Soil microorganisms impact biogeochemical structuring and nutrient cycling on a global level. It has been estimated that a gram of soil can contain as many as 5 x 10 11 bacterial cells and over 13,000 distinct phylotypes. However, only a small fraction of the microorganisms from most soil samples can be readily cultured. The fraction that has been cultured rarely includes the most abundant species and frequently is biased towards or against particular taxonomic or physiological types. Since most of what is known about soil microbes has come from studies of cultured species, we still know very little about soil microbial ecology. Therefore, culture-independent methods are an important tool in soil microbiology and promise major new insights into the roles of soil microflora in ecosystem function. Culture-independent methods have revealed that diverse members of two bacterial divisions, Acidobacterium and Verrucomicrobia, are abundant in soils. While they have a cosmopolitan distribution and can be numerically dominant, we know very little about their ecology and activity within the soil community. Using information derived from the application of newly developed genomic approaches, we will test hypotheses about the distribution and functions of these uncultured taxa in response to soil environmental gradients. First, we will sequence, annotate, and analyze large insert genomic DNA clones derived from bacterial taxa in these two divisions (Acidobacterium and Verrucomicrobia taxa). Each of these genomic DNA clones contains rRNA genes as well as functional information derived from sequence analysis and annotation. These data will be integrated into a publicly available and searchable database. We will next use the phylogenetic information contained within these clones to develop taxa-specific primers and probes. We will then use these molecular primers and probes to test hypotheses concerning the ecological niches inhabited by these taxa, shedding light on their distribution within various soil fractions, their size and membrane charge distributions, their relative abundance within plant rhizospheres, and their distribution along soil environmental gradients. The results of this research will provide hitherto unavailable information on specific uncultured soil microorganisms, including genomic organization, gene content, and environmental distributions. The knowledge gained from these studies will lead to further ecological hypotheses, and provide new approaches, tools and a database that will significantly advance microbial ecological studies of one of the most important microbial habitats on Earth-the soil. The long-term goal is to contribute to a platform of technologies and results that can be used to elucidate the variability, activity and significant ecological roles of uncultured soil microorganisms in their natural habitats.
