In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. with RRNA-targeted peptide nucleic acid probes

Abstract

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.

FIG. 1

Frequency distribution of oligonucleotide (DNA) probe-conferred fluorescence in Prochlorococcus strain SS120 cells hybridized with a positive probe, UNIV1392, and a negative control probe, NON338.

FIG. 2

Effect of hybridization time and PNA concentration (0.85 μg ml⁻¹ [●], 2.1 μg ml⁻¹ [▾], and 4.3 μg ml⁻¹ [■]) on mean cellular fluorescence (± standard errors) in Prochlorococcus strain SS120. (Top) Gross fluorescence of the positive (pEUB339) (closed symbols) and negative (pNEG1198) (open symbols) probes. (Bottom) Negative-probe-conferred fluorescence has been subtracted from that of the positive probe.

FIG. 3

Frequency distribution of PNA-conferred fluorescence of Prochlorococcus strain SS120 cells and Synechococcus strain WH8101 cells hybridized with the positive probe (pEUB339), the negative probe (pNEG1198), or no probe.

FIG. 4

Effect of RNase on PNA-conferred fluorescence in Prochlorococcus strain SS120 (EUB) pEUB339 and NEG (pNEG1198). Error bars show standard errors.

FIG. 5

Comparison of DNA and PNA probes in Synechococcus strain WH8007. Mean fluorescence conferred by pEUB339 (○) and EUB338 (■) in cells growing at four different light-limited growth rates is shown. Data were corrected for fluorescence from corresponding negative probes. Error bars indicate standard errors for pEUB339 and pNEG1198 fluorescences; standard errors for EUB338 are not shown, but they averaged 7% of the mean.

FIG. 6

In situ hybridization of natural Prochlorococcus population. (A) Flow cytometric signature of Prochlorococcus (with 0.474-mm-diameter latex beads) showing forward angle light scatter (related to size) versus red fluorescence (from chlorophyll). (B) Probe-conferred fluorescence of Prochlorococcus cells as defined from panel A, hybridized with positive probe (pEUB339), negative probe (pNEG1198), and no probe. Water was collected from the Pacific Ocean in an oligotrophic region off the California coast.