Proteomic analyses of Sulfolobus solfataricus, an extremophilic archaeon.

摘要

Microorganisms which inhabit extreme environments are known as extremophiles. Sulfolobus is a ubiquitous archaeon that can be found in hyperthermophilic and acidophilic habitats, such as the hot springs of Yellowstone National Park.; The proteins an organism uses to cope with environmental variables are known as its functional proteome and are a dynamic physiological response limited by the available genes in the organism. In this dissertation, the techniques of proteomics were applied toward studying Sulfolobus solfataricus .; Using high resolution two-dimensional gel electrophoresis, fluorescent protein stains, gel image analysis software, mass spectrometry, and bioinformatics, the practices and procedures of proteomics were developed. A quantitative and statistical evaluation was used to determine the reproducibility and confidence levels of using gel-separated proteins to monitor cellular protein expression levels. This evaluation strongly indicated the need for statistical rigor when using 2-D gels to determine protein expression differences.; The first proteomic mapping of a Crenarchaeota was performed on Sulfolobus solfataricus P2. A total of 867 protein spots (325 different gene products) from 2-D gels were mapped using MALDI MS and bioinformatic software. The proteomic techniques were extended to Sulfolobus solfataricus P1, where the genome is not known, and it was demonstrated that P1 proteins could be identified using the P2 genomic database, by mapping 420 P1 spots (224 gene products). Some of the proteins identified provide evidence of biochemical pathways that have not yet been explored for this microorganism. Additionally, protein expression differences obtained from using different carbon sources during cultivation revealed the possible presence of a novel respiratory pathway, previously unknown in archaea. Finally, lists of protein candidates have been created which can be used to guide further research to better understand the mechanisms used by Sulfolobus to cope with different carbon sources, or arsenate and arsenite stress.; A foundation has now been created for using a fully integrated proteomics approach to study a wide variety of complex biological systems. The new discoveries about Sulfolobus have already stimulated additional research projects to illuminate the mechanisms involved in its response to environmental variables and can be further used to complement genomic studies being performed by other research groups.