White paper.
Basic statements concerning molecular microbial ecology.
Donald A. Klein, Colorado State University. 15 Jan., 2007; rev. 18 June 2007 D.A. Klein © 2007
A fundamental concern, in what is described as molecular microbial ecology, is that nucleic acid recovery procedures, and the information they generate, have not been considered in an adequate manner, or considered at all in too many publications and presentations. A disproportionately greater emphasis has been given to the more mechanical aspects of processing the derived molecular information, while the more fundamental aspects of nucleic acid recovery, needed to assure that this molecular information is relevant to microbial ecology, the study of microbial communities, and/or microbial diversity, largely has been neglected. To assume that all molecular information derived using the bulk extraction of complex assemblages is relevant to microbial ecology, the study of microbial communities and biodiversity, cannot be justified. The reasons for this suggestion are noted in the following statements:
1. The underlying problem is that the practitioners of molecular microbial ecology, in most cases, where bulk nucleic acid extractions are used, simply don't know where their nucleic acids come from. Particularly in soils, aquatic, intestinal environments, as major examples, these sequences can be derived from active microbes, inactive potentially functional cells, as well as from cells that are not capable of functioning in the particular environment (including those that are dead), viruses; these sequences also can be derived from free nucleic acids. The methods that are used also can influence the sequences obtained (extraction, PCR primers, etc. ).
2. We are now in the post-genomic era; nucleic acid sequences, of themselves, are not sufficient to describe a microbe. With the recognition of the role of epigenetics in influencing the phenotype of a microbe, independent of a particular nucleic acid sequence, the reductionist view of a nucleic acid sequence describing a microbe, the foundation for most molecular microbial ecology carried out to date, no longer can be considered to be valid or sufficient in terms of understanding microbial ecology. As an additional point of concern, intragenomic heterogeneity impacts the entire field of molecular microbial ecology. Multiple and different 16S rRNA sequences can occur in the same individual microbe
3. Only active microbes are part of a microbial community, microbial ecology, and microbial diversity. The active microbes (and their derived molecular sequences) are a subset of the microbes sampled by molecular techniques based on bulk sample extraction procedures. Thus much, and often most, of the molecular information generated by bulk extractions of materials such as soils or aquatic environments is not relevant to microbial ecology. Stated in another way, all 16S sequences recovered from a complex environment (soil, water, intestine, etc., ) are not part of microbial ecology, a microbial community or biodiversity. The real challenge, usually not considered, is to determine which sequences are derived from active, functioning microbes, and thus of interest in terms of microbial ecology, microbial communities and/or microbial diversity.
4. The simpler the biological system one is studying, the more confidence one can have in the results of molecular analyses based on bulk-extracted nucleic acids. In less complex natural biological systems, or with known individual or mixed microbes, greater confidence can be placed in the use of bulk-extracted nucleic acids. In these simpler systems, one has a greater chance of having active microbes being the major or sole source of nucleic acids derived from the biological system.
5. One needs to return to direct observational microbial ecology, to be sure organisms being studied are actually functioning in the original environment, the essence of microbial ecology. It is now possible to characterize individual active microbes in situ or after removal from matrices such as soils and aquatic environments (including by the use of molecular techniques). There is no need to continue the use of largely meaningless bulk nucleic acid extractions for molecular analyses of microbes present in natural microbial assemblages.
6. The oft-stated concern with cultivable vs. non-cultivable cells is not directly relevant to questions of microbial ecology. A cell that may be "cultivable" in the laboratory may not be functioning, or capable of functioning, in the particular environment it was recovered from; a "non-cultivable" microbe, however, may be functioning in the environment and thus be a part of microbial ecology.
7. These basic statements, directed towards studies based on 16S-derived sequences, also are applicable to the consideration of studies that are described as "metagenomics," where direct cloning of larger extracted nucleic acid fragments, recovered by bulk extraction, is used without PCR-based amplification. Without knowing the source(s) of the nucleic acids used in such studies, particularly with complex assemblages such as soils, waters, and materials from the intestinal tract, such studies largely are compromised and of lesser significance. Only a subset of the recovered molecular information, derived from active, functioning microbes, will be relevant to microbial ecology, the analysis of microbial communities and/or microbial diversity.
