Diversity, Ecology and Evolution of Microorganisms Inhabiting Hot Spring Microbial Mats

David M. Ward

Molecular analysis of community composition and structure.

In the mid-1980s we began to develop and use methods for examining the genetic variation exhibited by the gene encoding 16S ribosomal RNA molecules, using the strategy depicted in Fig. 2. DNA is extracted from mat samples, then PCR amplified to produce a mixture of 16S rRNA sequences from community members. These are separated by cloning or gel methods (called denaturing gradient gel electrophoresis) to allow purification of individual 16S rRNA sequences. Purified 16S rRNAs are then sequenced and compared to a large and growing database on the evolution of life based on sequence variation in this molecule http://rdp.cme.msu.edu/html/. As expected, our community contained 16S rRNA sequences related to cyanobacteria and green nonsulfur bacteria, as shown by the blue- and red- highlighted Kingdom-level lineages in the 3-domain tree. The three red-highlighted lineages in Domain Eukarya are the traditional Animal, Plant and Fungal Kingdoms. Since line length separating organisms in the tree are proportional to genetic difference between the organisms, it is easy to note that microorganisms (all other lines) contain much more genetic diversity as assayed by 16S rRNA sequence variation.

Molecular Analysis of Microbial Mat Communities:
				Native Population
			Extract DNA
			PCR
				Mixed rRNA
	Separate by cloning into E. coli				Separate by DGGE

		Sequencing

Phylogenetic Identification

Figure 2. Approaches to 16s rRNA diversity analysis.

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