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Applied and Environmental Microbiology, July 2004, p. 4303-4317, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4303-4317.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Detection of Genes Involved in Biodegradation and Biotransformation in Microbial Communities by Using 50-Mer Oligonucleotide Microarrays

Sung-Keun Rhee ,{dagger},{ddagger} Xueduan Liu, Liyou Wu, Song C. Chong, Xiufeng Wan, and Jizhong Zhou{dagger}*

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

Received 12 December 2003/ Accepted 19 March 2004

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°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 ~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 107 cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r2 = 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 (r2 = 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.

* Corresponding author. Mailing address: Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6038. Phone: (865) 576-7544. Fax: (865) 576-8646. E-mail: zhouj{at}ornl.gov.

{dagger} S.-K.R. and J.Z. contributed equally to this work.

{ddagger} Present address: BioDiversity Information Laboratory, Biological Resources Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejon 305-333, South Korea.

Applied and Environmental Microbiology, July 2004, p. 4303-4317, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4303-4317.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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