ToxR/S and Unsaturated Fatty Acid Regulation in Photobacterium profundum Strain SS9

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BartlettThe ToxR/S regulon is widely distributed among bacteria of the family Vibrionaceae. However, little is known about the nature or even the number of genes regulated by these proteins, except for some genes involved in human colonization and pathogenesis which have been acquired by horizontal gene transfer in Vibrio cholerae. Recent experiments in the investigator's lab indicate that ToxR and ToxS influence the ability of the deep-sea bacterium Photobacterium profundum strain SS9 to respond to changes in pressure and to grow at elevated pressure. Furthermore, in this organism, ToxR/S, together with the cyclic AMP receptor protein (CRP), appears to modulate the levels of unsaturated fatty acids (UFA's). It has previously been suggested that UFA's may be important factors in the adaptation of the membrane to elevated pressure and to decreased temperature. During this project the investigator will characterize the ToxR/S regulon in general, and the pressure and temperature regulation of UFA's in particular, in SS9. The connection between toxRS gene expression and growth inhibition at elevated pressure will be clarified. ToxR/S regulated genes will be identified using both genetic and molecular screening strategies. A subset of these genes will then be sequenced, their transcriptional regulation characterized, and their functions ascertained through mutant studies. Additional studies will specifically explore the possibility of transcriptional, translational, and posttranslational regulation of UFA's by pressure and temperature. Transcriptional regulation of genes whose products govern the levels of cis-vaccenic acid and eicosapentaenoic acid will be determined as a function of temperature and pressure in various regulatory protein mutant backgrounds. Finally, the regulation of the activity of 0-ketoacyl-ACP synthase II, which is required for high pressure and low temperature regulation of cis-vaccenic acid levels, will be examined through kinetic analyses.Despite the fact that the deep sea represents the largest part of our planet where life is found, little is known about the adaptations of organisms to this cold environment where the water pressure can exceed 15,000 pounds per square inch. In this project a bacterial species isolated from the bottom of the ocean will be used for genetic and physiological experiments to address the control of membrane components (unsaturated fatty acids) which are required for its growth and survival at low temperature and high pressure. The possibility that an enzyme responsible for making unsaturated fatty acids in this microorganism possesses unique characteristics suiting it to deep-sea conditions will be explored. The investigator will seek to identify additional cell functions which help the organism adjust to high pressure. The results will be of use in understanding the adaptation of life to extreme environments and will provide new information about how cells maintain proper membrane function.