During the last 15 years aquatic microbial ecology has been shaped by a diversification of methods for the cultivation-independent study of microbial identity, activity, and genomic constitution (11). This trend is reflected in the recent foundation of a scientific journal on methods in limnology and oceanography that is edited by a microbial ecologist (http://aslo.org/lomethods/editor.html). The following section does not give an exhaustive listing of tools for the study of microbial populations in situ. Instead, it covers a suite of complementary approaches which have been applied in various combinations to analyze bacteria in environmental samples. They can provide a coherent framework for ecological investigations about the abundant microbes in the water column, i.e., taxa that contribute approximately 1% or more to the total picoplankton assemblages. Other approaches, e.g., quantitative PCR (17, 283), might be more appropriate to study microbial populations that are substantially smaller.
In order to define the important populations in a new environment or at a particular time point, it is often necessary to conduct a prior study about microbial diversity, most commonly of 16S rRNA genes. Full or partial sequences can be amplified from extracts of environmental DNA (75, 301) or directly from cells concentrated on membrane filters (21, 145) by PCR with appropriate primer sets (84, 194). These fragments are then ligated into plasmids, cloned into Escherichia coli, and the sequences of vector inserts from an appropriate number of clones are determined. The PCR step is omitted altogether in so-called shotgun clone libraries (305). Since such libraries require a drastically greater screening effort, they are used for obtaining metagenomic information rather than for the mere collection of 16S rRNA genes.
The diversity of sequences in PCR-generated clone libraries may often not quantitatively reflect the diversity of the sequence types that are present in the original sample (242, 294). Already the DNA extraction may introduce biases, e.g., against bacteria with gram-positive cell walls (69). Primers that are designed to target the majority of known bacterial 16S rRNA gene sequences may exhibit mismatches to unknown sequence types (45, 287), and the presence of particular sequence types in mixed DNA may influence the PCR amplification efficiencies of other templates (292).
As a consequence some phylogenetic groups of aquatic microorganisms are overrepresented in clone libraries, whereas others are absent. For example, bacteria affiliated to the Cytophaga/Flexibacter/Flavobacterium group of the Bacteroidetes were rarely found in 16S rRNA gene clone libraries from coastal marine water samples, yet they may represent one third of all bacteria in such habitats (47, 63). In a library of coastal North Sea surface waters 80% of sequence types were related to the marine SAR86 clade, whereas this group formed 63). In addition, PCR may result in chimeric sequences from two templates, and it may thus even produce artificial sequence diversity (124, 155). These biases may affect not only clone libraries, but also PCR-based methods for the genotypic fingerprinting of microbial assemblages (183, 193).
Sometimes it is desirable to limit the analysis of microbial diversity to a defined phylogenetic subset, e.g., to marine archaea or to freshwater actinobacteria (173, 309). This can be achieved by PCR with primers that are specific for the 16S rRNA genes of the group of interest (287). Such primers may cause an underestimation of the potential diversity within a particular phylogenetic group, e.g., some uncultured freshwater actinobacteria show one or more mismatches with the available sets of specific primers (309). Moreover, sequences generated with specific primers often cover only a part of the total 16S rRNA gene, typically less than 1,000 base pairs (55, 287). Partial sequence information negatively affects the accuracy of phylogenetic reconstruction (163), and it limits the range of potential signatures for subsequent in situ population studies by hybridization. Preferably, clone libraries of almost complete 16S rRNA gene sequences should be produced with general bacterial or archaeal primers, and they should then be screened for particular groups of interest (173, 184, 309).
The potential target groups for population studies are defined from sequence data by reconstruction of phylogenetic relationships. This analysis aims at placing the new environmental sequence types into different clades or clusters and to establish stable branching patterns. It forms the base for a phylogenetically meaningful definition of single populations and for the design of the corresponding oligonucleotide probes. A discussion of the reconstruction of microbial phylogenies from 16S rRNA gene sequences would go beyond the range of this review, and we draw the reader's attention to specific publications on this subject (163, 164).
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