ABSTRACT
Structure and function analysis of microbial communities is central to our understanding of microbial ecology. Given that the majority of environmental microorganisms cannot be cultured in vitro (1), nucleic-acid-based identification methods are commonly used to identify and phylogenetically assign organisms from the environment (Chapters 1 and 2). Identification of uncultured microorganisms is routinely carried out via the so-called rRNA cycle (2), a combination of PCR amplification followed by cloning and sequencing (Chapter 2) or alternatively by specific in situ probing (Chapter 9). Direct or indirect analysis of taxonomically informative nucleic acid sequences by comparative sequence analysis or taxon-specific probe hybridization as well as via diagnostic PCR are now standard techniques in microbial ecology studies. In this context the most important molecules are the small subunit rRNA genes. The suitability of the small subunit rRNA genes as phylogenetic markers is discussed in more detail in Chapter 2, but their value is based in no small part on the fact that no other molecule or gene has such a comprehensive database available with respect to the number of entries (>100 000 entries) or with regard to phylogenetic spectrum. Consequently, the current taxonomy of the prokaryotes is based upon phylogenetic conclusions derived from comparative rRNA analyses as outlined in the most recent edition of Bergey’s Manual of Systematic Bacteriology (3). However, against a background of 3-4 billion years of evolution of cellular life, the information content and taxonomic resolution of any phylogenetic marker is rather limited (4) especially at the lower taxonomic ranks such as the species and strain level. Now in the age of genomics, alternative conserved markers for supplementing and evaluating the higher-level systematics are increasingly considered. For species definition and identification of closely related strains, multi-locus sequencing is increasingly important and will replace genomic DNA-DNA hybridization as the criterion for species definition (5). In order to deal with, analyze and manage the wealth of information from large-scale sequencing methods, databases and software tools for data analysis have to be regarded as essential tools in microbial ecology studies. This not only holds true for identification based upon conserved and housekeeping genes but also for functional analyses. The presence or absence of genetic information on cellular molecules with physiological relevance may allow estimates of the physiological capacities of the organisms in microbial communities. In this context the rapid development of techniques for full genome sequencing of uncultured organisms-environmental genomics (Chapters 10-11)—will provide valuable knowledge on the potential physiological properties of uncultured organisms. This is of even greater importance against the background of continuous improvement in microautoradiography techniques (6) as well as in situ mRNA,
and plasmid and genomic DNA targeting hybridization methods (Chapter 8). Thus in the near future in situ studies on the function of uncultured members of microbial communities will become as routine as rRNA-based identification.
