Abstract:Sulfur, as an essential element for organisms, is involved in cell energy and substance metabolism, i.e. protein, vitamins, antibiotics. In nature, sulfur exists in multiple valence states, represented respectively by element sulfur, reduced inorganic sulfur compounds (RISC) and sulfate as well as sulfur-containing organics. Sulfur oxidation is of significance in the biogeochemical sulfur cycle, which refers to the oxidation process of element sulfur and RISC by various microorganisms. Among sulfur-oxidizing microorganisms, sulfur-oxidizing bacteria are highly diverse, and their genes, enzymes and pathways of sulfur oxidation are varied. In recent years, significant progresses have been gained in many aspects, but some important issues at different levels still need to be solved. In this paper, we summarized the progresses of sulfur-oxidizing bacterial taxa and their sulfur oxidation pathways.

Abstract:Rhizobia are Gram-negative bacteria that can form a nodule with specific host plants, which can transform nitrogen in the air to ammonia nitrogen for plant utilization. Studying on the biogeographical pattern of the rhizobia is not only theoretically meaningful, but also helpful to guide the selection of rhizobial inoculant. At present, with the development of molecular biotechnology, and the accumulation of rhizobia diversity research data, rhizobial biogeography has got great progress. This paper summarizes the current researches of rhizobial biogeography, and indicates the direction in future research.

Abstract:Clustered regularly interspaced short palindromic repeats (CRISPR) is the genetic structure found in most of bacteria and archaea. The function of CRISPR is defending against the invasion of exogenous DNA (plasmid, phage, etc.), preventing heterologous gene transfer. [Objective] This study is to study the structural differences of CRISPR loci in several Salmonella sp.[Methods] The molecular structure of CRISPR and the relationship between CRISPR and plasmid was analyzed in 30 strains, including Salmonella gallinarum, S. typhimurium, S. choleraesuis and S. enteritidis by bioinformatics.[Results] CRISPR structure existed in the 30 strains of Salmonella, totally 61 conformed CRISPR loci and 12 possible ones. Neither repeat sequence nor cas1 gene could be used for the classification of these strains.[Conclusion] Although the number of CRISPR loci and spacer sequence have no statistical relationship with the number of plasmid, the spacer sequence has genetic homology with plasmid, transposons, integron and resistant genes. This suggests that Salmonella sp. are attacked by exogenous genes constantly during evolution, and CRISPR loci of Salmonella sp. play a role in resisting exogenous genes.

Abstract:[Objective] We studied the biological characteristics of the fliD mutant strain to clarify the function and mechanism of fliD gene.[Methods] The fliD mutant was constructed through homologous recombination, and biological characteristics of the fliD mutant were identified in comparison with the wild type of Campylobacter jejuni.[Results] Mutant NCTC11168△fliD presented the same biochemical indexes and similar growth curves of the wild type. After being inoculated into semisolid Mueller Hinton ager plate, the wild strain presented diffusion growth phenotype, but fliD mutant could only grow in the site inoculated, indicating that its motility was significantly reduced. The adhesion rate and invasion rate of fliD mutant were 164.00±19.49 and 55.00±6.09 respectively, which means that the mutation of fliD gene significantly reduced the rates of adhesion and invasion.[Conclusion] fliD gene was not only an important molecular basis for bacterial motility but also necessary for the adhesion and invasion of Campylobacter jejuni infection cells.

Abstract:[Objective] To discover the community structure and diversity of aerobic anoxygenic phototrophic bacteria (AAPB) in different types of soil crusts (BSCs) and soils under them.[Methods] The pufM gene was sequenced via the Illumina MiSeq second-generation high-throughput sequencing platform, and the community structure and diversity of AAPB were analyzed by bioinformatics analysis.[Results] In the BSCs and soils under them, Proteobacteria and Alpha-Proteobacteria were the main phylum, and the main genus were Bradyrhizobium (9.69% to 90.02%), Brevundimonas (0.83% to 16.04%), Methylobacterium (1.74% to 12.56%), Rhodospirillum (0.91% to 32.87%), Roseiflexus (0.02% to 1.79%) and Sphingomonas (0.13% to 11.23%). Biological soil crusts and soil under them had the similar community structure, but different in the abundance. With the development of BSCs, the species richness and diversity of the crusts and their underlying soils increased.[Conclusion] The community structure of AAPB in BSCs of Hunshandake sandy land is relatively complex, which is significant different from that in water and general soil environment. The diversity of AAPB is high and the diversity increases with the BSCs development, which suggests that AAPB plays an important role in the stability of desert ecosystem.

Abstract:[Objective] LuxS/AI-2-type quorum sensing participates in regulation of bacterial biological properties by AI-2, which is synthesized by the enzymes LuxS (encoded by luxS). The aim of this study is to elucidate luxS effects on biological characteristics of different serotypes in Avian Pathogenic Escherichia coli (APEC).[Methods] The biological characteristics of 3 APEC strains, including APECO₁ (serotype O₁), DE17 (serotype O₂) and E940 (serotype O₇₈) and their luxS mutant strains were analysed by growth curve, biofilm-producing, rdar morphology, bacterial motility and sensitivity to antibiotics.[Results] luxS knockout, eliminating AI-2 formation, had no influence on bacterial growth, but significantly decreased biofilm-producing in E940 and APECO₁ (P<0.05), without effect on DE17. Results of rdar morphology indicate that the APECO₁ strain showed remarkable change in rdar morphology compared with luxS mutant, but no change in DE17 and E940. Moreover, deletion of luxS lead to decreasing the motility of APECO₁ and DE17, but no influence on E940 strain. The transcriptional levels of fliG and fliI were significantly decreased in APECO1, DE17 and E940 (P<0.05). Furthermore, results of drug-resistance indicate that luxS inactivation caused APECO₁ resistant to cefepime, amikacin, and sensitive to chloramphenicol. luxS inactivation also caused E940 strain sensitive to chloramphenicol, but no significant effect on drug-resistance of DE17 strain.[Conclusion] LuxS plays a significant role in regulation of biological properties in APEC.

Abstract:Planktonic bacteria are indispensable in marine ecosystems and play a key role in the process of marine biogeochemical cycling. [Objective] The aim of this study was to analyze the bacterial community structure in different functional sea areas of Zhoushan archipelago.[Methods] Samples were collected in surface seawater from Zhoushan Archipelago of the East China Sea in 8 typical sites representative of 8 functional zones in the summer (August) of 2016. We used high throughput sequencing to study bacterial diversity and community structure in seawater of the of Zhoushan archipelago based on the bacterial 16S rRNA gene. Flow cytometry was used to reveal the abundance of bacteria in each site. The technique of Canonical correspondence analysis was used to analyze the effects of the environmental factors on bacterial community composition.[Results] In this research, 305487 raw reads were obtained. OTU cluster analysis was performed at the 97% similarity level, and all the sequences were grouped into 1088 OTUs, including 29 phyla, 62 classes, 138 orders, 239 families, and 416 genera. The bacterial community structure of each station were different, but they all included three dominant strains of Flavobacteria, Alphaproteobacteria and Gammaproteobacteria. A canonical correspondence analysis showed that different stations and different Phyla were affected by different ecological factors, Cyanobacteria was most affected by nitrate, Parcubacteria was most affected by temperature, and phosphate had little effect on all bacteria in this study area. The prediction of the potential function of marine microflora showed that the sea areas of each site were more prominent in Amino Acid Metabolism, Carbohydrate Metabolism, Membrane Transport and so on, which provided a new direction for marine microbial research in the future.[Conclusion] High throughput sequencing analysis can accurately reveal the bacterial structure information of marine bacteria. This research provides references for the association of bacterial composition and diversity with environmental factors. The large amount of data that was obtained not only as a response of Zhoushan marine function zoning purposes of the case but also lay foundations for further bacterial community structure research in the east China sea.

Abstract:[Objective] To isolate phosphate-solubilizing strains from crop rhizosphere soil for functional composite microbial community and bio-fertilizer production.[Methods] The phosphate-solubilizing capacity was evaluated by the halo zone of dissolving phosphate and molybdenum antimony colorimetric. Phosphate-solubilizing fungi were identified based on their morphological and cultural characteristics and β-tubulin sequence analysis. Acid producing substances of phosphate solubilizing fungus were analyzed by GC-MS and the biological compatibility of microbial strains was tested by medium method.[Results] Two phosphate-solubilizing fungi were screened from soybean rhizosphere soil, P1-1, P2-2, which were identified as Aspergillus niger and A. tubingensis. The volatile acids (oxalic acid, gluconic acid, lactic acid and glycerol acid) were detected in both fungi. They were compatible with a group of nematicidal microorganisms (Purpureocillium lilacinum + Streptomyces olivaceus + Ochrobactrum pseudogrignonense). The D/d (diameter of the halo zone/diameter of the colony) value of P1-1 and P2-2 was detected after culturing on three media with Ca₃(PO₄)₂, Zn₃(PO₄)₂ and phosphorite at 25℃ for 5 days, and the available phosphorus contents were detected after 5 days culturing in liquid medium containing Ca₃(PO₄)₂, Zn₃(PO₄)₂ and phosphorite. The results indicated that the two phosphate-dissolving fungi had similar capability in dissolving phosphorus.[Conclusion] Two high-efficient phosphate solubilize fungi were obtained, and they could activate a variety of insoluble phosphorus sources accompanied by the production of volatile acidic substances. The two strains showed good biological compatibility with a group of nematicidal microorganisms.

Abstract:[Objective] Cytochrome P450 (CYP) plays a significant role in mycotoxin metabolism of necrotrophic fungus and may be related to pathogenicity. The objective of the study was to reveal the function of CYP genes in infection process.[Methods] The knockout cassette was constructed using Double-joint PCR and mutants were obtained and confirmed by PEG-mediated transformation of protoplasts, PCR and Southern blotting analysis. Gene complemented mutants were constructed by gap repair technology. The PDA routine culture was selected to analyze the vegetative growth of mutants. In vitro inoculation to apple twigs was used to detect the pathogenicity. Salient differences were analyzed by SPSS and qRT-PCR was used to detect the expression of melanin gene cluster.[Results] Compared with the original strain 03-8, the colony color of deletion mutant changed from light yellow to white, and the amount of propagulum reduced by 51.3%. The relative expression level of Vmcyp5-knockout mutant was assayed by qRT-PCR, and mutant's melanin gene cluster was down-regulated. More importantly, the pathogenicity of deletion mutant was decreased by 24.5%. Complementation mutant was almost back to the level of the original strain 03-8 in colony color, propagulum and pathogenicity.[Conclusion] Vmcyp5 may be related to melanin biosynthesis and propagulum formation, and participates in pathogen process of Valsa mali.

Abstract:[Objective] We studied the effect of different nitrogen source on bacteria from porcine small intestine.[Methods] Digesta samples from different segments of the small intestine were inoculated into media containing enzyme-treated protein (soybean meal, rapeseed meal or fish meal) and incubated for 12 h.[Results] After incubation of jejunal and ileal microbes, the contents of ammonia and microbial crude protein increased in groups with nitrogen resources, especially the groups with rapeseed meal which had the highest increment of microbial crude protein. However, concentrations of microbial crude protein and ammonia decreased in duodenal groups. Besides, concentrations of volatile fatty acid and lactate increased in all groups, and acetate concentrations were more than 50% of the total volatile fatty acid concentration. The contents of propionate and butyrate increased in duodenal groups, whereas the contents of lactate decreased during the last four hours. However, propionate and butyrate were not changed in jejunal and ileal groups. The copy numbers of total bacteria, Firmicutes, Bacteroides and Lactobacillus increased during the experiment, but it was not significantly different between groups with different enzyme-treated protein.[Conclusion] Protein after digestion would be utilized by jejunal and ileal microbes, which mostly used to produce microbial crude protein. Duodenal microbes would utilize lactate and produce propionate and butyrate.

Abstract:[Objective] To compare the inhibition effects of antibodies against extracellular fibrinogen-binding protein (EfB) and fibronection binding protein (FnBPA) to clumping factor A (ClfA) of Staphylococcus aureus (SA) to bovine mammary epithelial cells (bMEC) by immunization method.[Methods] The antibodies were obtained by immunizing rabbits with the recombinant plasmids Efb or FnBPA in-combination with ClfA. The antibodies inhibition effects on adereing of 2 SA strains to bMEC were tested using plate counting method and observed by fluorescent staining of SA and bMEC, respectively.[Results] Our results indicated that the antibodies had different inhibition effects on adhesion of SA (GY278 and GY309). ClfA antibody showed the highest inhibition ability. FnBPA-A showed greater inhibition enhancement to ClfA than FnBPA-D.[Conclusion] The in-combination immunization of FnBPA-A, FnBPA-D and EfB with ClfA could affect the antibodies inhibition to various degrees and this result provided experiment basis for the development of bovine mastitis vaccine.

Abstract:[Objective] This study aimed to investigate the effect of environmental factors on a monoethylhexyl phthalate hydrolase (MehpH) activity, model the 3-D structure of the enzyme and the interaction of the catalytic amino acid residues with the substrate.[Methods] The effect of environmental factors was determined by the standard enzyme assay. Primary structure analysis and 3-D model prediction were completed by DNAMAN (Version 2.1) and SWISS-MODEL server respectively and the results visualized by PyMOL software. Autodock tools, Swiss-PDB viewer and PyMOL were used to investigate the interactions between the enzyme and monoethylhexyl phthalate (MEHP).[Results] The primary structure of this enzyme was similar to MehpH from Gordonia sp. P8219 with different optimum temperature and pH (40℃ and 8.0, respectively). The enzyme was stable in presence of organic solvents, detergents and ions. However, it was inhibited by 2 mol/L of Ni²⁺, Fe³⁺, Cu²⁺, Zn²⁺ ions, 1 mol/L phenylmethylsulfonyl fluoride (PMSF), 0.5 mol/L paraoxon, 1 mol/L phenyl glyoxal (PGO), 2 mol/L diethyl pyrocarbonate (DEPC) and 5 mol/L eserine. The pentapeptide motif GXSXG and catalytic triad HSD conserved in serine hydrolases were present in the sequence. The docking result showed that the amino residues Thr152 and Ser230 were much conserved among the hydrolases and closely associated with MEHP (5.8 Å and 3.6 Å respectively) and thus may play important roles in the catalytic process. However, MEHP was not very close to the catalytic triad acid residues Ser125, His291, Asp259.[Conclusion] This study showed that MehpH in YC-RL2 was fairly stable in presence of organic solvents, detergents and metal ions indicating its application potential. The structural and catalytic analysis provides important information for further investigation of catalytic mechanism and enzymatic modification.

Abstract:[Objective] To investigate the prevalence, antimicrobial susceptibility, and genetic characteristics of Staphylococcus aureus from ready-to-eat foods (stewed meat, roast, salad and pasteurized milk) and vegetables from 15 representative cities of China and to provide baseline information for effective tracing S. aureus source and controlling food contamination.[Methods] All samples were subjected to qualitative and most probable number (MPN) analysis for S. aureus according to the National Food Safety Standard-Food microbiological examination:S. aureus. Antimicrobial susceptibility of all isolates was evaluated using the Kirbye-Bauer disk diffusion and mecA-positive isolates was obtained by PCR. The sequence types of S. aureus were performed via multilocus sequence typing (MLST).[Results] In total 540 food samples, 9.3% (50/540) were tested positive for S. aureus, of which the most polluted foods were stewed meat (16.3%, 30/184) followed by roast (9.2%, 6/65), and vegetable showed lowest prevalence (4.0%, 6/150). Most probable number (MPN) analysis showed that 62.0% samples were ranged from 0.3 to 1 MPN/g, and three samples exceeded 110 MPN/g. 82.0% isolates were resistant to ampicillin and penicillin G, and 64.0% isolates were multi-drug resistant. In addition, the mecA-positive isolates were both belonged to the SCCmecIVa subtype using staphylococcal cassette chromosome mec (SCCmec) typing. Furthermore, 14 sequence types (STs) were obtained by MLST, including two novel STs (ST3595 and ST3847).[Conclusion] The general multi-drug resistance exhibited by S. aureus was still the most serious issue of common concern, which posing a health risk for consumers. In addition, the antimicrobial susceptibility of S. aureus was highly associated with STs. Therefore, it is necessary to provides scientific data for further analysis of epidemic trends and risk assessment of bacteria prevalent in foods.

Abstract:[Objective] We studied the regulatory role of rasfonin in mediating sunitinib induced autophagy and apoptosis.[Methods] We used both methanethiosulfonate assay and colony growth assay to detect the cell viability and proliferation. In addition, we used electronic and fluorescence microscopy to examine the formation of autophagosome, as well as carried out immunofluorescence or immunoblotting to determine autophagy and apoptosis.[Results] Both rasfonin and sunitinib could induce autophagy and caspase-dependent apoptosis in renal carcinoma cells. Notably, low dose of rasfonin enhanced sunitinib-dependent autophagy and apoptosis, meanwhile sunitinib and rasfonin synergistically inhibited cell viability. In addition, both sunitinib and rasfonin inhibited the phosphorylation of mammal target of rapamycin and increased the activity of extracellular regulated protein kinases.[Conclusion] Rasfonin promotes sunitinib-induced autophagy and caspase dependent apoptosis, and strengthens the cytotoxic effect of sunitinib in renal carcinoma cells.

Abstract:[Objective] The present study was to clone a novel alpha-amylase from marine bacterium Pseudoalteromonas sp. K8 isolated from the sediment of Kongsfjorden and characterize the enzyme.[Methods] We cloned a cold-active and salt-tolerant α-amylase Amy3, from Pseudoalteromonas sp. K8. The protein was expressed in Escherichia coli, purified by Ni-NTA and characterized.[Results] Using soluble starch as substrate, the optimum pH of Amy3 was about pH 8.5, and more than 40% of the maximal activity maintained in the pH range of 6.5 to 10. Amy3 showed the maximum activity at 25℃, and retained more than 50% activity at 0℃. It exhibited improved catalytic activity and thermostability in NaCl solution, with maximal activity occurring at 800 mmol/L NaCl/KCl, and more than 50% of maximal activity retained after incubation in 2 mol/L NaCl for 80 h at 25℃. NaCl did not cause significant change of the global tertiary structure of Amy3, whereas influenced the catalysis efficiency. Amy3 hydrolyzed amylopectin preferentially, and could also hydrolyze wheat starch, corn starch and tapioca starch.[Conclusion] The results indicated that the alpha-amylase Amy3 is a cold-active and salt-tolerant α-amylase with potential use in basic research and industry.

Abstract:[Objective] The aim of this experiment was to study the effects of zinc-bearing palygorskite (Zn-Pal) on rumen bacterial diversity in vitro.[Methods] We prepared Zn-Pal by the ion-exchange, and evaluated the compositions of bacterial communities in 60 samples based on 16S rDNA genes.[Results] We obtained a total of 1490959 effective sequences and 87662 OTUs. The bacterial diversity in the treatment IV group increased at 24 h, and the abundance of the treatment IV group increased at 48 h. Bacterial community composition analysis shows that the dominant phyla were Bacteroidetes, Firmicutes, Proteobacteria and Lentisphaerae. Compared with the control group, Firmicutes in treatment groups significantly increased (P<0.05) at 24 h and 48 h, whereas Bacteroidetes in the treatment IV group was decreased (P<0.05) at 48 h. At the genus level, the sequences could be assigned to 124 different genera. The content of Prevotella had no significant difference between the control and treatments. The relative abundance of Treponema in the treatment IV group was significantly higher at 48 h than in the control group (P<0.05), whereas the relative abundance of Victivallis and Pseudobutyrivibrio in treatment IV was lower.[Conclusion] Zn-Pal may affect rumen fluid bacterial diversity in dairy cows, and the degrees of this influence varied with the dose and time of Zn-Pal.