title: Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays.
abstract: To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50 degrees C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was approximately 5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 10(7) cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r(2) = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r(2) = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.
authors.affiliation: Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6038, USA.
authors.forename: Sung-Keun
authors.initials: SK
authors.lastname: Rhee
authors.forename: Xueduan
authors.initials: X
authors.lastname: Liu
authors.forename: Liyou
authors.initials: L
authors.lastname: Wu
authors.forename: Song C
authors.initials: SC
authors.lastname: Chong
authors.forename: Xiufeng
authors.initials: X
authors.lastname: Wan
authors.forename: Jizhong
authors.initials: J
authors.lastname: Zhou
mesh_terms: D001419:Bacteria / Q000235:genetics*; D001673:Biodegradation, Environmental; D001711:Biotransformation; D009281:Naphthalenes / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
chemical_list: D009281:Naphthalenes; C031721:naphthalene