Use of Bioluminescence for Detection of Genetically Engineered Microorganisms Released into the Environment

The persistence and movement of strain JS414 of Xanthomonas campestris pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent X. campestris pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mist- and wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. Our bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Our results demonstrate that transgenic incorporation of the luxCDABE operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.     

Stimulation of Native Microorganisms for Improving Loose Salty Sand

Prolonged droughts and excessive water harvesting in western Asia has accelerated desertification and caused longer dry seasons of salt lakes. The Aral Sea experience has proven that dust from saline soil is a serious health issue. Various stabilization techniques to reduce wind erosion have been used in the past. However, in recent years, a potentially viable method has been developed; microbial induced calcite precipitation (MICP) has been introduced as a method of soil stabilization, though its effectiveness in saline soils remains to be examined. The effect of salt content in loose sandy soil on calcite precipitation of calcite through stimulation of native bacteria is investigated in this article. Samples with salinity up to 30% salt content were prepared and treated with different culture medium compounds. A number of tests were used to evaluate the effect of the mentioned parameters. The results show that improvement increases with increasing salinity up to 5% salt, and further increase in salinity reduces the effectiveness of improvement. It is also shown that the addition of urea in the culture medium has a significant effect on the urea hydrolysis which resulted in a five-fold increase in compressive strength. Four native strains of halotolerant urease-positive bacteria were also identified.     

Comparison of rRNA and Polar-Lipid-Derived Fatty Acid Biomarkers for Assessment of 13C-Substrate Incorporation by Microorganisms in Marine Sediments

We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in ¹³C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of ¹³C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the ¹³C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the ¹³C-labeled compounds. Similar results were obtained with the ¹³C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the ¹³C analysis of the captured SSU rRNA.     

Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.     

微生物在水环境中的存活和生长

在灭菌自来水模拟水体中,研究了7种细菌的存活和生长规律。Klebsiella pneumo-niae,Enterobacter aerogenes,Agrobacterium tumefatciens,在7天内平板计数降至0,而水体中镜检细菌总数(AODC)和活菌直接计数(DVC)结果无大变化,说明细菌已变成活的非可培养状态。Micrococcus,flavus 和 Streptococcus faecalis 的可培养菌数也可降至0。Pseudomonas sp.在48小时内由10~5降至10~2cfu/ml,随即升至10~6 cfu/ml 并持续到实验终了(41天)。Bacillus subtilis 在48小时平板计数降至10~2cfu/ml 并维持在该水平至实验结束(38天)。研究结果表明仅用涂布平板法检测多种细菌在水环境中的生存和分布是不合适的。     

Methods for Detection and Quantification of Polyphosphate and Polyphosphate Accumulating Microorganisms in Aquatic Sediments

It has been speculated that the microbial P pool is highly variable in the uppermost layer of various aquatic sediments, especially when an excessive P accumulation in form of polyphosphate (Poly‐P) occurs. Poly‐P storage is a universal feature of many different organisms and has been technically optimised in wastewater treatment plants (WWTP) with enhanced biological phosphorus removal (EBPR). In the recent past, new insights into mechanisms of P elimination in WWTP almost exclusively depended on the development and application of novel methods like ³¹P‐NMR spectroscopy and molecular methods for identifying Poly‐P accumulating microorganisms (PAO). The aim of the present review is to compile current methods potentially available for detection and quantification of Poly‐P in sediments and to complement it with yet unpublished results to validate their application in natural sediments. The most powerful tool for reliable Poly‐P quantification in sediments is the liquid ³¹P‐NMR technique which has been successfully used for Poly‐P measurements in a variety of aquatic sediments. But the microorganisms as well as mechanisms involved in Poly‐P storage and cycling are largely unknown. Therefore, we also intend to stimulate future studies focusing on these encouraging topics in sediment research via the implementation of novel methods. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Silica Gel Medium for Enumeration of Petroleumlytic Microorganisms in the Marine Environment

A silica gel medium was developed for the enumeration of petroleumlytic microorganisms in the marine environment. The medium was satisfactorily used for the investigation of the vertical distribution of bacteria in seawater from the surface to 1,000 m depth of western north Pacific central water as well as the neritic region of Japan.     

油藏极端环境中的微生物

油藏属于高温、高压、高矿化度以及油、气、水共存的极端环境,与之相适应,油藏中孕育着丰富多样的微生物.介绍了油藏极端环境、油藏微生物的一般特征、典型的微生物生理群以及随油田开发微生物分布的变化.     

Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).     

A Comparison of Methods for Clustering 16S rRNA Sequences into OTUs

Recent studies of 16S rRNA sequences through next-generation sequencing have revolutionized our understanding of the microbial community composition and structure. One common approach in using these data to explore the genetic diversity in a microbial community is to cluster the 16S rRNA sequences into Operational Taxonomic Units (OTUs) based on sequence similarities. The inferred OTUs can then be used to estimate species, diversity, composition, and richness. Although a number of methods have been developed and commonly used to cluster the sequences into OTUs, relatively little guidance is available on their relative performance and the choice of key parameters for each method. In this study, we conducted a comprehensive evaluation of ten existing OTU inference methods. We found that the appropriate dissimilarity value for defining distinct OTUs is not only related with a specific method but also related with the sample complexity. For data sets with low complexity, all the algorithms need a higher dissimilarity threshold to define OTUs. Some methods, such as, CROP and SLP, are more robust to the specific choice of the threshold than other methods, especially for shorter reads. For high-complexity data sets, hierarchical cluster methods need a more strict dissimilarity threshold to define OTUs because the commonly used dissimilarity threshold of 3% often leads to an under-estimation of the number of OTUs. In general, hierarchical clustering methods perform better at lower dissimilarity thresholds. Our results show that sequence abundance plays an important role in OTU inference. We conclude that care is needed to choose both a threshold for dissimilarity and abundance for OTU inference.     

新疆特殊环境微生物资源的研究与发展

新疆特殊环境微生物资源丰富、代谢类型多样。目前,已发现了放线菌新属2个,放线菌新种24个,根瘤菌新属1个,嗜盐古生菌2个,食用真菌195种,药用真菌97种。通过对新疆特殊环境微生物资源的分析,论述了新疆特殊环境微生物资源研发对策,明确了重点研发领域为新疆特殊环境极端环境微生物研究、新疆特色药用真菌研究和新疆维吾尔医药用植物内生菌的研究,同时建立新疆特殊环境微生物资源库及功能基因库和生物信息系统,从而实现新疆特殊环境微生物资源最大限度的开发、保护及持续利用。     

Use of 4-Nitrophenoxyacetic Acid for Detection and Quantification of 2,4-Dichlorophenoxyacetic Acid (2,4-D)/(alpha)-Ketoglutarate Dioxygenase Activity in 2,4-D-Degrading Microorganisms

Purified 2,4-dichlorophenoxyacetic acid (2,4-D)/(alpha)-ketoglutarate dioxygenase (TfdA) was shown to use 4-nitrophenoxyacetic acid (K(infm) = 0.89 (plusmn) 0.04 mM, k(infcat) [catalytic constant] = 540 (plusmn) 10 min(sup-1)), producing intensely yellow 4-nitrophenol. This reagent was used to develop a rapid, continuous, colorimetric assay for the detection of TfdA and analogous activities in 2,4-D-degrading bacterial cells and extracts.     

Rapid Quantification of Phototrophic Microorganisms and their Physiological State through their Colour

Quantification of phototrophic organisms on solid substrata together with their metabolic activity can be assessed easily, reliably and quickly through measurement of the organisms' colour. For that purpose only a chroma meter for solid substrata able to quantify the three components of colour is needed. A correlation between these three components and the number of organisms and their physiological state was demonstrated. The methodology developed here makes it possible to save time and materials in comparison with traditional microbiological methods. Moreover, it is a non-destructive method which can be used directly on site and in site. This characteristic is important when microbial environmental monitoring of cultural heritage is involved.     

Native Useful Plants of Chile: A Review and Use Patterns

Economic Botany - Native Useful Plants of Chile: A Review and Use Patterns. We compiled an inventory of the uses of the native flora of Chile by extracting uses cited in the literature until 2015....     

叶际微生物及其与外界互作的研究进展

叶际微生物及其生存环境共同形成了一个复杂的生态系统。建立在纯种分离和纯培养技术基础之上的传统研究方法只能了解其中部分叶际微生物,但对物种组成、种群结构和生态学作用等方面的认识都比较片面。近年来随着分子生物学和生物信息学的进步,人们对叶际微生物总群落的分析逐渐揭示了叶际微生物组成的多样性及其特点,以及与外界互相作用的复杂性。研究表明,植物种类、地理位置和季节差异等都不同程度地影响着叶际微生物群落的构成。本文综述了近年来国内外叶际微生物群落结构组成及其与外界互作方面的研究进展,有利于加深对叶际微生物的了解,也有助于深入理解叶际微生物与植物生长和植物病虫害防治的关联关系。     

Use of Colicin E3 for Biological Containment of Microorganisms

The genetic determinant of the lethal antibiotic colicin E3 was cloned under the control of a tightly regulated promoter in the absence of the gene for its cognate inhibitor. Combination of this killing cassette with a stringent regulatory element provided a substrate-dependent conditional suicide system that was exploited for the biological containment of a Pseudomonas putida strain. The lethality of a single gene copy and the distinct and universal cellular target of the antibiotic suggest colicin E3 as an ideal candidate for combination with other lethal functions to design highly efficient containment systems for microorganisms.     

Use of Trinitrophenylation for Quantification of Protease and Peptidase Activities

A sensitive and precise method for quantifying protease and peptidase activities is suggested. N-Terminal amino groups of peptides which are formed during hydrolysis of the substrates react with trinitrobenzenesulfonic acid (TNBS), and the trinitrophenyl (TNP) derivatives are determined spectrophotometrically. Spontaneous hydrolysis of TNBS is considerably diminished on trinitrophenylation at pH 7.4 rather than at pH 9-10 as is usually used. The trinitrophenylation method can be used to determine the initial rate of hydrolysis and the kinetics of reactions catalyzed by proteases and peptidases.