The importance of calcium (Ca2+) as a cell regulator is well established in eukaryotes. However, the role ofCa2+ in prokaryotes still remains elusive. Recent experimental work has suggested that Ca2+ ions may playa regulatory role in prokaryotic organisms. An essential step toward an increased understanding of the roleof Ca2+ is the identification and characterization of Ca2+ binding proteins (CaBPs). In eukaryotes, CaBPsare involved in the regulation of multiple cellular events including: cell differentiation, gene expression,transport mechanisms and others. Our preliminary data indicates that several CaBPs are present in bacteria(E. coli, B. subtilis and B. pertussis). These proteins share similar biochemical characteristics with eukaryoticCaBPs, including calmodulin (CaM). In an effort to isolate and sequence these proteins we analyzed crudecell lysates by 2D-electrophoresis followed by mass spectrometry. Most of the proteins found with CaBPcharacteristics are associated with stress responses. Based on these initial findings and other published datathe purpose of this application is: 1) to map changes in protein expression as a function of cytosolic Ca2+levels during both optimal growth conditions and during induced stress responses (heat/cold/pH shock). 2) tostudy the role of Ca2+ ions in transcription and establish the nature of the calcium regulon. The long-termgoal of this research is to illuminate the role of Ca2+ in bacteria. We hypothesize that CaBPs play animportant role in Ca2+ homeostasis and that Ca2+ ions are involved in the regulation of several intracellularprocesses in bacterial cells. In particular, we hypothesize that the intracellular concentration of Ca2+ ions areinvolved in the regulation of stress response proteins with associated changes in gene expression. Thefollowing specific aims were developed: 1) To characterize further the previously identified CaBPs and toidentify new CaBPs that are involved in the bacterial response to changes in calcium levels 2) To identifyand characterize the adaptive mechanisms utilized by cells to maintain calcium homeostasis 3) To determinethe role of calcium in stress-related proteomic changes 4) To investigate the specific genomic changes thatoccur in bacteria in response to changing calcium levels. This study will lead to better understanding of basiccell biology and physiology of both pathogenic and environmental microorganisms and may lead todevelopment of new therapeutics for the control of pathogenic bacteria.
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