Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

Abstract

Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.Ribosomal RNA genes (rDNAs) are extensively used to study the diversity of microorganisms in environmental samples. To date, most surveys of microbial diversity in environmental samples have relied upon cloning and sequencing of rDNAs. These studies have shown that the diversity of microorganisms in natural ecosystems has been severely underestimated in culture collections and have led to the discovery of several new microbial lineages (5,10,20,28). Despite the increased knowledge of the phylogenetic diversity of indigenous microbes, less is known about the abundance of particular groups or their spatial and temporal dynamics.Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Recoveries of PCR-amplified rDNAs from clone libraries are subject to several quantitative biases (see reference 26 for a review). rRNAbased quantification therefore has relied mainly on radiolabeled oligonucleotide hybridization to extracted rRNA (11,16,21) or whole-cell hybridization using fluorescence-labeled oligonucleotides (1, 7). However, in many ecosystems the abundance of microorganisms is relatively low and many cells may have a low rRNA content, limiting the sensitivity of oligonucleotide hybridization techniques for quantification of specific microorganisms.In individual cells, the number of copies of rDNAs may be several orders of magnitude lower than that of rRNAs; in DNA, rRNA coding sequences exist in a context of much higher genetic complexity. It is therefore difficult to quantify rDNAs by oligonucleotide hybridization. However, rDNA is suitable for amplification by the PCR. Provided that the amplification efficienc...

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