Regulation of carbon and nitrogen metabolism: Adding regulation of ion channels and another second messenger to the mix

The regulation of metabolism in bacteria is accomplished by a complex array of signal transduction systems, metabolic interactions, and gene regulatory relationships. For example, maintenance of the balance of carbon and nitrogen metabolism involves (as a partial list) ppGpp, cAMP, the NtrBC two-component system and the coupled Nac-controlled regulon, the leucine-responsive protein LRP, the carbohydrate phosphotransferase system (PTS), and a parallel PTS that has been referred to as the nitrogen PTS. Understanding how these signaling and regulatory systems function to coordinate metabolism remains a significant challenge. In a recent issue of PNAS, Lee et al. (1) show for the first time one of the ways that the nitrogen PTS plays a role in regulating gene expression and metabolism. It does so by regulating an ion channel that is the major K⁺ transporter, TrkA. Apparently, K⁺ is a second messenger controlling gene expression and enzyme activity in bacteria.

Lessons From the Carbohydrate PTS

Control of the activity of membrane permeases is a well known function of the carbohydrate PTS, explaining, for example, one of the early problems in the regulation of gene expression, namely, the catabolite repression of lacZYA operon expression by glucose in the famous diauxy experiments of J. Monod (17). In those experiments, cells were grown in the presence of glucose and lactose; as long as glucose was available, it prevented the induction of the lacZYA operon. The mechanism responsible for this regulation is referred to as inducer exclusion; glucose prevents the entry of lactose into the cell (2). It does so indirectly, by bringing about the dephosphorylation of the key regulatory factor EIIA^glc, which forms part of the glucose PTS.

The glucose PTS consists of the general PTS components enzyme I (EI) and histidine phosphocarrier protein (HPr), the glucose-specific EIIA^glc protein, and the glucose permease referred to as EIIBC^glc. Phosphoryl groups from phosphoenolpyruvate (PEP) are transferred to EI, then to HPr, then to EIIA^glc, then to the EIIB portion of the permease and to the incoming sugar. Because the components are at equilibrium and the rate of vectoral phosphorylation of glucose can exceed the rate of EI phosphorylation by PEP, the phosphorylation state of the components is controlled by the presence of glucose. Dephosphorylated EIIA^glc is a signal that glucose is being rapidly internalized, whereas EIIA^glc∼P is a signal that glucose is absent. Both forms of EIIA^glc play important regulatory roles (3). The dephosphorylated EIIA^glc is directly responsible for certain examples of inducer exclusion. For example, it forms a complex with the lacY product, galactoside pemease, in the presence of external substrates for the permease, and blocks permease activity. EIIA^glc∼P is an activator of adenylate cyclase and thus plays a role in the regulation of gene expression by the global regulator cAMP–cAMP-binding protein. The carbohydrate PTS system of Escherichia coli also provides an example of regulation of transcription by a membrane permease (4). Glucose controls the expression of the PTS components indirectly, by interacting with its permease, the EIIBC^glc protein. When glucose is being rapidly internalized, the IIBC^glc sequesters the repressor Mlc at the membrane, derepressing the Mlc regulon.

Glucose controls the expression of the phosphotransferase system components indirectly.

The Nitrogen PTS

In addition to the carbohydrate PTS, E. coli contains a parallel PTS (5, 6). This system consists of an EI protein encoded by ptsP, an HPr homologue (Npr) encoded by ptsO, and an EIIA^Ntr encoded by ptsN (5, 6). The ptsN and ptsO genes are found within the same operon as the rpoN gene encoding σ⁵⁴, involved in expression of nitrogen assimilation genes and other genes (5). The association in an operon with rpoN suggested some involvement with nitrogen regulation, leading to the designation “nitrogen PTS.” In retrospect, this designation may be too narrow.

The initial phenotype associated with ptsN in E. coli was suppression of the conditional lethality of a temperature sensitive mutation in the small G protein Era (5). Era is involved in regulating traverse of a cell division checkpoint that occurs after DNA replication and before cytokinesis (7). It is also implicated in ribosome biogenesis and interacts directly with RNA and ribosome subunits (8). Finally, Era appears to regulate utilization of certain tricarboxylic acid cycle intermediates and pyruvate and to affect the starvation response of cells that are grown on succinate and then starved (9). Thus, the roles of ERA are somewhat confusing but suggest that it participates in metabolic regulation of the cell cycle, perhaps by multiple mechanisms.

As far as a “classical” Ntr phenotype, as might result, for example, from ptsN having a role in controlling the activity of σ⁵⁴, the results have been somewhat confusing. The ptsN mutation did not seem to have a significant effect of nitrogen control of glnA expression, which requires σ⁵⁴ and the NtrBC signaling system and its associated sensory system, implying that these processes were not perturbed (5). Similarly, the ptsN mutation had little discernable effect on the ability of E. coli to grow on certain amino acids as the sole source of carbon and nitrogen (5). On the other hand, the ptsN mutation had a very significant effect on utilization of certain amino acids as a nitrogen source when glucose was added to the growth medium. Specifically, the ptsN strain showed a mucoidy character in the presence of glucose and either Arg, His, or Pro (5). A mutant lacking NtrC shows a very mucoidy phenotype when glucose is the carbon source and either of these three amino acids is the nitrogen source, so perhaps there is some connection, directly or indirectly, with NtrC function or a parallel mechanism of controlling Ntr gene activation. The effect of glucose in inhibiting utilization of amino acids as nitrogen source was surpassed by even more dramatic effects of succinate, fumarate, glycerol, and citrate in blocking growth when adenosine or alanine was used as a source of carbon and nitrogen. These carbohydrates are poorly used by E. coli (citrate is not used at all), and the severity of the growth inhibition appeared to correlate with the poorness of the sugar, which is the opposite of what one would expect for catabolite repression (5). This growth inhibition by poorly used carbohydrates was eliminated by inclusion of the preferred nitrogen source, ammonium sulfate, in the medium (5). Thus, the carbohydrate inhibitors acted by affecting utilization of alanine or adenine as a nitrogen source.

Phenotypic analysis of the ptsP mutant, lacking the EI^Ntr, revealed a dramatic resistance of the strain to leucine-containing peptides (LCPs) and a less dramatic resistance to leucine (10). [It is thought that LCPs lead to elevated intracellular leucine, and it is not understood why leucine itself is less effective in raising intracellular leucine.] Mutation of ptsO also led to increased resistance to LCPs, and, in contrast, mutation of ptsN resulted in a dramatically increased sensitivity to LCPs. These results suggested that the dephosphorylated form of EIIA^Ntr was required to prevent leucine toxicity. As for the mechanism of leucine toxicity, it was traced to the absence of acetohydroxy acid synthetase I (AHAS I), which is one of two functional AHAS enzymes in E. coli (10). Leucine inhibition of AHAS I results in isoleucine pseudoauxotrophy, and prior studies had shown that one mechanism for LCP resistance is by mutation of AHAS I to insensitivity to leucine inhibition. It was observed that the ptsN mutation prevented derepression of the ilvBN genes encoding AHAS I (10). Thus, it was imagined that EIIA^Ntr should interact with a transcription factor that regulated ilvBN expression (10), and factors that interacted with EIIA^Ntr were sought (1).

Regulation of an Ion Channel

Remarkably, the factor that EIIA^Ntr interacts with to regulate ilvBN expression was not a transcription factor in the typical sense, but rather was TrkA, the main permease for K⁺ under normal growth conditions where K⁺ is available at relatively high concentration. Dephospho EIIA^Ntr bound tightly to TrkA and appears to diminish the internalization of K⁺, lowering the internal K⁺ concentration. Apparently, elevated intracellular K⁺ blocks both expression of ilvBN and the activity of AHAS I (1). Together, the results of Lee et al. (1) present a remarkable parallel to the process of inducer exclusion mediated by EIIA^glc, which, because K⁺ is an inhibitor or ilvBN expression, can be thought of as “inhibitor exclusion.” Signals controlling the phosphorylation state of EIIA^Ntr control the exclusion of K⁺, which acts as a second messenger to control gene expression and activity. The analogy to the signaling by regulation of ion channels in eukaryotes could not be sharper.

The dephosphorylated form of EIIA^Ntr was required to prevent leucine toxicity.

Moving from Clues to Mechanisms

What are the signals that control the phosphorylation state of EIIA^Ntr? In a carbohydrate PTS, it is the transported carbohydrate that dephosphorylates the specific PTS components. A compound that serves as a “traditional” substrate for phosphorylation by the nitrogen PTS remains to be identified. Yet, there almost certainly has to be a mechanism for dephosphorylation of EIIA^Ntr. One possibility is that EIIA^Ntr∼P may transfer its phosphoryl group to components of another PTS perhaps by way of reverse transfer to NPr. But, biochemical analysis so far suggests that the nitrogen PTS did not significantly cross-react with carbohydrate PTS in vivo or readily cross-talk with general and glucose PTS components in vitro (5, 6).

The unusual structure of the ptsP-encoded EI^Ntr may provide some clues. Unlike EI, EI^Ntr contains a GAF domain at its N terminus, similar to the Azotobacter vinelandi NifA protein that activates nif gene expression in response to signals of nitrogen status (6). Little and Dixon (11) have shown that the GAF domain of this NifA protein plays a key role in sensation. Specifically, the GAF domain is directly controlled by α-ketoglutarate and in addition binds to the histidine kinase-like domain of the NifL protein, which itself interacts with the GlnK signal transduction protein. Thus, perhaps EI^Ntr is allosterically regulated by a histidine kinase, such as NtrB, which also interacts with and is regulated by GlnK. Because NtrB, GlnK, and the related PII protein all are directly involved in regulation of σ⁵⁴ and NtrC-dependent nitrogen assimilation genes, this would be a remarkable connection.

The extensive results from the study of ptsN function in Pseudomonas putida should also be considered. In this organism, global expression analysis using two-dimensional gel electrophoresis has indicated that ptsN affects numerous proteins, comprising ≈5–9% of all proteins analyzed (12). Of these, a small number are regulated by glucose, and, of this small set, some are regulated from σ⁵⁴-dependent promoters. A much larger number of proteins regulated by ptsN were unrelated to control by glucose or σ⁵⁴. Thus, the broad conclusion is that ptsN controls numerous genes, most likely by a variety of different mechanisms. On the other hand, the EIIA^Ntr of P. putida clearly has a role in mediating glucose repression of certain promoters that depend on σ⁵⁴ and the transcriptional activator XylR (13, 14). Furthermore, the phosphorylated form of EIIA^Ntr was apparently required for repression (15), and some evidence suggests that repression involves blocking the interaction of XylR with its binding site (16). Whether these effects are direct or mediated by additional components remains to be determined.