Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays

• Published in: Applied and Environmental Microbiology Vol. 70; no. 7; pp. 4303 - 4317
• Main Authors: Rhee, S.K, Liu, X, Wu, L, Chong, S.C, Wan, X, Zhou, J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.07.2004
• Subjects: Bacteria - genetics; benzene; Biodegradation; Biodegradation, Environmental; Biological and medical sciences; bioremediation; Biotransformation; Burkholderia; Comamonas; DNA probes; ethyl benzene; Fundamental and applied biological sciences. Psychology; gene expression; genes; Hybridization; Methods; microarray technology; Microbiology; naphthalene; Naphthalenes - metabolism; Oligonucleotide Array Sequence Analysis - methods; polluted soils; polycyclic aromatic hydrocarbons; Polymerase Chain Reaction; Pseudomonas; Quantitative genetics; Ralstonia; Rhodococcus; Sensitivity and Specificity; soil bacteria; Soil contamination; Soil Microbiology; Soil microorganisms; soil pollution; Test methods; toluene; xylene; Biodegradation; Analysis method; Gene; Biotransformation; Oligonucleotide; Detection; Microbial community
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.70.7.4303-4317.2004

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 10(7) 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.