MINIREVIEWS Identifying the Dominant Soil Bacterial Taxa in Libraries of 16S rRNA and 16S rRNA Genes

In 1909, H. Joel Conn (25) expressed the hope that methods would soon be at hand by which the significance of the different bacteria present in any soil could be determined. However, by 1918 he was pointing out that the methods available to him, which relied on cultivation of bacteria on artificial media, resulted in the formation of colonies by only 1.5 to 10% of the bacterial cells in soil (26). Fifty years later, Vagn Jensen concluded a review of cultivation-based methodologies by stating his suspicion that those cells that were forming colonies were unrepresentative of the total bacterial community (59). This was confirmed when cultivation-independent methods began to be used to study soil bacteria (see below). Even so, in the absence of better methods, the pure cultures derived from the colonies that did form were extensively and successfully studied throughout the 20th century. Much of our basic knowledge of soil bacteria, as well as the discovery of many important antibiotics, came from investigations of pure cultures (2, 86, 104). Cultured isolates are still very important in developing our understanding of bacterial physiology, genetics, and ecology (85, 122). Beginning in the 1990s, the application of molecular ecological methods, especially those based on surveys of genes after PCR amplification, has allowed cultivation-independent investigations of the microbial communities of soils to be made. The power of these methods has largely rendered obsolete the plate count approach to detecting and enumerating subsets of soil bacteria, and a range of diagnostic and quantitative methods that target functional genes, phylogenetically informative genes, or RNAs has been developed (49, 69). In particular, 16S rRNA and its gene have proven to be useful and powerful markers for the presence of bacteria in samples (36, 56, 88). The utility of these markers is facilitated by the availability of primers that allow amplification of almost the complete gene or its RNA product (66) and by the phylogenetic inferences that can be made from the resultant nucleotide sequences, permitting placement of the host organism within a phylogenetic framework even if closely related cultured organisms are lacking (36, 56, 74). Since the initial pioneering studies to survey soil bacterial communities using molecular ecological surveys (13, 68, 79, 91, 111), a number of libraries of 16S rRNA and 16S rRNA genes derived from soils have become available (Table 1). It is important to realize that libraries of PCR-amplified 16S rRNA and 16S rRNA genes may not represent a complete or accurate picture of the bacterial community. Firstly, the species diversity is so great (28, 46, 109) that libraries of 400 cloned sequences must represent only an incomplete sampling. Even all of the currently published sequences combined would seem to constitute an incomplete census of all of the 16S rRNA genes on earth (98). In addition, there may be biases in the contributions of the various bacterial groups to libraries. The efficiencies of nucleic acid extraction may be different for different bacteria, the number of copies of 16S rRNA or 16S rRNA genes per cell varies, and there may be preferential amplification of some sequence types relative to others by PCR (36, 43, 113). Some sequences may arise from contaminating DNA and may not represent bacteria actually present in the sample being studied (108). Assigning physiologies and functions to the hosts of 16S rRNA gene sequences is complicated in many cases by the lack of characterized close relatives (e.g., see references 31, 57, 81, and 88) and by the diversity of phenotypes among close relatives in some groups (1, 95). Some, but not all, of these biases may be overcome as metagenomic data sets accumulate (71, 87, 110). In the meantime, the available libraries of 16S rRNA and 16S rRNA genes permit an initial survey of the global soil bacterial community structure.