Cyclic AMP (cAMP) dependent catabolite repression effect in E. coli is among the most intensely studied regulatory processes in biology. However, the physiological function(s) of cAMP signalling and its molecular triggers remain elusive. Here we use a quantitative physiological approach to show that cAMP signalling tightly coordinates the cell’s protein expression program with its metabolic needs during exponential cell growth: The expression of carbon catabolic genes increased linearly with decreasing growth rates upon limitation of carbon influx, but decreased linearly with decreasing growth rate upon limitation of nitrogen or sulfur influx. In contrast, the expression of biosynthetic genes exhibited the opposite linear growth-rate dependence as the catabolic genes. A coarse-grained mathematical model provides a quantitative framework for understanding and predicting gene expression responses to catabolic and anabolic limitations. A scheme of integral feedback control featuring the inhibition of cAMP signalling by metabolic precursors is proposed and validated. These results reveal a key physiological role of cAMP-dependent catabolite repression: to ensure that proteomic resources are spent on distinct metabolic sectors as needed in different nutrient environments. Our finding underscores the power of quantitative physiology in unravelling the underlying functions of complex molecular signalling networks.

Transcription by54 RNA polymerase depends on activators that contain ATPase domains of the AAA + class. These activators, which are often response regulators of two-component signal transduction systems, remodel the polymerase so that it can form open complexes at promoters. Here, we report the first crystal structures of the ATPase domain of an activator, the NtrC1 protein from the extreme thermophile Aquifex aeolicus. This domain alone, which is active, crystallized as a ring-shaped heptamer. The protein carrying both the ATPase and adjacent receiver domains, which is inactive, crystallized as a dimer. In the inactive dimer, one residue needed for catalysis is far from the active site, and extensive contacts among the domains prevent oligomerization of the ATPase domain. Oligomerization, which completes the active site, depends on surfaces that are buried in the dimer, and hence, on a rearrangement of the receiver domains upon phosphorylation. A motif in the ATPase domain known to be critical for coupling energy to remodeling of polymerase forms a novel loop that projects from the middle of an ␣ helix. The extended, structured loops from the subunits of the heptamer localize to a pore in the center of the ring and form a surface that could contact 54 .

Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH 4 ؉ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH 4 ؉ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium͞methylammonium transport B (AmtB) protein [called methylammonium͞ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH 4 ؉ ͞NH 3 in bacteria as well as eucarya. Because NH 3 is the species limiting at low pH for a given total concentration of NH 4 ؉ ؉ NH 3 , results with both organisms indicate that AmtB͞MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [ 14 C]methylammonium depends on its conversion to ␥-Nmethylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH 4 ؉ ͞NH 3 . Hence, contrary to previous views, we propose that AmtB͞MEP proteins increase the rate of equilibration of the uncharged species, NH 3 , across the cytoplasmic membrane rather than actively transporting-that is, concentrating-the charged species, NH 4 ؉ .

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Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein.Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate͞nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains. Together with their cognate autokinases, response regulators dominate signal transduction in bacteria (1-5) and are also found upstream of protein kinase cascades in eukarya (6-9). Autokinase͞response regulator pairs, which are referred to as ''two-component'' systems, control bacterial cell division (10), development (11), chemotaxis (12), virulence (13)(14)(15)(16), and the responses to many changes in nutrient availability (17,18). Phosphorylation of an Asp in receiver domains of response regulators is used to modulate the function of their corresponding output domains (19), many of which activate or repress transcription (2, 3). The structures of six nonphosphorylated receiver domains have been determined (20-25), and it has been shown that a substantial conformational change occurs after phosphorylation in several cases (26-28). However, the lability of the acyl phosphate linkages in these domains (half-lives of seconds to hours) (2, 3) has hindered structural studies of their phosphorylated active forms.The response regulator nitrogen regulatory protein C (NtrC), an enhancer-binding protein, functions as a molecular machine to activate transcription by the 54 -holoenzyme form of RNA polymerase (29-31). The NtrC protein of Salmonella typhimurium is composed of three functional domains (32-34): an (amino) N-terminal receiver or regulatory domain that is phosphorylated on Asp-54 (D54), a central output domain that hydrolyzes ATP and activates transcription, and a C-terminal DNA-binding domain. The central domain of NtrC apparently adopts a mononucleotide-binding fold characteristic of a large group of purine nucleotide-binding proteins (35,36). Phosphorylation of D54 allows NtrC to form large oligomers that are essential for ATP hydrolysis and hence, transcriptional activation (30,31,37,38).The receiver domain of NtrC (NtrC r ) is the only such domain for which the structures of both the phosphorylated and nonphosphorylated forms have been determined (D. Kern, B. F. Volkman, P. Luginbuhl, M. J. Nohaile, S.K., and D.E.W., unpublished material; ref. 21). Although it was possible to maintain phosphorylated NtrC r (P-NtrC r ) for long enough to allow determination of its structure b...