When the ammonia supply to the bacteria is adequate, it is the preferred source of nitrogen for the bacteria, and it acts to repress the genes required for the assimilation of other nitrogenous compounds. When the ammonia supply in the growth medium becomes limiting, then the genes which are required for ammonia production from other external nitrogen sources are activated. It is also well known that, during limiting ammonia conditions, glutamine synthetase becomes more active, with the transcription of the gene for glutamine synthetase also being stimulated. Thee overall result is the production of more glutamine synthetase to scavenge all of the ammonia present for the synthesis of glutamine and glutamate.
The genes which are regulated by the ammonia supply belong to the Ntr regulon, a regulon being a set of non-contiguous operons or genes which are all controlled by a common regulator, the common response regulator in the Ntr regulon being a response regulator protein, NRI. The Ntr regulon contains the genes for glutamine synthetase, NRII (a histidine kinase, which is sometimes called NtrB) and NRI (the response regulator, also called NtrC), amongst others. The other genes which may be present will have different functions, depending upon the type of bacteria in which it is found.
Glutamine can be considered as the intracellular signal which informs the cell whether the ammonia levels are adequate or not, as the case may be. This ability of the glutamine is as a consequence of the nature of the nitrogen assimilation reaction. When there is plentiful ammonia in the medium, that ammonia is incorporated into glutamate by the action of glutamate dehydrogenase, ammonia is also incorporated by the ATP-dependent reaction catalysed by glutamine synthetase. This combination of reactions is important as, of all the nitrogen containing molecules in the cell; glutamate provides 85% of the nitrogen, while glutamine provides the remaining 15%. However, under limiting conditions, the glutamate dehydrogenase reaction is not significant in the cell’s glutamate synthesis. In its place, glutamine is produced by transferring an amino group from the amide nitrogen of glutamine to α-ketoglutarate by glutamate synthase. This results in two molecules of glutamate, one of which is changed back to glutamine by glutamine synthetase. Thus glutamine becomes the nitrogen source for glutamate. This means that glutamine levels within the cell will mirror the levels of ammonia in the medium and can so be used as an indicator of external ammonia levels.
The regulation of the Ntr regulon is a complex signalling pathway. The transcription of the Ntr regulon is inhibited in situations with excess nitrogen, and this inhibition is relieved when the nitrogen concentration drops below 1mM. What happens is that excess ammonia results in an increase in the concentration of the active form of the PII, which indirectly represses transcription of the Ntr regulon by inactivating a positive transcription regulator protein (NRI) via dephosphorylation. When ammonia concentrations decrease, the amount of active PII also decreases, and transcription of the Ntr regulon is stimulated because the response regulator is phosphorylated.
Ammonia controls the levels of active PII in the following way. As the ammonia concentration is increased, the intracellular concentration of glutamine rises. It is actually the glutamine that causes active PII levels to increase, and, as a consequence, transcription to be repressed. This is due to the fact the glutamine is able to activate and enzyme that converts PII-UMP to PII, the active form of PII. The increase in PII represses the Ntr regulon.
When ammonia levels fall to below 1mM, the opposite happens, the concentration of glutamine falls. This is due to the level of glutamine synthetase, the enzyme that synthesises glutamine from ammonia, glutamate, and ATP, is low in cells grown in the presence of high ammonia. In addition to this, glutamate dehydrogenase, the enzyme that synthesises glutamate from α-ketoglutarate, ammonia, and NADPH, has a high Km for ammonia and is therefore relatively inactive when the ammonia concentrations fall below 1nM. The consequence of all of this is that when the ammonia concentrations sharply decrease, there occurs a drop in the intracellular concentration of glutamine. When these glutamine levels drop, the concentration of α-ketoglutarate increases because it is the glutamine that donates an amino group to α-ketoglutarate via the glutamate synthase reaction when the ammonia concentrations are low. The increased amounts of α-ketoglutarate and decreased amounts of glutamine lower the concentration PII by stimulating its conversion to PII-UMP. The decrease in PII stimulates transcription of the Ntr regulon.
The reason why increasing the amount of PII inhibits transcription of the Ntr regulon is that PII activates the phosphatase activity of the histidine kinases, NRII, and the phosphatase activity of NRII inactivates the response regulator (positive transcription factor), NRI-P. When the levels of PII decrease, NRII acts as a phosphatase and phosphorylates NRI, which then activates transcription. The sequence of events can be summarised as…
- Low ammonia results in an increase in the concentrations of α-ketoglutarate because its conversion to glutamine and glutamate is slowed;
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The α-ketoglutarate lowers the concentration of PII by stimulating its conversion to PII-UMP;
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When PII levels fall, NRII no longer acts as a kinase and phosphorylates NRI; and
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NRI-P stimulates transcription.
When ammonia levels are high, then transcription is repressed because glutamine stimulates the conversion of PII-UMP to PII, and PII stimulates the phosphatase activity of NRII, which results in a decrease in NRI-P.
The central operon involved in these processes is called the glnALG operon. This encodes for three genes. They are;
- glnA, the gene for glutamine synthetase;
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glnL, the gene for NRII, which is a bifunctional histidine kinase / phosphatase whose substrate is NRI; and
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glnG, the gene for NRI, a response regulator.
Under nitrogen excess (high ammonia), the operon is transcribed at a low level from the glnAp1 and glnLp promoters by sigma 70 RNA polymerase. The small amount of transcription from glnAp1 is sufficient to guarantee the synthesis of enough glutamine synthetase to meet the cell’s needs for glutamine when the ammonia concentrations are high. Part of the reason is that only a small amount of transcription takes place from these promoters is that transcription from them is repressed by NRI. Under nitrogen-limiting conditions, the glnALG operon is transcribed at a high frequency from the glnAp2 promotor by the sigma 54 RNA polymerase. Transcription from the glnAp2 promoter requires the phosphorylated form of the response regulator NR1, reflecting the requirement of sigma 54 RNA polymerases for the binding of a transcription regulator upstream to the promoter in order to form an open complex. Since NRI-P binds more than 100 bp upstream of the promote region, the DNA must bend in order for NRI-P to make contact with the RNA polymerase. The protein kinase, NRII, is itself regulated by another protein, called PII. When PII levels are high, NRII acts like a phosphatase rather than a kinase, and the levels of NRI-P drop. Hence PII inhibits transcription of the glnALG operon. Under conditions of excess ammonia, PII is generated from PII-UMP because glutamine stimulates the removal of UMP from PII-UMP catalysed by the bifunctional enzyme uridylyl transferase-uridylyl removal (UT/UR). When ammonia levels fall, the PII is converted to PII-UMP in a reaction catalysed by UT/UR and stimulated by α-ketoglutarate (and inhibited by glutamine). Under these circumstances, NRII acts as a kinase rather than as a phosphatase, and transcription is stimulated.
Thus high ammonia leads to the dephosphorylation of the response regulator, hence repression of the glnALG operon. A similar situation exists for the regulation of the PHO operon. In this case, a high concentration of inorganic phosphate leads to the dephosphorylation of the response regulator and a consequent repression of the PHO operon.
A key enzyme in the signal transduction pathway is the bifunctional enzyme UT/UR that either adds or removes UMP from PII. It can be considered a sensor protein that responds to the α-ketoglutarate/glutamine ratio and modifies PII, which in turn regulates the activity of the histidine kinase/phosphatase (NRII). Alternatively, glutamine synthetase may be considered to be the sensor as it responds to the ammonia supply and determines the levels of glutamine and α-ketoglutarate, which signal the UT/UR.
Within enteric bacteria there exist other nitrogen control genes, for example, nac and gluF. Within bacteria there also exist many novel nitrogen regulation systems. For example, nitrogen control in cyanobacteria and in gram-positive bacteria is different to that which has already been discussed. The methods by which bacteria are able to sense extracellular nitrogen, along with nitrate and nitrite sensing also differs between the different genera of bacteria, as does the regulation of nitrogen fixations.
