微生物学报最新文献

Objective: A thermostable esterase EstZ1 from Bacillus sp. HJ14 able to degrade diethyl-phthalate (DEP) was heterologously expressed in Escherichia coli BL21(DE3) and characterized.

Methods: Full-length EstZ1 was obtained based on specific amplification and genome sequencing, and amino acid sequence of EstZ1 was analyzed. EstZ1 was expressed in Escherichia coli BL21(DE3) using the pEASY-E2 expression system. EstZ1 was purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography, and the enzyme was characterized. The degradation products from DEP were detected by high-pressure liquid chromatography and electrospray ionization mass spectrometry.

Results: The 903 bp full-length EstZ1 encoded 300 amino acid residues (EstZ1:33.84 kDa). EstZ1 showed the highest identity of 98% with hormone-sensitive lipase (HSL)-like family in NCBI databases. The optimal temperature and pH was 50℃ and 9.0, respectively, with p-NP butyrate as the best substrate. Meanwhile, it was stable between 40 and 70℃, pH 7.0 to 9.5. Most of metal ions, chemical agents had little impact. DEP could partially be degraded by EstZ1 to its corresponding monoalkyl and alcohol.

Conclusion: Our findings may serve as reference for phthalate esters degradation.

Objective: Marine microorganisms have a great potential in producing biologically active secondary metabolites. In order to study the diversity and antimicrobial activity, we explored 9 sediment samples in different observation sites of Jiaozhou bay.

Methods: We used YPD and Z2216E culture medium to isolate bacteria from the sediments; 16S rRNA was sequenced for classification and identification of the isolates. Then, we used Oxford cup method to detect antimicrobial activities of the isolated bacteria against 7 test strains. Lastly, we selected 16 representatives to detect secondary-metabolite biosynthesis genes:PKSI, NRPS, CYP, PhzE, dTGD by PCR specific amplification.

Results: A total of 76 bacterial strains were isolated from Jiaozhou bay; according to the 16S rRNA gene sequence analysis. These strains could be sorted into 11 genera belonging to 8 different families:Aneurinibacillus, Brevibacillus, Microbacterium, Oceanisphae, Bacillus, Marinomonas, Staphylococcus, Kocuria, Arthrobacters, Micrococcus and Pseudoalteromonas. Of them 34 strains showed antimicrobial activity against at least one of the tested strains. All 16 strains had at least one function genes, 5 strains possessed more than three function genes.

Conclusion: Jiaozhou bay area is rich in microbial resources with potential in providing useful secondary metabolites.

DNA phosphorothioate modification was the first reported physiological modification in the DNA backbone. Five putative proteins encoded by the five-member dndABCDE gene cluster replaced the non-bridging oxygen in the sugar-phosphate backbone with a sulfur. Phosphorothioate modification occurrs in sequence-selective and Rp stereo-specific manner in diverse bacterial stains. In recent years, researchers have made systemic achievements in this area. To have a comprehensive understanding of this unusual modification, we reviewed the discovery and research progress in DNA phosphorothioate modification and also discussed opportunities and challenges in the future.

Objective: We assessed the phylogenetic relationship of Sugarcane streak mosaic virus (SCSMV) according to NIa sequences, to infer the prevalence and variation of SCSMV and to prevent and control this virus.

Methods: Leaf samples with mosaic symptom were collected from sugarcane-growing areas in Yunnan province and the Chinese national nursery of sugarcane germplasm resources (NNSGR). NIa sequences of SCSMV were determined by RT-PCR, and analyzed by Splits Tree, RDP, PhyML and DnaSP softwares, in aspect of phylogenetic, selection, and gene flow.

Results: We obtained 23 NIa sequences; clear recombination site was not found in NIa; a novel cluster formed by SCSMV Yunnan isolates determined here was found; strong purifying selection was found in NIa of SCSMV; and the gene flow of SCSMV subpopulations between sugarcane-growing areas in Yunnan province and the NNSGR was not frequent.

Conclusion: Similar with P1, HC-Pro and CP genes, SCSMV isolates could be divided into five clusters. NIa of SCSMV Yunnan isolates showed high genetic diversity and clear geographical distribution.

Objective: To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3).

Methods: We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy.

Results: We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3.

Conclusion: This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.

Objective: Under conventional cultivation conditions zebrafish harbors numerous microbes from the environment, leading to activation of its innate immune systems and interfering the results of relevant studies. We aimed to establish a germ-free zebrafish embryo model suitable for studies of host immune responses to infections.

Methods: A germ-free cultivation process including simple disinfection of the fertilized eggs and growth in a positive-pressured thermostatic isolator. Sterility testing of germ-free zebrafish embryos and water samples was done according to the national standards. The transcriptional level of TLRs, the mark genes indicating activation of the innate immune system, was detected by qPCR. Listeria monocytogenes was used as an infection model.

Results: The cultivation system and disinfection process could ensure germ-free status as shown by absence of microbes in zebrafish embryos and egg water. TLRs were barely detectable in zebrafish raised in the germ-free system, but highly induced in conventionally raised zebrafish or in germ-free zebrafish immersion-infected with pathogenic Listeria monocytogenes. The germ-free fish was sensitive to infection by L. monocytogene EGDe at a 100-CFU dose with 100% mortality in one week, while its isogenic mutants Δmpl and ΔplcB exhibited reduced death (70% and 40%, respectively). Macrophages were recruited around the intestine in EGDe immersion infected fish, but not in Δmpl and ΔplcB infected fish.

Conclusion: Zebrafish embryos produced by this simple process were free of microbes and could be used to study the innate immune responses and the pathogenesis of microbial pathogens.

Carotenoids, as a group of over 700 valuable unsaturated terpene compounds classified as carotene and xanthophyll family, are endowed with powerful nutritional value. Phytoene dehydrogenase is the key rate-limiting enzyme in carotenoids biosynthesis pathway, involved in catalyzing the conversion from colorless hydrocarbon phytoene to other pigmented carotenoids, and plays an essential central regulation role. The function of phytoene dehydrogenases from different organisms exist diversity. CrtP, CrtQ and isomerase CrtH are essential for the formation of lycopene in most Cyanobacteria, whereas PDS, ZDS and isomerase Z-ISO, CrtISO are in charge of producing lycopene in most algae and plants. Nevertheless, there is only one CrtI-type for the formation of neurosporene, lycopene or dehydrolycopene in most bacteria and fungi. In this review, isolation, characterization, functional diversity, transcription regulatory mechanisms and phylogenetic analysis of phytoene dehydrogenase from different organisms are illustrated. This paper will provide insights into phytoene dehydrogenase and may facilitate the optimization of carotenoids production in genetic engineering strategy.

Objective: This experiment was conducted to study different metabolic patterns of pig hindgut bacteria on aromatic amino acids by an in vitro fermentation method.

Methods: Ileum, cecum and colon chyme in Duroc, Landrace and Yorkshire goods hybridization pigs were taken as inoculum. The single aromatic amino acid concentration was kept 10 mmol/L in fermentation flask. Then the fermentation flask was incubated at 37℃ for 24 h. Gas production was measured at 4, 8, 12, 16 and 24 h, and samples of fermentation collected at 0 h and 24 h were used to measure ammonia nitrogen NH3-N and microbial crude protein (MCP). Denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor and quantify the development of bacteria community in zymotic fluid.[

Results: The concentrations of NH3-N and MCP were significantly affected by aromatic amino acids and intestinal segments (P<0.01). Intestinal segments also affected gas production (GP) significantly (P0.01). NH3-N, MCP and GP were affected by interaction of aromatic amino acids and intestinal segments. DGGE analysis showed bacteria of aromatic amino acids shared amount of bands together, especially similarity analysis of DGGE profile of Phe and Tyr in ileum, Tyr and Trp in colon were 87.9% and 80.5% separately. Shannon diversity indices analysis revealed that aromatic amino acids in cecum and colon varied significantly (P<0.05). Real-time PCR results showed that the quantity of total bacteria were affected by aromatic amino acids and intestinal segments significantly (P<0.05).

Conclusion: The potential as proportion of different aromatic amino acids are different. Compared with Trp and Phe, the diversity of bacteria utilizing Tyr in cecum or colon is low; compared with Tyr and Trp, a large number of Phe participated in synthesizing bacteria.The fermentation pattern of specific aromatic amino acids in different intestinal segment was unique. Compared with ileum and cecum, much more aromatic amino acids participated in the synthesis of bacteria in colon.

We compared different methods used in single-cell isolation and whole genome amplification of microbial cells, and summarized research progresses in single-cell genomics (SCG) in studying environmental microbes. The studies of single cell and SCG have become one of the hot topics in life sciences in recent years. As a newly developed technology following microbial metagenomics, microbial SCG can efficiently explore large amounts of information of the uncultivable microbial genomics from environment. Generally, following steps need to be taken to obtain microbial SCG:isolation of single microbial cells, whole genome amplification of the single cells, amplicon sequencing, and data analysis. Microbial SCG can be widely used to explore new functional genes that are not detectable by metagenomics and other traditional methods, detect uncultured microbes with extremely low abundance, and study the life evolution of microbial cells.

Objective: The aim of this study was to isolate and characterize manganese (Ⅱ)-oxidizing bacteria from surrounding area of manganese mine.

Methods: Mn (Ⅱ)-oxidizing strains were isolated based on the ability to produce brown Mn oxides on agar plates. The presence of Mn oxides was confirmed by using the leucoberbelin blue (LBB) assay. The isolate was identified by morphological and physiological characterization and sequence analyses of 16S rRNA gene, gyrB gene and gyrA gene. The phylogenetic relationship between the isolated strain and reported Mn (Ⅱ) oxidizers was also analyzed. LBB assay was used to indicate the kinetics of Mn (Ⅱ) oxides formation. The Mn oxides morphology and chemical contents were determined by scanning electron microscope with energy dispersive spectrometer and X-ray diffraction.

Results: An isolate, named strain CP133, with high manganese oxidizing activity was obtained and identified as Bacillus cereus. There were some phylogenetic differences between strain CP133 and other Mn (Ⅱ)-oxidizing Bacillus species isolated from deep sea and soils. Strain CP133 produced amorphous manganese oxides that adhered to spore surface after the stationary phase of the cell culture. About 0.3 mmol/L manganese oxides was obtained after 12 days.

Conclusion: A Mn (Ⅱ)-oxidizing Bacillus cereus was successfully isolated. Our results suggest the diversity of Mn (Ⅱ)-oxidizing Bacillus species, and help understanding biogeochemical cycles in manganese mine and surrounding soils.