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°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 ϳ5 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 ؋ 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.The transformation of environmental contaminants is a complex process that is influenced by the nature and amount of the contaminant present, the structure and dynamics of the indigenous microbial community, and the interplay of geochemical and biological factors at contaminated sites (1,14,26,36). A better understanding of the processes inherent in natural bioremediation requires, in part, a better understanding of microbial ecology. However, conventional molecular methods (PCR-based technologies, such as gene cloning, terminal-restriction fragment len...

Environmental microarray analyses of Antarctic soil microbial communities

“…Furthermore, probe signals were a summation of signals derived from multiple specific signals within a phylum, whereas real-time PCR results were generated by the use of group-specific primers. Even when using probes and primers targeting the exact same site, correlations between real-time PCR and microarray intensity are not perfect (for example, r ¼ 0.87 in Rhee et al, 2004). Here again, both methods obviously rely on available sequence data for primer and probe design and may either miss some members of the target phyla (incomplete coverage) or overlap with the related phyla (incomplete specificity).…”

GeoChip: a comprehensive microarray for investigating biogeochemical, ecological and environmental processes

“…Microarrays are a recently developed, powerful genomic technology and are widely used to study gene expression (Schena et al, 1995;Lockhart et al, 1996;DeRisi et al, 1997;Liu et al, 2003;Gao et al, 2004), monitor environmental processes (Loy et al, 2002;Taroncher-Oldenburg et al, 2003;Zhou, 2003;Bodrossy and Sessitsch, 2004;Rhee et al, 2004;Steward et al, 2004;Tiquia et al, 2004;Zhou et al, 2004;Wu et al, 2006a), and potentially apply to clinic diagnosis (Lesko et al, 2003). Similar to the situation in which microprocessors have increased the speed of computation, microarraybased genomic technologies have revolutionized genetic analyses of biological systems.…”

“…Similar to the situation in which microprocessors have increased the speed of computation, microarraybased genomic technologies have revolutionized genetic analyses of biological systems. Although microarray technology has been used successfully to analyze global gene expression in pure culture studies (Schena et al, 1995;Lockhart et al, 1996;DeRisi et al, 1997;Liu et al, 2003;Gao et al, 2004;Mukhopadhyay et al, 2006), adapting microarray technology for use in environmental studies presents numerous challenges in terms of probe design, the coverage of gene sequences, specificity, sensitivity and quantitation (Loy et al, 2002;TaroncherOldenburg et al, 2003;Rhee et al, 2004;Steward et al, 2004;Tiquia et al, 2004;Wu et al, 2006a).…”

“…To overcome such obstacles for studying microbial communities in natural settings, a particular type of microarrays, called functional gene arrays (FGAs), has been developed and used (TaroncherOldenburg et al, 2003;Rhee et al, 2004;Steward et al, 2004;Tiquia et al, 2004). This type of arrays contains probes from the genes involved in key microbially mediated biogeochemical processes, such as C, N and S cycling, phosphorus utilization, organic contaminant degradation and metal resistance and reduction, and is particularly powerful for studying various biogeochemical processes.…”

Introduction to Microarray‐Based Detection Methods

“…However, somewhat higher background signals may occur when low-quality coatings are used. Substrates based on amino silane surface chemistry are widely used for deposition of PCR products [40,68,141,145,167,175]. …”