16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales".
For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.
Loy
Alexander
A
Department of Microbial Ecology, Institute of Ecology and Conservation Biology, University of Vienna, Vienna, Austria. loy@microbial-ecology.net
Schulz
Claudia
C
Lücker
Sebastian
S
Schöpfer-Wendels
Andreas
A
Stoecker
Kilian
K
Baranyi
Christian
C
Lehner
Angelika
A
Wagner
Michael
M
D001483:Base Sequence; D020562:Betaproteobacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004783:Environmental Microbiology; D004784:Environmental Monitoring; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000706:statistics & numerical data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D012722:Sewage / Q000382:microbiology
D015342:DNA Probes; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012722:Sewage