Characterization of the reconstituted UTase/UR-PII-NRII-NRI bicyclic signal transduction system that controls the transcription of nitrogen-regulated (Ntr) genes in Escherichia coli.

A reconstituted UTase/UR-PII-NRII-NRI bicyclic cascade regulated PII uridylylation and NRI phosphorylation in response to glutamine. We examined the sensitivity and robustness of the responses of the individual cycles and of the bicyclic system. The sensitivity of the glutamine response of the upstream UTase/UR-PII monocycle depended upon the PII concentration, and we show that PII exerted substrate inhibition of the UTase activity of UTase/UR, potentially contributing to this dependence of sensitivity on PII. In the downstream NRII-NRI monocycle, PII controlled NRI phosphorylation state, and the response to PII was hyperbolic at both saturating and unsaturating NRI concentration. As expected from theory, the level of NRI∼P produced by the NRII-NRI monocycle was robust to changes in the NRII or NRI concentrations when NRI was in excess over NRII, as long as the NRII concentration was above a threshold value, an example of absolute concentration robustness (ACR). Because of the parameters of the system, at physiological protein levels and ratios of NRI to NRII, the level of NRI∼P depended upon both protein concentrations. In bicyclic UTase/UR-PII-NRII-NRI systems, the NRI phosphorylation state response to glutamine was always hyperbolic, regardless of the PII concentration or sensitivity of the upstream UTase/UR-PII cycle. In these bicyclic systems, NRI phosphorylation state was only robust to variation in the PII/NRII ratio within a narrow range; when PII was in excess NRI∼P was low, and when NRII was in excess NRI phosphorylation was elevated, throughout the physiological range of glutamine concentrations. Our results show that the bicyclic system produced a graded response of NRI phosphorylation to glutamine under a range of conditions, and that under most conditions the response of NRI phosphorylation state to glutamine levels depended on the concentrations of NRI, NRII, and PII.

Jiang P ，Ventura AC ，Ninfa AJ 《-》

Sensory components controlling bacterial nitrogen assimilation.

In enteric bacteria, the transcription of the Ntr regulon is regulated by a signal transduction system that measures and transmits information on the nitrogen status of the cell. Four of the components of this signal transduction apparatus have been previously identified, and the roles of these are known, to a first approximation, from studies with purified components. The sensor is a uridylyltransferase/uridylyl-removing enzyme (UTase/UR) that controls the uridylylation state of the PII protein. PII indirectly regulates the transcription of the Ntr regulon by acting through the kinase/phosphatase protein NRII. In the absence of unmodified PII, NRII autophosphorylates on a histidine residue, and these phosphoryl groups are transferred to the transcription factor NRI, resulting in the conversion of NRI to the form able to activate transcription. In the presence of PII and NRII, NRI approximately P is rapidly dephosphorylated, preventing the activation of Ntr transcription. This PII-dependent dephosphorylation of NRI approximately P is referred to as the regulated phosphatase activity. In this report, we describe improved methods for the purification of the UTase/UR and PII, and the crystallization of PII. We also present improved methods for the assay of the activities of the UTase/UR protein and PII. The results of our assays indicate that purified PII is effective in eliciting the regulated phosphatase activity, but does not affect the autophosphorylation of NRII or affect the transfer of phosphoryl groups from NRII approximately P to NRI. In addition, we demonstrate that the elicitation of the regulated phosphatase activity by PII is strongly dependent on the ratio of NRI approximately P to NRI, and that the isolated N-terminal domain of NRI, once phosphorylated, is dephosphorylated by the regulated phosphatase activity.

Kamberov ES ，Atkinson MR ，Feng J ，Chandran P ，Ninfa AJ ... -  《-》

Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli.

Nitrogen-regulation of gene transcription in Escherichia coli results from the regulation of the phosphorylation state of the enhancer-binding transcription factor NRI (NtrC). We examined the regulation of NRI phosphorylation in a reconstituted bicyclic cascade system containing four regulatory proteins: NRI, the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate the signal transduction protein PII, and the kinase/phosphatase NRII (NtrB), which is a PII receptor that phosphorylates and dephosphorylates NRI. In this reconstituted system, the phosphorylation state of NRI was regulated reciprocally by the small molecule effectors glutamine, which prevented the accumulation of NRI-P, and 2-ketoglutarate, which caused accumulation of NRI-P. Regulation of the bicyclic system by glutamine was exclusively due to sensation and signal-transduction by the UTase/UR-PII monocycle, which was observed to function essentially as a glutamine-sensing apparatus. In contrast, regulation of NRI phosphorylation by 2-ketoglutarate, which binds to PII, was due to direct regulation of the NRII-PII interaction and the rate of NRI-P dephosphorylation. Thus, the PII protein transduces the glutamine signal to the NRII-NRI monocycle in the form of its uridylylation state and is also the receptor of the antagonistic 2-ketoglutarate signal, which blocks the activity of unmodified PII.

Jiang P ，Peliska JA ，Ninfa AJ 《BIOCHEMISTRY》

Reversible uridylylation of the Escherichia coli PII signal transduction protein regulates its ability to stimulate the dephosphorylation of the transcription factor nitrogen regulator I (NRI or NtrC).

We have reconstituted the signal transduction system responsible for the negative regulation of the transcription of the Escherichia coli glnA gene, encoding glutamine synthetase, by glutamine. This signal transduction system consists of four proteins: the transcription factor NRI (NtrC), which activates glnA transcription when it is phosphorylated, the kinase/phosphatase protein NRII (NtrB) that directly controls the extent of NRI phosphorylation, the PII signal transduction protein that controls the phosphatase activity of NRII, and the uridylyltransferase/uridylyl-removing (UTase/UR) enzyme that is regulated by glutamine and controls the activity of PII. In the reconstituted system, the removal of uridylyl groups from the PII protein, catalyzed by the UTase/UR protein in the presence of glutamine, resulted in the stimulation of NRI approximately P dephosphorylation. In contrast, the uridylylated form of the PII protein had no discernible effect on NRI phosphorylation. The uridylylation of the trimeric PII protein by the monomeric UTase/UR protein is a non-cooperative reaction in which the partially modified species accumulated and were readily observed. Partially modified PII trimers were partially active in stimulating the dephosphorylation of NRI approximately P. Thus, both the PII-UTase/UR and PII-NRII interactions display the continuous variability characteristic of rheostats as opposed to the binary variability characteristic of toggle switches.

Atkinson MR ，Kamberov ES ，Weiss RL ，Ninfa AJ ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》

A source of ultrasensitivity in the glutamine response of the bicyclic cascade system controlling glutamine synthetase adenylylation state and activity in Escherichia coli.

Glutamine synthetase (GS) activity in Escherichia coli is regulated by reversible adenylylation, brought about by a bicyclic system comprised of uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate, PII, adenylyltransferase (ATase), and its substrate, GS. The modified and unmodified forms of PII produced by the upstream UTase/UR-PII cycle regulate the downstream ATase-GS cycle. A reconstituted UTase/UR-PII-ATase-GS bicyclic system has been shown to produce a highly ultrasensitive response of GS adenylylation state to the glutamine concentration, but its composite UTase/UR-PII and ATase-GS cycles displayed moderate glutamine sensitivities when examined separately. Glutamine sensitivity of the bicyclic system was significantly reduced when the trimeric PII protein was replaced by a heterotrimeric form of PII that was functionally monomeric, and coupling between the two cycles was different in systems containing wild-type or heterotrimeric PII. Thus, the trimeric nature of PII played a role in the glutamine response of the bicyclic system. We therefore examined regulation of the individual AT (adenylylation) and AR (deadenylylation) activities of ATase by PII preparations with various levels of uridylylation. AR activity was affected in a linear fashion by PII uridylylation, but partially modified wild-type PII activated the AT much less than expected based on the extent of PII modification. Partially modified wild-type PII also bound to ATase less than expected based upon the fraction of modified subunits. Our results suggest that the AT activity is only bound and activated by completely unmodified PII and that this design is largely responsible for ultrasensitivity of the bicyclic system.

Jiang P ，Ninfa AJ 《-》