16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"

Abstract

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.

FIG. 1.

16S rRNA-based phylogenetic tree of the “Rhodocyclales” and selected type strains of other betaproteobacterial orders. The consensus tree is based on maximum-likelihood analysis (AxML) performed with a 50% conservation filter for the “Betaproteobacteria.” The bar indicates 10% estimated sequence divergence. Polytomic nodes connect branches for which a relative order could not be determined unambiguously by applying neighbor-joining, maximum-parsimony, and maximum-likelihood treeing methods. Numbers at branches indicate percent parsimony bootstrap values. Branches without numbers had bootstrap values of less than 50%. The minimum 16S rRNA sequence similarity for each “Rhodocyclales” lineage is shown.

FIG. 2.

Frequency distribution of ΔG values for positive (black bars) and negative (white bars) probe-target combinations having up to five mismatches. The horizontal lines indicate the 5th and 95th percentiles and the median value (M). The difference between the ΔG values of positive and negative probe-target combinations was highly significant (analysis of variance, P < 0.001).

FIG. 3.

(A) DNA microarray diversity analysis of “Rhodocyclales” in activated sludge from the industrial WWTP Kraftisried. Three RHC-PhyloChips were hybridized separately with fluorescently labeled 16S rRNA gene PCR amplicons that were retrieved from the activated sludge sample by using either bacterial or “Rhodocyclales” subgroup-selective (R or Z) primer pairs (Table 3). Each probe was spotted in triplicate. For each microarray position, the probe sequence and specificity are depicted in Table 4. Probe spots having a signal-to-noise ratio (SNR) equal to or greater than 2.0 are indicated by boldface boxes and were considered positive. In the composite microarray pattern, probes which were positive in any of the three individual RHC-PhyloChip hybridizations are indicated by black boxes. (B) Flow chart illustrating the presence of distinct “Rhodocyclales” groups in the activated sludge from Kraftisried as inferred from the composite microarray pattern. For each probe, the position on the microarray is indicated in the superscript text.

FIG. 4.

16S rRNA gene phylogenetic consensus tree based on maximum-likelihood analysis (Tree-puzzle) performed with a 50% conservation filter for the “Betaproteobacteria.” The tree shows the affiliation of clone sequences (boldface type) retrieved from the sewage treatment plant Kraftisried by using “Rhodocyclales” subgroup-selective primer pairs A (KRA clones), R (KRR clones), and Z (KRZ clones) for PCR. The grey box shows affiliation to a “Rhodocyclales” lineage. The bar indicates 10% estimated sequence divergence. Polytomic nodes connect branches for which a relative order could not be determined unambiguously by applying neighbor-joining, maximum-parsimony, and maximum-likelihood treeing methods. The percent reliability value of each internal branch indicates how often the corresponding cluster was found among 50,000 intermediate trees during quartet puzzling. Values below 70% are not shown. Parentheses indicate the perfect-match target organisms of the probes. Probe S-*-OTU1-1415-a-A-20 (OTU1-1415) (Table 5) is depicted in bold and was used for quantitative FISH analysis. The microarray position is depicted after the probe name. Probes RHC630, RHC143, RHC222, RHC175a, and RHC175b, perfectly matching some of the Kraftisried clones, are not shown to enhance clarity.