Characterization of factors regulating PII function in Rhodospirillum rubrum.

by David M. Wolfe

The need for coordination of nitrogen metabolism with other cellular metabolic processes is universal. One of the most widely distributed and evolutionarily conserved systems for regulating nitrogen assimilation involves the PII protein. This work uses Rhodospirillum rubrum, a photosynthetic nitrogen-fixing member of the alpha-proteobacteria, as a model organism in understanding PII function in vivo. PII interacts with a wide range of receptor proteins involved in the transcriptional and posttranslational control of nitrogen metabolism. PII functions as a signal integrator, sensing relative cellular nitrogen, carbon, and energy status, by directly or indirectly detecting the levels of glutamine, 2-ketogluratate (2-KG), ATP and ADP. PII integrates these signals into two basic indicators of cell status: the cell is nitrogen-sufficient regardless of energy or carbon status, or the cell is nitrogen-deficient AND energy- and carbon-sufficient.;In this thesis, I explore the role that the integral membrane protein AmtB1 and ATP or ADP binding have on PII function, and examine PII variants perturbed in their equilibrium between the nitrogen-sufficient and nitrogen-deficient conformations. AmtB1 was necessary for the switch-off of nitrogenase activity in response to conditions where PII signals nitrogen sufficiency. Both PII and AmtB1 were required to sequester and inactivate dinitrogenase reductase-activating glycohydrolase (DraG), preventing aberrant reactivation of nitrogenase. The interaction of PII and AmtB1 favored the PII homolog G1nJ, and was disrupted by 2-KG and ATP but stabilized by ADP. This led to an analysis of the role nucleotide binding plays in PII function. Two conserved residues of the PII homolog G1nB, K90 and R101, were randomized and screened for altered activity in vivo. G1nB variants with altered activity were less readily modified both in vivo and in vitro and were unable to function like wild-type in the uridylylated nitrogen-deficient state. Finally, several variants were analyzed that shifted G1nB towards a nitrogen-sufficient conformational state as determined by interactions with receptor proteins in a yeast two-hybrid system. Both sets of variants were altered in both modification states and activities in vivo and support a model of PII function where global protein conformation responds small molecule binding to regulate PII activity.

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