The biosphere is dominated by microorganisms (32), yet most microbes in nature have not been studied. Traditional methods for culturing microorganisms limit analysis to those that grow under laboratory conditions (14,25). The recent surge of research in molecular microbial ecology provides compelling evidence for the existence of many novel types of microorganisms in the environment in numbers and varieties that dwarf those of the comparatively few microorganisms amenable to laboratory cultivation (7,13,31). Corroboration comes from estimates of DNA complexity and the discovery of many unique 16S rRNA gene sequences from numerous environmental sources (8,10,28). Collectively, the genomes of the total microbiota found in nature, which we termed the metagenome (11), contain vastly more genetic information than is contained in the culturable subset. Given the profound utility and importance of microorganisms to all biological systems, methods are needed to access the wealth of information within the metagenome.Cloning large fragments of DNA isolated directly from microbes in natural environments provides a method to access soil metagenomic DNA. Previously, we investigated the use of the bacterial artificial chromosome (BAC) vector to express Bacillus cereus genomic DNA (20). The advantage of BAC vectors is that they maintain very large DNA inserts (greater than 100 kb) stably in Escherichia coli (23), facilitating the cloning of large fragments of DNA. Our results demonstrated that expression of heterologous DNA from B. cereus in an E. coli BAC system was detectable at a reasonable frequency (20), validating the idea that the low-copy BAC vector (one to two per cell) (23) could be used to express foreign DNA from foreign promoters in E. coli.Here we describe the construction and initial screening of two BAC libraries made with DNA isolated directly from soil. We found detectable levels of several biochemical activities from BAC library clones. Sequence analysis of selected BAC plasmids encoding such activities and of 16S rRNA genes in one of the libraries confirms the novelty of the genomic information cloned in our libraries. The results show that DNA extracted directly from soil is a valuable source of new genetic information and is accessible by using BAC libraries. Our results demonstrate that both traditional and functional genomics of uncultured microorganisms can be carried out by this approach and that screening of metagenome libraries for activities or gene sequences can provide a basis for conducting genomic analyses of uncultured microorganisms. MATERIALS AND METHODSBacterial strains and plasmids. E. coli strain DH10B and the BAC vector pBeloBAC11 were provided by H. Shizuya (15). Bacillus subtilis strain BR151(pPL608) is strain 1E32 (lys-3 metB10 trpC2) from the Bacillus Genetic Stock Center, Ohio State University. -TnphoA was used as described before (20).
Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.
We combine a scanning near-field microwave microscope with an atomic force microscope for use in localized thin film dielectric constant measurement, and demonstrate the capabilities of our system through simultaneous surface topography and microwave reflection measurements on a variety of thin films grown on low resistivity silicon substrates. Reflection measurements clearly discriminate the interface between approximately 38 nm silicon nitride and dioxide thin films at 1.788 GHz. Finite element simulation was used to extract the dielectric constants showing the dielectric sensitivity to be Deltaepsilon(r)=0.1 at epsilon(r)=6.2, for the case of silicon nitride. These results illustrate the capability of our instrument for quantitative dielectric constant measurement at microwave frequencies.
The terahertz (THz) regime (0.1-10 THz) is rich with emerging possibilities in sensing, imaging and communications, with unique applications to screening for weapons, explosives and biohazards, imaging of concealed objects, water content and skin. Here we present initial surveys to evaluate the possibility of sensing plastic explosives and bacterial spores using field-deployable electronic THz techniques based on short-pulse generation and coherent detection using nonlinear transmission lines and diode sampling bridges. We also review the barriers and approaches to achieving greater sensing-at-a-distance (stand-off) capabilities for THz sensing systems. We have made several reflection measurements of metallic and non-metallic targets in our laboratory, and have observed high contrast relative to reflection from skin. In particular, we have taken small quantities of energetic materials such as plastic explosives and a variety of Bacillus spores, and measured them in transmission and in reflection using a broadband pulsed electronic THz reflectometer. The pattern of reflection versus frequency gives rise to signatures that are remarkably specific to the composition of the target, even though the target's morphology and position is varied. Although more work needs to be done to reduce the effects of standing waves through time-gating or attenuators, the possibility of mapping out this contrast for imaging and detection is very attractive.
By illuminating the sample with a broadband 10-300 GHz stimulus and coherently detecting the response, we obtain reflection and transmission spectra of common powdered substances, and compare them as a starting point for distinguishing concealed threats in envelopes and on personnel. Because these samples are irregular and their dielectric properties cannot be modulated, however, the spectral information we obtain is largely qualitative. To show how to gain quantitative information on biological species at micro- and millimetre-wave frequencies, we introduce thermal modulation of a globular protein in solution, and show that changes in single-wavelength microwave reflections coincide with accepted visible absorption spectra, pointing the way towards gaining quantitative chemical and biological spectra from broadband terahertz systems.
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