockout mutant approximately to the wild type 03-8.[Conclusion] VmNRPS12 contributes positively to the pathogenicity of V. mali.

Abstract:[Objective] Salmonella enterica serovar enteritidis is an important food-borne pathogen of human and animal. To further study the function of SlyD associated with virulence and regulation in stress responses of Salmonella Enteritidis, we constructed slyD gene-deletion mutant,, expressed it in E. coli, and characterized the PPIase enzyme obtained.[Methods] The slyD gene-deletion mutant of Salmonella enteritidis C50041 was constructed by suicide plasmid mediated homologous recombination. Salmonella enteritidis slyD prokaryotic expression vector was carried out in E. coli, and PPIase activity of recombination SlyD was measured in protease-coupling assay with chymotrypsin. For amino acids conservation studies, functional domain searches and secondary structure predictions, the BLAST, SMART, TMHMM, SignalP, PHD and SWISS MODEL were used.[Results] Salmonella enteritidis C50041 ΔslyD mutant strain was successfully constructed. The growth rate of slyD-deleted strain was identified consistent with its parent strain C50041. A soluble recombinant SlyD protein was expressed in Escherichia coli BL21(DE3) cells and confirmed by SDS-PAGE. Catalytic activity confirmed that the SlyD protein was biologically active. Bioinformatic analysis showed that Salmonella Enteritidis SlyD as a multifaceted protein including three separated domains, the FKBP type peptidal-prolyl cis-trans isomerase domain, the IF chaperone domain and the metal-binding domain.[Conclusion] Salmonella enteritidis C50041 ΔslyD mutant strain and soluble SlyD protein was obtained, and the present study may provide a basis for further study of the role of SlyD in Salmonella enteritidis.

Abstract:[Objective] The objective of the study was to study the effects of Lactobacillus plantarum and Lactobacillus casei on the changes in composition and quantity of microbial populations and the concentrations of short chain fatty acids in the gut digesta of piglets before and after weaning, to explore the mechanisms of the tested two strains to relieve weaning stress.[Methods] Fifteen litters of piglets (Duroc×Yorkshire×Landrace) at the age of 7 days were randomly allocated to 3 groups (5 each), including the control group with an oral administration of saline, LP group with L. plantarum and and LC group, with L. casei. On day 21, 24 and 35, the piglets were slaughtered, and the ileum and colon digesta were analyzed for microbial populations and short chain fatty acids.[Results] In the ileum and colon, the test strains significantly increased the microbial diversity (P<0.05), promoted the growth of Lactobacilli spp. and Bifidobacteria spp. after 2 weeks of post-weaning. The two test strains increased the concentration of acetate, propionate, butyrate and total short chain fatty acids both in ileum and colon before weaning, and the concentration of acetate, total short chain fatty acids after weaning. Correlation analysis showed that the decreased diarrhea rate before weaning in the LP and LC groups was significant associated with the increased short chain fatty acids concentration and total bacteria of ileum and colon, the increased height of the ileum villi. The improved average daily gain presented a correlation with the increased concentration of acetate and total short chain fatty acids of colon.[Conclusion] The findings imply that the tested strains are contribute to increase the microbial diversity, the quantities of beneficial bacteria and the production of short chain fatty acids in the gut intestinal tract of weaning piglets.

Abstract:[Objective] This study aimed to study the phylogenetic diversity and community structure of bacteria in petroleum contaminated soils from Karamay oil field, and to analyze the relationship between the community variation and the environment parameters, to provide a reference for bioremediation of petroleum contaminated soils.[Methods] We collected samples from petroleum contaminated soils in 5 cm, 20 cm and 50 cm depth layers, and measured the environment parameters subsequently. We constructed three 16S rRNA gene clone libraries of these soil samples, and then determined the operation taxonomy units (OTUs) restriction fragment length polymorphism method, and finally sequenced the representative clones of every OUT. The diversity, richness and evenness index of the bacteria communities were calculated by using Biodap software. Neighbor-Joining phylogenetic tree was constructed based on 16S rRNA gene sequences of bacteria from Karamay oil field and the references from related environments. Canonial correspondence analysis (CCA) was used to analyze the relationship between environment parameters and species by using CANOCO 4.5 software.[Results] Environment parameters showed that 50 cm dee_p soil contained the highest amount of total nitrogen (TN) and total phosphorus (TP), whereas the 20 cm depth soil contained the lowest amount. The 5 cm depth soil contained the highest amount of total organic carbon (TOC), whereas the 50 cm depth soil contained the lowest amount. Among the 3 layers, 20 cm depth had the highest diversity and richness of bacteria, whereas the bacteria in 50 cm depth was the lowest. Phylogenic analyses suggested that the bacteria in Karamay oil field could be distributed into five groups at the level of phylum, Cluster I to V, respectively belong to Proteobacteria, Actinobacteria, Firmicute, Bacteroidetes, Planctomycetes. Cluster I accounts for 78.57% of all tested communities. CCA results showed that TN, TP, TOC significantly affected the bacteria community structure. Especially, TOC content is significantly related to the distribution of Pseudomonas.[Conclusion] The petroleum-contaminated soil inhabited abundant of bacteria. The diversity index and spatial distribution of these communities were affected by the environment parameters in the soil.

Abstract:[Objective] We isolated an aerobic denitrifier with affinity for nitrogen removing and low accumulation of nitrous oxide, and analyzed its denitrification activity to provide technical support for controlling eutrophic wetland lakes.[Methods] An aerobic denitrifier named C3 was isolated from phragmites wetland soil in Liaohe estuary by BTB medium preliminary screening and denitrification activity analysis. We used morphological and physiological characteristics as well as sequence analysis of the 16S rRNA to identify the strain. Effects of culture conditions, such as temperature, carbon source, initial pH, C/N ratio were evaluated regarding removal of nitrite nitrogen and accumulation of nitrous oxide under aerobic condition.[Results] The highly effective strain C3 was identified as Pseudomonas sp., for which the best carbon source was sodium citrate, optimal growth and denitrification occurred at 30℃, pH 7.0, and a C/N ratio of 10. Under these conditions, the concentration of nitrate was reduced from 179.55 mg/L to 5.08 mg/L by strain C3 in 36 hours, a reduction in NO₃^- of more than 97.17%. In the process of denitrification, nitrous oxide as an intermediate accumulated at a level of 0.22 mg/L.[Conclusion] Strain C3, an aerobic denitrifying agent, was isolated from wetland soil and has obvious advantages in efficient removal of nutrients with low accumulation of nitrous oxide, representing a great potential to treat eutrophic wetland lakes.

Abstract:[Objective] The purpose of this study is to reduce the colonization level of Campylobacter jejuni in chicken intestine by oral immunization of recombinant Lactococcus lactis expressing the ferric enterobactin receptor CfrA of C. jejuni.[Methods] The whole cfrA gene and its N-terminal fragments were amplified by PCR, inserted into the expression vector pNZ8149 and transformed into L. lactis NZ3900. Based on the expression of CfrA in recombinant L. lactis by Western blot, the expression level was optimized by screening nisin concentration, induction temperature and time. Then the recombinant L. lactis strains were used to orally immunize specific-pathogen-free chickens. After oral immunization, the duration of recombinant L. lactis in chickens was determined by PCR, and the antibody levels of anti-CfrA serum IgG and intestinal mucosal sIgA were measured by ELISA. Finally, the immunized chickens were orally inoculated with C. jejuni to evaluate the inhibitory effect of recombinant L. lactis on colonization of C. jejuni.[Results] Western blot results determined that the whole cfrA gene and its N-terminal fragments were both expressed in recombinant L. lactis in soluble forms whereas no secreted CfrA protein was detected outside bacterial cells. The optimal conditions for inducing the expression were grown at 37℃ for 1 h with nisin concentration of 25 ng/mL. Detection of chicken cloacal swabs showed that the duration of oral L. lactis was less than 10 days in chicken. The immunized groups produced higher antibody titers of anti-CfrA specific serum IgG and mucosal sIgA than the control groups. Moreover, the colonization rate of C. jejuni in the immunized groups was significantly lower than that in the control groups.[Conclusion] Oral immunization of chickens with recombinant L. lactis expressing CfrA inhibited the colonization of C. jejuni. Our findings can be useful to develop oral vaccines with recombinant Lactobacillus for control of C. jejuni infection in chickens.

Abstract:[Objective] We screened and identified a strain capable of enantioelectively hydrolyzing methyl (R,S)-N-(2,6-dimethylphenyl) alaninate (MAP), a key intermediate for the synthesis of metalaxyl, followed by cloning and expressing the esterase in E. coli.[Methods] We used MAP as the sole carbon source in the medium inoculated with an active sludge specimen to enrich the target microorganism. The strain with the highest hydrolysis activity and enatioelectivity was identified by 16S rRNA sequence analysis, morphological observation and physiological and biochemical properties. From the gene library of the strain, the DNA sequence fragment containing the target gene was found. By DNA sequence analysis and PCR amplification, the esterase gene was obtained. It was ligated with plasmids pET28a (+), then transformed into E. coli BL21Gold (DE3) plysS.[Results] We isolated a gram-negative bacterial strain capable of enantioelective hydrolyzing MAP. It was identified as Achromobacter denitrificans. From its gene library, the esterase gene named EHest was found. The recombinant EHest-pET28a(+)-BL21Gold (DE3) plysS was constructed. The recombinant expressed esterase (EHesterase) capable of catalyzing enatioelective hydrolysis of methyl (R,S)-N-(2,6-dimethylphenyl) alaninate. Its size was 27 kDa. The expression activity was 27.1 times as high as that in the original strain. Hydrolysis of MAP (5% M/V) by EHesterase for 1 h at 37℃, the substrate conversion was 29.5% and ee p of the product acid (major in R configuration) was 85.1%. The optimum pH was 9.0 and temperature 50℃.[Conclusions] A new isolate Achromobacter denitrificans 1104 capable of enantioelective hydrolyzing MAP was found and identified.

Abstract:[Objective] This study was conducted to evaluate the effects of nisin on in vitro fermentation, methanogenesis and functional microbial populations of the rumen.[Methods] The negative control did not contain any additives. Monensin (5 μmol/L) was added as positive control. Nisin was added at 3 doses:3 (NI-3), 9 (NI-9), and 27 mg/100 mL (NI-27). Each treatment contained 4 replicates. Gas and methane production was measured at 0, 3, 6, 9, 12, and 24 h after incubation. Samples of culture were collected at 24 h, and used to measure rumen fermentation parameters and functional microbial populations.[Results] Compared with negative control, both nisin and monensin addition dramatically reduced gas and methane production (P<0.05). Nisin addition had no effect on pH, dry matter degradability, and organic matter degradability (P0.05). Ammonia concentration was reduced by NI-9 (P<0.05), but was not influenced by NI-3 and NI-27 (P0.05). In contrast, monensin addition significantly lowered dry matter degradability, organic matter degradability, and ammonia concentration (P<0.05), but had no influence on pH (P0.05). Compared with negative control, both nisin and monensin addition significantly reduced acetate concentration and acetate-propionate ratio (P<0.05), and increased propionate concentration (P<0.05). Quantitative real-time PCR results showed that both nisin and monensin addition had no effects on the populations of total bacteria and Bacteroides (P0.05). Compared with negative control, the populations of protozoa, methanogens, fungi and Firmicutes were not influenced by nisin (P0.05), but were significantly reduced by monensin addition (P<0.05). Both nisin and monensin addition significantly increased the populations of sulfur-reducing bacteria and Clostridium aminophilum (P<0.05), but had no influence on the population of Clostridium sticklandii (P0.05).[Conclusion] Appropriate nisin addition could reduce methanogenesis and ammoniagenesis, while had no adverse effect on feed digestion. These effects are probably associated with the variation of rumen functional microbial populations and communities.

Abstract:[Objective] Mycobaterium neoaurum MN4 is a substrate-resistant mutant strain with high-yield androstenedione. In order to further study MN4 strain substrate-resistant mechanism and androstenedione biosynthetic pathway, it is necessary to decipher the MN4 strain genome.[Methods] The genome was sequenced using highthroughput sequencing technology, and analyzed using relevant software for genome assembly, gene prediction and functional annotation, COG cluster analysis and secondary metabolite biosynthesis gene clusters prediction.[Results] The whole genome is assembled into 33 contigs, and the genome size is 5.39 Mb, GC content of 66.9% with encoding 4920 protein genes. The genome sequence was deposited in the GenBank database under the accession number JXYZ00000000.[Conclusion] This study is the first report of androstenedione producing strain Mycobacterium neoaurum MN4 genome sequence, and provides a theoretical basis for further heterologous expression of secondary metabolites on Mycobacterium neoaurum MN4.