Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology.
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.
Koizumi
Yoshikazu
Y
Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-Ohsawa, Hachioji, Tokyo 192-0397, Japan.
Kelly
John J
JJ
Nakagawa
Tatsunori
T
Urakawa
Hidetoshi
H
El-Fantroussi
Saïd
S
Al-Muzaini
Saleh
S
Fukui
Manabu
M
Urushigawa
Yoshikuni
Y
Stahl
David A
DA
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D001555:Benzene Derivatives / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013440:Sulfides / Q000378:metabolism; D014050:Toluene / Q000378:metabolism*
D001555:Benzene Derivatives; D013440:Sulfides; D014050:Toluene; C004912:ethylbenzene