A key issue facing astrobiology is assessing what subset of environments could be hospitable to life. Of particular interest are the upper and lower temperature limits for species growth ('habitable zone') and the adaptation strategies to thrive in those extreme temperatures. In both the Solar System and the Galaxy, cold environments are much more common than hot environments. For example, Mars, and the icy moons of Jupiter and Saturn, which are the prime Solar System targets of searches for life beyond Earth, present relatively hostile environments to mesophilic terrestrial life. However, they may be capable of supporting liquid water, which is likely to be of high salinity. At their best, Martian conditions are not vastly dissimilar from the most-extreme cold regions of the terrestrial biosphere, such as the Dry Valleys of Antarctica. Those regions were regarded as essentially barren of life in the past; however, more recent investigations have revealed considerable microbial activity. Microorganisms that have adapted to these extreme conditions are some of the best candidates for terrestrial analogues of potential extraterrestrial life; understanding their biology and adaptive strategies will provide deeper insight on fundamental constraints on the range of habitable environments. The current project will focus on key aspects of the biology of Halorubrum lacusprofundi, an extremely halophilic archaeon isolated from Deep Lake, Antarctica. The temperature of Deep Lake is below zero for more than half of the year, but it does not freeze due to its extremely high salinity (~3.5 M NaCl). In order to capture the scientific potential of H. lacusprofundi, its genome was completely sequenced. H. lacusprofundi therefore is an excellent model organism to study the effects of extreme and potentially extraterrestrial conditions on an earthly life form. At the protein level, its enzymes have adapted to work at both high salinity and cold temperatures. At the gene level, transcription is regulated in response to multiple extremes, including temperature extremes. At the cell level, a physiological hallmark of H. lacusprofundi is the formation of multicellular assemblages, or biofilms. Multicellularism is of great importance in the evolution of higher life forms, but biofilms have not been studied in detail in the Archaea. We propose to investigate these key aspects of the biology of the Antarctic H. lacusprofundi using a combination of genetic, biochemical, and transcriptomic approaches and employing the genetic system of the related mesophilic haloarchaeaon Halobacterium sp. NRC-1. The project will address three main questions using the following methods: 1) What is the basis of polyextremophilic enzyme function (cold-activity and halophilicity)? Site-directed mutagenesis of a cold-active beta-galactosidase functioning over a broad temperature range (-20 to 70 degrees Celsius) and high salinity (>4 M salts) will be conducted. 2) What is the transcriptional mechanism for temperature regulation? The roles of general transcription factors (TBP/TFB) in regulating gene expression in response to cold temperature will be tested. 3) What are the genetic requirements for biofilm formation under low temperature and high salinity conditions? A primarily transcriptomic approach will be used to address the basis of biofilm formation. To provide broader access to younger students, we plan to develop a hands-on teaching kit for astrobiology with Carolina Biologicals. Our ultimate goal is to expand understanding of H. lacusprofundi biology as a model for the possible existence of extraterrestrial life.