In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes

Abstract

Individual cyanobacterial cells are normally identified in environmental samples only on the basis of their pigmentation and morphology. However, these criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identification of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since fluorescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later, it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, TSA-based detection of HRP was more sensitive than that based on nonfluorescent substrates. Critical points of the assay, such as cell fixation and permeabilization, specificity, and sensitivity, were systematically investigated by using four oligonucleotides newly designed to target groups of cyanobacteria.

FIG. 1

Phylogenetic tree of cyanobacteria and probe specificities. Sequences with complementary target sites are identified by the symbols ● (CYA361), ■ (CYA762), ▴ (CYA664), and ⧫ (CIV/V1342). References for sequences not included in the public ARB datafile are as follows: Calothrix (Rivularia) sp. strain PCC7116, Calothrix sp. strain PCC7507, Prochlorococcus marinus PCC9511, Tolypothrix sp. strain PCC7101, and Tolypothrix sp. strain PCC7415 (all according to reference 12a), Limnothrix redekei MEF6705 (41), Pseudanabaena sp. strain PCC7403 (17a), Microcystis wesenbergii NIES111 (EBI accession no. D89034), M. aeruginosa NIES98 (accession no. D89032), M. aeruginosa NIES87 (accession no. D89031), Synechococcus sp. strain PCC7942 (accession no. D88288), M. holsatica NIES43 (accession no. D89036), Synechococcus elongatus (accession no. D83715), Merismopedia glauca (accession no. X94705), Synechocystis sp. strain PCC6803 (accession no. D90916), Synechococcus sp. strain PCC7002 (accession no. D88289), and Calothrix sp. strain D253 (accession no. X99213). Strain names refer to the database entries, in most cases regardless of taxonomic validity. The bar indicates 10% estimated sequence divergence.

FIG. 2

Difference alignments of the PCC reference strains, probe and target sequences, probe positions, and results of the different hybridization methods. Probe positions correspond to the E. coli 16S rRNA numbering of Brosius et al. (8). Dots in the difference alignments indicate bases that are identical to those in the target sequences. N stands for an unknown base in the respective sequence. Hybridization results are shown as follows: +, positive; −, negative; (+), positive signal but weaker than that obtained by hybridization of reference probe EUB338 with the same method.

FIG. 2

Difference alignments of the PCC reference strains, probe and target sequences, probe positions, and results of the different hybridization methods. Probe positions correspond to the E. coli 16S rRNA numbering of Brosius et al. (8). Dots in the difference alignments indicate bases that are identical to those in the target sequences. N stands for an unknown base in the respective sequence. Hybridization results are shown as follows: +, positive; −, negative; (+), positive signal but weaker than that obtained by hybridization of reference probe EUB338 with the same method.

FIG. 3

Micrographs of artificial mixtures of cyanobacteria and of a microbial mat after hybridization. (A) Artificial mixture of Nodularia sp. strain PCC73104 and Spirulina sp. strain PCC6313 hybridized with probe CIV/V1342, which was detected with fluorescein-tyramide. Shown are a phase-contrast image (left) and an epifluorescence image obtained with filter set 09 (right). Note that the heterocysts of Nodularia sp. were not stained, likely due to lack of probe permeability. (B) Artificial mixture of two Synechococcus sp. strains, WH8103 and PCC6301, hybridized successively with probes CYA664 and CYA762. Fluorescein-tyramide was used to detect the former probe, and tetramethylrhodamine was used to detect the latter probe. Shown are a phase-contrast image (left) and a double-exposure epifluorescence image obtained with filter sets 09 and 15, yielding green and red epifluorescence, respectively (right). Both mixtures were recorded at an original magnification of ×1,000. The bars represent 10 μm. (C) Left to right, autofluorescence of a section of a microbial mat as seen with filter set 09 and bright-field micrographs, respectively, the latter two obtained after hybridization with probes CYA762 and CYA664 and detection with TETON. The original magnification was ×100. (D) Part of the same mat after hybridization with probe CYA762 and detection with fluorescein-tyramide was recorded by confocal laser scanning microscopy and is shown as an all-in-focus image of selected optical sections. Both channels, the green for the fluorescein signal and the red for autofluorescence, were detected simultaneously. Yellow color results from the overlay of autofluorescence and signals within the same cells. The original magnification was ×630.