Project description:The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.
Project description:Increasing concern about pollution of our environment calls for advanced and rapid methods to estimate ecological toxicity. The use of gene expression microarrays in environmental studies can potentially meet this challenge. We present a novel method to examine soil toxicity. We exposed the collembolan Folsomia candida to soil containing an ecologically relevant cadmium concentration, and found a cumulative total of 1586 differentially expressed transcripts across three exposure durations, including transcripts involved in stress response, detoxification, and hypoxia. Additional enrichment analysis of gene ontology (GO) terms revealed that antibiotic biosynthesis is important at all time points examined. Interestingly, genes involved in the "penicillin and cephalosporin biosynthesis pathway" have never been identified in animals before, but are expressed in F. candida’s tissue. The synthesis of antibiotics can possibly be a response to increased cadmium-induced susceptibility to invading pathogens, which might be caused by repression of genes involved in the immune-system (C-type lectins and Toll receptor). This study presents a first global view on the environmental stress response of an arthropod species exposed to contaminated soil,and provides a mechanistic basis for the development of a gene expression soil quality test. Keywords: cadmium, soil, Collembola, environmental genomics
Project description:Pentachlorophenol (PCP) as a widely used pesticide is also considered to be an endocrine disruptor. Molecular effects of chemicals with endocrine disrupting potential on soil invertebrates are largely unknown. Collembola (Folsomia candida) has been used as a model organism in ecotoxicity and in this study we explored the transcriptional expression changes of Folsomia candida in response to PCP contamination. A total of 92 genes were significantly differentially expressed at all exposure time and majority of them were found to be down-regulated. In addition to the transcripts encoding cytochrome P450s and transferase enzymes, chitin-binding protein was also identified in the list of common differentially genes. Analyses of Gene Ontology (GO) annotation and enrichment revealed that cell cycle related transcripts were significantly induced by PCP, indicating it can stimulated the cell proliferation in springtail as reported in human breast cancer cells. We also observed enrichment of functional terms related to steroid receptor and particularly twenty significant differential expressed genes involved in Chitin metabolism in response to PCP exposure. Combined with the confirmation by qPCR, our results appears that the adverse effects on reproduction of springtails after exposure to PCP can be attributed to a chemical-induced delay in the molting cycle and molting associated genes may serve as possible biomarkers for toxicological effects. In general, analysis of changes in the gene expression profiles of springtails in response to PCP exposure is useful for obtaining information on endocrine disruptor exposure of soil invertebrate and may contribute to the classification and risk assessment of relative chemicals.
Project description:The Arecibo Observatory (AO) located in Arecibo, Puerto Rico, is the most sensitive, powerful and active planetary radar system in the world [1]. One of its principal components is the 305 m-diameter spherical reflector dish (AORD), which is exposed to high frequency electromagnetic waves. To unravel the microbial communities that inhabit this environment, soil samples from underneath the AORD were collected, DNA extracted, and sequenced using Illumina MiSeq. Taxonomic and functional profiles were generated using the MG-RAST server. The most abundant domain was Bacteria (91%), followed by Virus (8%), Archaea (0.9%) and Eukaryota (0.9%). The most abundant phylum was Proteobacteria (54%), followed by Actinobacteria (8%), Bacteroidetes (5%) and Firmicutes (4%). In terms of functions, the most abundant among the metagenome corresponded to phages, transposable elements and plasmids (16%), followed by clustering-based subsystems (11%), carbohydrates (10%), and amino acids and derivatives (9%). This is the first soil metagenomic dataset from dish antennas and radar systems, specifically, underneath the AORD. Data can be used to explore the effect of high frequency electromagnetic waves in soil microbial composition, as well as the possibility of finding bioprospects with potential biomedical and biotechnological applications.
Project description:Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome.