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The goal of this study was to develop and validate a novel fosmid-clone-based metagenome isotope array approach - termed the community isotope array ( CIArray) - for sensitive detection and identification of microorganisms assimilating a radiolabeled substrate within complex microbial communities. More specifically, a sample-specific CIArray was used to identify anoxic phenol-degrading microorganisms in activated sludge treating synthetic coke-oven wastewater in a single-sludge predenitrification-nitrification process. Hybridization of the CIArray with DNA from the 14C-phenol-amended sample indicated that bacteria assimilating 14C-atoms, presumably directly from phenol, under nitrate-reducing conditions were abundant in the reactor, and taxonomic assignment of the fosmid clone end sequences suggested that they belonged to the Gammaproteobacteria. The specificity of the CIArray was validated by quantification of fosmid-clone-specific DNA in density-resolved DNA fractions from samples incubated with 13C-phenol, which verified that all CIArray-positive probes stemmed from microorganisms that assimilated isotopically labeled carbon. This also demonstrated that the CIArray was more sensitive than DNA- SIP, as the former enabled positive detection at a phenol concentration that failed to yield a 'heavy' DNA fraction. Finally, two operational taxonomic units distantly related to marine Gammaproteobacteria were identified to account for more than half of 16S r RNA gene clones in the 'heavy' DNA library, corroborating the CIArray-based identification. [ABSTRACT FROM AUTHOR]

The use of randomly generated DNA fragment sequences as probes on DNA arrays offers a unique potential for exploring unsequenced microorganisms. In this study, the detection specificity was evaluated with respect to probe-target sequence similarity using genomic DNAs of four Pseudomonas strains. Genome fragments averaging 2000 bp were found to be specific enough to discriminate 85-90% similarity under highly stringent hybridization conditions. Such stringent conditions compromised signal intensities; however, specific signals remained detectable at the highest stringency (at 75 °C hybridization) with negligible false negatives. These results suggest that, without any probe design or selection, genomic fragments can provide a reasonable specificity for microbial diagnostics or species delineation by DNA- DNA similarities. [ABSTRACT FROM AUTHOR]

Diverse environmental genes have been identified recently. To characterize their functions, it is necessary to understand which genes and what combinations of those genes are responsible for the biodegradation of soil contaminants. In this article, a 60-mer oligonucleotide microarray was constructed to simultaneously detect di- and monooxygenase genes for benzene and related compounds. In total, 148 probes were designed and validated by pure-culture hybridizations using the following criteria to discriminate between highly homologous genes: ≤53-bp identities and ≤25-bp continuous stretch to nontarget sequences. Microarray hybridizations were performed using PCR products amplified from five benzene-amended soils and two oil-contaminated soils. Six of the probes gave a positive signal for more than six soils; thus, they may represent key sequences for benzene degradation in the environment. The microarray developed in this study will be a powerful tool for the screening of key genes involved in benzene degradation and for the rapid profiling of benzene oxygenase gene diversity in contaminated soils. [ABSTRACT FROM AUTHOR]