The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function

Abstract

A new microarray method, the isotope array approach, for identifying microorganisms which consume a (14)C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [(14)C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of (14)C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO(2) fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [(14)C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO(2) fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by (14)C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of (14)C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

FIG. 1.

DNA microarray evaluation. Hybridizations were performed with 10% (A) and 20% (B) formamide (FA) in the hybridization buffer. Individual hybridization results are shown for probes with the following AOB reference organisms (phylogenetic affiliations are as in references and 53): Nitrosospira sp. strain Nsp40 (belonging to Nitrosospira cluster 3); N. oligotropha Nm45 (affiliated with the N. oligotropha lineage); Nitrosomonas sp. strain Nm51 (affiliated with the N. marina lineage); N. europaea Nm50, N. eutropha Nm57, and N. mobilis Nm107 (all belonging to the N. europaea-N. mobilis lineage); Nitrosomonas sp. strain Nm41 (affiliated with the N. communis lineage); N. cryotolerans Nm55; and N. oceani Nc4 (a gamma-proteobacterial AOB). Shaded boxes indicate hybridization signals with a signal-to-noise ratio above the threshold value of 2, as indicated by the key below the panels. 0MM indicates hybridization to an AOB with a target sequence fully complementary to the probe. 1MM and 2MM indicate hybridizations to target sequences having one and two mismatches, respectively. False-negative signals represented the absence of duplex formation despite the presence of a fully matched target sequence. For each of the observed mismatch hybridizations, the position and type of the mismatch(es) within the duplex are shown.

FIG. 2.

Isotope array experiment with N. eutropha Nm57. rRNA was extracted from N. eutropha cultures after incubation with [¹⁴C]bicarbonate. After labeling of the rRNA with Cy5, a single slide carrying three prototype arrays with different spot sizes was hybridized, washed, and scanned for fluorescence and radioactivity. (A) Spot diameter, 125 μm. (B) Spot diameter, 500 μm. (C) Approximate spot diameter, 1 mm. Bars, 500 μm.

FIG. 3.

Phylogenetic tree showing the affiliations of the AmoA sequences obtained from the Aalborg West and Oberding wastewater treatment plants (wwtps). The tree was calculated based on the AmoA amino acid sequences deduced from the FITCH algorithm with global rearrangements and a randomized input order. The bar shows 10% estimated sequence divergence. AmoA sequences of gammaproteobacterial AOB were used as outgroups (not shown). The accession numbers for the clones from wwtps, estuaries, and lake sediment were as follows: estuary clone—AF367463 (Duc-27); freshwater clones—AJ388566 (pGtA.2), AJ388570 (pG5B.1), and AJ388571 (pG5B.2); wwtps clones—AF420298 (M-13), AF272442 (IA-32), AF272482 (BF1-1), AF489661 (S_3), and AF489660 (S_2); and lake sediment clone—AF489645 (T_2). amoA sequences of reference organisms were published elsewhere (30, 41, 51, 53, 65).

FIG. 4.

Incorporation of radioactive ¹⁴C into nitrifying activated-sludge biomass after incubation with [¹⁴C]bicarbonate. Filled symbols indicate incorporation in the absence of the inhibitor allylthiourea. Open symbols indicate incorporation in the presence of allylthiourea.

FIG. 5.

Isotope array experiment with activated sludge. rRNA was extracted from inhibited and noninhibited activated sludge from the Aalborg West and Oberding wastewater treatment plants after incubation with [¹⁴C]bicarbonate for 13 and 11 h, respectively. After labeling of the rRNA with Cy3, the manually spotted microarrays were hybridized and scanned for fluorescence and radioactivity. In order to enhance clarity, recorded spot signals were aligned by using Adobe Photoshop. Within each slide, blue indicates low signals and green, yellow, and red indicates increased signals. It should be noted that signal intensities in the radioactivity scans cannot be compared directly between slides. ATU, allylthiourea.

FIG. 6.

Incorporation of radioactivity into rRNA of phylogenetic subgroups, as measured by A-value determination for each probe (see the text for details). Isotope experiments (as displayed in Fig. 5) were performed after three different lengths of incubation of activated sludge with [¹⁴C]bicarbonate. A-values were determined for each probe and experiment. Error bars indicate standard deviations of the A-values from duplicate or triplicate experiments. (A) Aalborg West activated sludge. (B) Oberding activated sludge. (C) Aalborg West activated sludge inhibited by allylthiourea. (D) Oberding activated sludge inhibited by allylthiourea. In panels A and B, only probes which had a fluorescence signal-to-noise ratio above the threshold value of 2 are depicted.

FIG. 7.

Flowchart of the isotope array approach.