Quantitative tracing, by Taq nuclease assays, of a synechococcus ecotype in a highly diversified natural population

Abstract

Quantitative Taq nuclease assays (TNAs) (TaqMan PCR), nested PCR in combination with denaturing gradient gel electrophoresis (DGGE), and epifluorescence microscopy were used to analyze the autotrophic picoplankton (APP) of Lake Constance. Microscopic analysis revealed dominance of phycoerythrin (PE)-rich Synechococcus spp. in the pelagic zone of this lake. Cells passing a 3- micro m-pore-size filter were collected during the growth period of the years 1999 and 2000. The diversity of PE-rich Synechococcus spp. was examined using DGGE to analyze GC-clamped amplicons of a noncoding section of the 16S-23S intergenic spacer in the ribosomal operon. In both years, genotypes represented by three closely related PE-rich Synechococcus strains of our culture collection dominated the population, while other isolates were traced sporadically or were not detected in their original habitat by this method. For TNAs, primer-probe combinations for two taxonomic levels were used, one to quantify genomes of all known Synechococcus-type cyanobacteria in the APP of Lake Constance and one to enumerate genomes of a single ecotype represented by the PE-rich isolate Synechococcus sp. strain BO 8807. During the growth period, genome numbers of known Synechococcus spp. varied by 2 orders of magnitude (2.9 x 10(3) to 3.1 x 10(5) genomes per ml). The ecotype Synechococcus sp. strain BO 8807 was detected in every sample at concentrations between 1.6 x 10(1) and 1.3 x 10(4) genomes per ml, contributing 0.02 to 5.7% of the quantified cyanobacterial picoplankton. Although the quantitative approach taken in this study has disclosed several shortcomings in the sampling and detection methods, this study demonstrated for the first time the extensive internal dynamics that lie beneath the seemingly arbitrary variations of a population of microbial photoautotrophs in the pelagic habitat.

FIG. 1.

Target positions of PCR primers and probes in the ribosomal operon of Synechococcus spp. used in this study. The target sequence and the arrows indicating the 5′-to-3′ orientation of the oligonucleotides are not drawn to scale.

FIG. 2.

Separation of PE-rich Synechococcus spp. from Lake Constance with DGGE. Ten to 40% gradients were used; for details, see Materials and Methods. (A) Migration behavior of seven PE-rich Synechococcus isolates from Lake Constance, for establishment of a marker. (B) Band pattern determined for environmental samples 1 to 11 from 1999 (compare with Fig. 4A). (C) Band pattern with environmental samples 1 to 10 from 2000 (compare with Fig. 4B). M, marker, represents a mixture of fragments depicted in panel A. One microliter from PCR mixtures with isolated strains (panel A and each strain in the marker) and 5 μl from assays with environmental DNA were applied per lane.

FIG. 3.

Standard curves obtained by the threshold cycle method in real-time PCR. For TNAs, 10-ml-assay volumes contained approximately 10¹ to 10⁶ copies of Synechococcus sp. strain BO 8807 genomes, 5 μl of 2× TaqMan Universal PCR master mix (5 mM Mg²⁺ final concentration), 300 nM concentrations of primers, and 200 nM concentrations of probe. Primers used with probe S8807A were P8807AP and P8807AM; primers used with probe S100A were P100PA and P3. For PCR conditions, see Materials and Methods. Fluorescence threshold ΔRQ = 0.02; s = slope. Amplification efficiency was calculated as follows: ɛ_c = 10^−1/s − 1. Error bars represent the standard deviations of six experiments.

FIG. 4.

Abundance of cyanobacterial picoplankton (Synechococcus spp.) and Synechococcus sp. strain BO 8807 in the pelagic zone of Lake Constance during the growth periods 1999 (A) and 2000 (B). Genomes per milliliter were determined by TNAs; the results were not corrected for prospective filtration losses. The percentage of BO 8807 is the relative genome numbers of Synechococcus sp. strain BO 8807 (□) compared to the total genome number of Synechococcus spp. (▪), determined with probe S100A. Error bars represent standard deviations of three filter pieces analyzed in duplicate.