Most microbes live in a world that is limited by the very nutrients that are required for their growth and replication. In response to the harsh conditions of most natural environments, bacteria have adopted numerous strategies for survival. One solution that appears to be quite common is that of miniaturisation, and in fact, most of the microorganisms seen in marine and soil samples tend to be smaller than those capable of growth in the laboratory. At the very end of the size spectrum are marine bacteria that are known by various names, but which are frequently referred to as "ultramicrobacteria" due to their especially small size. These bacteria may well represent the lower limit of cellular life, since the amount of genetic material (DNA) that they contain per cell is much less than that typically seen in other bacterial cells. This proposal is a first step in testing whether the ultramicrobacteria represent a physiological group distinct from the larger marine microorganisms or are simply members of this group of larger bacteria that have adopted a miniature size for survival. In order to determine the evolutionary relationships among these organisms, the total marine microbe population will first be separated into various size classes by filtration. The DNA from each size class will be isolated and compared to that from the other filters using short radioactively labeled fragments of RNA to determine the diversity of species that comprise the ultramicrobacteria. The experiments will utilize probes produced from known marine bacteria and from species of any ultramicrobacteria that are capable of growing in pure culture. The initial research will focus on developing protocols to successfully prepare pure chromosomal DNA from the marine microbes captured on the filters. Later studies will test the ability of the various probes to recognize and bind to the DNA in the samples.
