Regulation of Cyst cell development in Rhodospirillum centenum

• Main Author: Marden, Jeremiah Nathaniel
• Format: Dissertation
• Language: English
• Subjects: biology, microbiology; biology, molecular; chemistry, biochemistry
• ISBN: 1124246703; 9781124246703

Rhodospirillum centenum is a photosynthetic bacterium that when stressed, forms metabolically quiescent, desiccation resistant cyst cells similar to those formed by Azotobacter and Azospirillum species. Several regulators of cyst cell development have been identified in R. centenum, including an operon of chemotaxis-like genes (che3). Encoded two component regulators of the Che3 cluster include the histidine kinase-response regulator hybrids CheA3 and CheS3, as well as a small receiver domain protein, CheY3. Strains deleted of either cheS3 or cheY3 form cysts prematurely, whereas a cheA3 deletion strain is delayed in cyst cell development. In this study, we purified and biochemically characterized recombinant His-tagged versions of these three proteins. The results of this work demonstrate that CheS3 is capable of autophosphorylation, and that intramolecular phosphotransfer between kinase and receiver domains does not occur, but that intermolecular phosphotransfer to CheY3 rapidly takes place. Preliminary studies with CheA3 suggest that while it is capable of autophosphorylation, intramolecular phosphotransfer is possible, but not intermolecular phosphotransfer to CheY3. Through transposon mutagenesis of a strain deleted of the che3 operon, we identified several novel regulators, including two hybrid sensor kinase response regulators, a serine/threonine phosphatase, a σ70 transcription factor and a class III nucleotidyl cyclase. Genetic and biochemical studies of the nucleotidyl cyclase demonstrate that it is a novel guanylyl cyclase whose product (cGMP) is shown to be a crucial signal molecule which regulates R. centenum cyst-cell development. A homolog of the E. coli cAMP receptor protein linked to the guanylyl cyclase is shown to be involved in this process, as it preferentially binds cGMP in vitro, and a strain deleted of this homolog fails to form cysts.