Measurement of microbial biodiversity

To estimate the number of species that are present in a specific habitat is more difficult than it may seem. The situation is aptly described by the following phrase from the classical book by Charles Elton (Elton 1927), the founder of animal ecology:

Two boys of rather good powers of observation were sent into a wood in summer to discover as many animals as they could, returned after half an hour and reported that they had seen two birds, several spiders, and some flies— that was all. When asked how many species of all kinds of animals they thought there might be in the wood one replied after a little hesitation 'a hundred', while the other said 'twenty'. Actually there were probably over ten thousand.

Now it is obvious that if Elton's boys had been asked to include microorganisms, their estimate would have been even more inaccurate. For obvious reasons, estimating the biodiversity of microorganisms is more difficult than estimating species richness of plants or animals. In addition, micro-biologists struggle with an even more fundamental question; that is, it is often not clear what constitutes a microbial species.

According to classical bacteriological taxonomy, an isolate is recognized as a proper species if its morphology is described plus some key aspects of its metabolism (trophic system, substrate use, etc.). Two isolates are considered to belong to the same species if their DNAs are similar by more than 70% or if there is less than a 5°C difference in the melting temperature of a DNA-DNA hybridization (Wayne et al. 1987). Obviously, species that cannot be put into pure culture cannot be characterized in this way. It is estimated that anything between 50 and 99% of microorganisms may belong to this group of unculturables and these remain undescribed as species, although parts of their genome may be sequenced from the environment. Why so many organisms cannot be cultured in the laboratory is unclear and probably there are many reasons, including specific growth conditions, unknown nutrient requirements, very slow growth, and special surfaces to which cells must attach. Recently, microbiologists have discovered that some 'uncultivable' bacteria can be brought into culture when placed in close proximity to other species, from which they are separated only by a membrane; apparently, chemical signals from other members of the community are sometimes crucial to induce growth (Kaeberlein et al. 2002). We will see later in this chapter that genomics approaches provide another solution to the problem: the DNA of species in the environment can be assembled and its functions characterized without even attempting to put them into a culture tube.

Microorganisms have been given little attention in ecological studies until recently. The last decade has produced a new awareness of microbial diversity and the suitability of microorganisms to address questions of fundamental ecological importance (0vreas 2000; Horner-Devine et al. 2004; Kassen and Rainey 2004; Jessup et al. 2004). Microorganisms have been reported from extreme habitats in which they are the only type of organism surviving, such as hot springs, deep ocean vents, volcanic crater lakes, and sediments under permanent ice cover. Such extreme habitats hold many surprises in store. For example, a completely new phylum of Archaea, the Nano-archaeota, was discovered in a hot submarine vent north of Iceland and a new division of Euryar-chaeota was found in a hypersaline anoxic basin in the Mediterranean Sea (Huber et al. 2002; Van der Wielen et al. 2005). The development of universal phylogenetic trees on the basis of genes that are common to all life forms has demonstrated that the biodiversity of the Bacteria and Archaea is at least as large as that of the whole domain of the Eukarya (Fig. 4.2).

4.2.1 Diversity of small-subunit rRNA genes

In the so-called polyphasic taxonomy of current microbiology a species is differentiated on both genetic and phenotypic grounds. The genetic characterization is derived from the sequence of the small-subunit rRNA gene. From basic biochemistry we know that the size of ribosomes may be characterized by Svedberg units (S), a measure of sedimentation velocity during ultracentrifuga-tion (1 S corresponds to 10~13s). The prokaryotic ribosome measures 70 S and is made up of a small subunit (SSU) of 30 S, consisting of 21 proteins and an RNA molecule of 16 S, and a large subunit (LSU), measuring 50 S, consisting of 34 proteins and two RNA molecules, one 23 S and the other 5 S. In the prokaryotic genome, the genes encoding these RNAs are organized in an rRNA transcription unit (rrn region), with the 16, 23, and 5S rRNA genes lying behind each other, separated by spacers and being transcribed as one unit. The 16 S rRNA gene (also called the SSU rRNA gene) has been chosen as the basic diagnostic instrument of prokaryote phylogeny and classification. The gene is assumed to fall into the category of essential genes, which are not, or at least infrequently, subjected to lateral transfer (see Section 3.2).

Bacteria

Chlor°bium

Leptonema Clostridium Bacillus

Helio'

Methanospirillum

Marine Gp. 1 low temp GPS/

Archaea

Bacteria

Chlor°bium

Phylogenetic Tree Archaeota

0.1 changes per site

Eukarya

Figure 4.2 The universal phylogenetic tree of life, based on small-subunit rRNA gene sequences. Reprinted with permission from Pace (1997). Copyright 1997 AAAS.

0.1 changes per site

Eukarya

Figure 4.2 The universal phylogenetic tree of life, based on small-subunit rRNA gene sequences. Reprinted with permission from Pace (1997). Copyright 1997 AAAS.

The size of ribosomal components is slightly different in eukaryotes (Table 4.1). The SSU rRNA is 17-18 S in eukaryotes (18 S in vertebrates) and the LSU rRNA molecule, which is 23 S in prokar-yotes, is enlarged to 28 S. In addition, eukaryotes have the prokaryote rRNA genes in their mitochondria and chloroplasts. The fact that all life forms have the same basic organization of rRNA

genes allows comparison across domains and the development of phylogenies such as that shown in Fig. 4.2.

The reason why the 16 S rRNA is particularly suitable as an anchor for prokaryote classification is that it shows a mosaic of conserved and variable regions. The molecule is shown in Fig. 4.3. A characteristic feature is that the RNA molecule

Table 4.1 Composition of ribosomes of prokaryotes and eukaryotes
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