The Escherichia coli signal transducers PII (GlnB) and GlnK form heterotrimers in vivo : Fine tuning the nitrogen signal cascade

Abstract

The PII protein is Escherichia coli's cognate transducer of the nitrogen signal to the NRII (NtrB)͞NRI (NtrC) two-component system and to adenylyltransferase. Through these two routes, PII regulates both amount and activity of glutamine synthetase. GlnK is the recently discovered paralogue of PII, with a similar trimeric x-ray structure. Here we show that PII and GlnK form heterotrimers, in E. coli grown in nitrogen-poor medium. In vitro, fully uridylylated heterotrimers of the two proteins stimulated the deadenylylation activity of adenylyltransferase, albeit to a lower extent than homotrimeric PII-UMP. Fully uridylylated GlnK did not stimulate, or hardly stimulated, the deadenylylation activity. We propose that uridylylated PII͞GlnK heterotrimers fine-regulate the activation of glutamine synthetase. The PII͞GlnK couple is a first example of prokaryotic signal transducer that can form heterotrimers. Advantages of hetero-oligomer formation as molecular mechanism for fine-regulation of signal transduction are discussed.I n enteric bacteria, the PII protein occupies a pivotal position in the network regulating the activity of glutamine synthetase (GS), itself a key enzyme in the assimilation of ammonia. The nitrogen status of the cell, as sensed by uridylyltransferase (UTase), is transferred to PII by adjusting the degree of uridylylation of the latter. Native PII signals a nitrogen-rich status whereas PII-UMP flags a nitrogen-poor status. From PII, the signal is transferred to two targets, i.e., adenylyltransferase (ATase) and NRII (NtrB), involved in the covalent modification of GS and in the transcriptional regulation of the gene encoding GS, respectively (refs. 1 and 2; Fig. 1).Escherichia coli can also contain a PII-like protein: i.e., GlnK (3, 4). PII and GlnK are similar, both structurally and functionally. The two proteins are each composed of 112 amino acid residues that are 67% identical and have virtually the same trimeric crystal structure (5-7). Each protein has been purified as homotrimer (5, 8). Both PII and GlnK can be modified by UTase at tyrosine-51 (4, 9-12). Functionally, PII and GlnK can address the same targets; i.e., in vitro, they can both regulate the activities of ATase and NRII (4, 11). However, the in vivo response of either target to PII exceeds that to GlnK. This may in part be because of a difference in the effect of the small molecule 2-oxoglutarate on the two signal proteins (11, 13).One functional aspect differs drastically between PII and GlnK; i.e., the expression of the glnB gene (which encodes PII) is constitutive with respect to the intracellular nitrogen status (14) whereas the expression of the glnK gene is regulated by it (4, 15). There is little or no GlnK after growth in nitrogen-rich conditions. This report describes the identification of PII͞GlnK-heterotrimers in E. coli after growing the cells in nitrogen-poor media and also shows that in vitro generated heterotrimers have lower activity than PII homotrimers. It suggests that GlnK may moderate the regulation of ammoni...

A mutant GlnD nitrogen sensor protein leads to a nitrogen-fixing but ineffective Sinorhizobium meliloti symbiosis with alfalfa

“…In the Rm1021⌬glnB mutant, GlnK-UMP might induce nitrogen stress response activities leading to a high level of GS expression and catabolism of various nitrogen sources, but in a way that differs from GlnB-UMP. Regulatory cross-talk between different PII proteins has been shown in other bacteria (24,25). The rpoN mutant results are more difficult to explain but suggest that an alternate sigma factor can induce catabolic activities.…”

Metabolic Context and Possible Physiological Themes of ς54-Dependent Genes inEscherichia coli

“…Purified GlnK and P II have similar activities, but the regulation of these activities is different (9,58,160,161). However, the physiological relevance of many differences has not been established and is sometimes refuted by mutant phenotypes.…”

“…One property of purified GlnK that accounts for this suppression is the relatively slow uridylylation of GlnK compared to that of P II (9). This property is accentuated by the formation of GlnK-P II heterotrimers (58,161) and the inactivation of P II -UMP by GlnK in such heterotrimers (161). The net effect is enhanced dephosphorylation of NR I ϳP and lower expression of Ntr genes.…”

Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum

“…Both proteins can also interact with NtrB to regulate NtrC activity, and with adenylyltransferase (ATase, the glnE product) to modify glutamine synthetase (GS) (4)(5)(6). However, they also display some distinct properties, such as the ability of GlnB-UMP, but not GlnK-UMP, to strongly stimulate the deadenylylation of GS-AMP (the adenylylated form of GS) (7). In K. pneumoniae, GlnK, but not GlnB, regulates the interaction of NifL and NifA to control the expression of the nif operons (8,9).…”