Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

Abstract

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Effects of soil type and farm management on soil ecological functional genes and microbial activities

“…Studies using FGAs for the elucidation of specific ecological questions are still rare. While several studies have compared results from FGAs with other molecular techniques with a small total number of samples (Koizumi et al, 2002;Loy et al, 2002;Bodrossy et al, 2003;Rhee et al, 2004), we know of only one study comparing FGAs with soil processes and enzyme activities on the landscape level (Yergeau et al, 2007) and none employing a randomized replicated statistical design to investigate the impact of management and soil type on soil function and microbial diversity. We sampled soils in 2005 from eight adjacent pairs (replicates) of commercial organic and conventional strawberry fields.…”

Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

“…False-negative signals are a common phenomenon in microbial microarray analysis [28,33] and can be explained by parameters such as secondary structures in the target molecule or steric hindrance, causing a reduction in the hybridization efficiency in a probe-binding site-specific way [37].…”

Microbial community study of the iron ore concentrate of the Sishen Iron Ore Mine, South Africa

“…Denaturing gradient gel electrophoresis (DGGE) is an ideal molecular method for monitoring microbial community ecology since it is PCR based and does not employ classical methods of culturing the microbes within a sample. DGGE relies on variation in genetic sequence of a specific amplified region to differentiate between species within microbial communities (Banks and Alleman 2002;Koizumi et al 2002). By determining the closest relatives of unknown microorganisms, the known characteristics can be inferred upon them (Ueda et al 1995).…”