Microorganisms adapted to growth at high temperature (thermophiles) have evolved a variety of unique mechanisms to cope with these extreme conditions. This demands a repertoire of "adaptation" mechanisms to cope with fluctuating environmental temperature ("adaptability"). There have been numerous reports suggesting that thermophilic microorganisms (e.g. Bacilli) exhibit a variety of biochemical adaptations in response to a change in the growth temperature. This is accomplished by employing a variety of adaptation mechanisms whose components are encoded by genes that are differentially regulated by temperature (temperature-responsive genes). Temperature-responsive gene products can include one or more enzymes for catabolic reactions, for energy generation, or for the formation or modification of lipids and/or other cell components which exhibit increased temperature stability or which allow the cell to survive or exhibit enhanced growth at the higher temperature (extrinsic protection of critical cell macro molecules). This was confirmed by genetic and biochemical studies on the unique glutamine transport operon (glnQH) of the genetically well characterized Bacillus stearothermophilus NUB3621. The normal temperature-regulated transcription pattern for glnQH is inactive at low temperature (50 degrees C) and active at high temperature (65 degrees C). One of the goals of this project is to determine the molecular mechanisms of temperature-regulated transcription of glnQH. Reporter gene technology in combination with deletion analyses, site specific mutagenesis, gel mobility shift analysis, and DNAase I footprint analysis will provide detailed knowledge about the elements (promoters and transcription factors) that function in the differential regulation of this operon. Another goal of this project is to identify other genes/operons that are differentially expressed by temperature. The identification and characterization of other loci that are transcriptionally regulated by temperature will establish if there is more than one mechanism for the regulation of temperature-responsive genes. and enhance our understanding of the thermoadaptation mechanisms that allow B. stearothermophilus to survive at high temperature. This will be accomplished by subtraction differential hybridization and by Tn9 17-mediated operon fusions that place a reporter gene (bgaB) under the transcriptional control of contiguous genes. The temperature-responsive genes will be characterized using reporter gene technology, in silico analyses, and by the phenotype of null mutants. Thermoadaptation studies with B. stearothermophilus have opened up a relatively unexplored area of gene regulation. This is a challenging frontier of current biology that will have wide spread interest in both the scientific and the biotechnology communities. Research on thermoadaptation in the moderate thermophile B. stearothermophilus will not only contribute to our understanding of how this organism has adapted but will serve as the paradigm for studies with microorganisms that exist near the temperature extremes of planetary conditions. The long-term goal of this project is to find the right tools and approaches so that this knowledge can be applied to these novel microorganisms.
