1. Most processes in bacteria are too complex to be encoded on a single operon. Control becomes to complex or inefficient to be practical.

2. An additional reason is that many processes must be subject to multiple levels/types of regulation.

For example, dual regulation of the lactose or galactose or arabinose operons is desirable to that cells don't waste energy producing enzymes for less desirable substrate utilization.

REGULON: an operon network in which the member operons are associated with a single pathway, function, or process and regulated by a common regulatory protein and its effector(s). Example: arginine regulon.

MODULON: an operon network concerned with multiple pathways or functions, in which operons may be under individual controls as well as a common, pleiotropic regulatory protein. Example: catabolite repression = crp modulon = those operons/genes under the regulation of the Catabolite Repressor Protein and its effector, cAMP.

STIMULON: All operons that respond to a common environmental stimulus, no matter what regulons or modulons might be involved. Examples: heat-shock and cold-shock stimulons, phosphate stimulon.

Others: GLOBAL CONTROL SYSTEM (stringent response); MULTIGENE SYSTEMS (cell division, DNA replication, sporulation); ADAPTIVE RESPONSE (networks that respond to specific environmental stresses such as heat shock, UV light (SOS response)).

A common procedure to recognize proteins belonging to regulatory groups is to radiolabel proteins after a shift to the new conditions. Proteins that become specifically labeled are identified by 2-D gel electrophoresis. Proteins are separated accoding to their charge in one direction (isoelectric point) and according to their mass in a second dimension. Proteins that change in abundance are characterized by protein sequencing, and the sequence is used to prepare a DNA hybridization probe (or compared to the translation of the entire genome now for E. coli and other bacteria). The process of using protein sequence to predict a gene sequence to isolate a gene is called REVERSE GENETICS. Another method is to use a promoter lacZ gene borne on a transposable element. The element is allowed to randomly insert in the genome, and then colonies are screened for color reaction for b-galactosidase after change in growth conditions. Genes are easily isolated, since the transposon serves as a hybridization "tag" to identify the site of insertion.

1. Carbon limitation. Catabolite Repression in enteric bacteria. Regulates many catabolic operons for for carbon/energy source utilization.

2. Amino acid or Energy limitation. Stringent Response in enteric bacteria.

3. Nitrogen limitation. Ntr system of enteric bacteria or nif system in other bacteria.

4. Phosphate limitation. Pho system of many bacteria.

1. Presence of oxygen. Arc (aerobic respiration)

2. Presence of electron acceptors other than O₂. Fnr system for anaerobic respiration

3. Absence of usable electron carriers = fermentation conditions.

1. UV and other DNA damages. SOS Response

2. Alkylation of DNA. Ada system.

3. Oxidative stress/oxidative response.

4. Heat-Shock

5. Cold-Shock

6. High Osmolarity/Osmotic Shock.

1. Growth Rate Control

2. Stationary Phase/Nutrient limitation

3. Starvation/Competence in Bacillus subtilis

4. Starvation/Sporulation in Gram-positives

5. Temperature-shifts/Virulence in some pathogens.

6. Heterocyst Differentiation, other cellular differentiation systems in many bacterial systems

1. Constitutive synthesis of glucose utilizing enzymes.

2. Glucose prevents the entry of some other inducing substrates by inactivation of their permeases. This is mediated by EnzIIA^Glu binding directly to LacY permease in case of lactose. Phosphotransferase substrates are preferred over non-PTS substrates.

3. Transient Repression. Mechanism unknown

4. Catabolite Repression. Inhibition of synthesis of enzyme synthesis even in the presence of appropriate inducers.

Catabolite Repression is best understood part of these controls.

CRP (Catabolite Repressor Protein) or CAP (Catabolite Activator Protein) is an allosteric DNA binding protein that is a dimer of 22.5 kDa. Each subunit can bind one cAMP, and when cAMP is bound, the protein binds to DNA at specific sequences, where it serves as a positive control element. Controls ability to utilize lactose, galactose, arabinose, maltose, tryptophan, D-serine, and histidine. CRP/cAMP binding only provides the POTENTIAL to express the appropriate catabolic genes, which also require an inducer to be present that is specific for each operon. In the case of the lac operon, CRP/cAMP binding to operator causes a ~10-fold increase in frequency of lacZYA operon transcription. EnzIIA^Glu becomes phosphorylated as glucose supply runs out. The phosphorylated form of this protein is a positive modulator of ADENYLATE CYCLASE, which converts ATP into cAMP. CRP/cAMP is a negative regulator of crp and cya transcription.

System is very important. Mutants in crp or cya (adenylate cyclase) genes have growth defects even on glucose. The regulation of enzyme

levels, etc. must optimize the cell metabolism in ways that are still not understood completely.

Because PTS substrates (glucose, mannose, fructose, mannitol, etc.) will indirectly cause less phosphorylation of EnzIIA^Glu, such subtrates will be preferred over non-PTS substrates (lactose, galactose, arabinose, etc.).

Stimulus: level of EnzIIA^Glu

Sensor: Adenylate Cyclase

Signal: cAMP

Regulator: cAMP/CRP complex

Response: catabolic operons produce enzymes that allow the utilization of secondary substrates for growth.

Return: not well understood, but includes control of crp and cya transcription by cAMP/CRP

Reduction in levels of precursors for protein synthesis leads to a rapid depletion of pool metabolites causing "starvation" for charged tRNAs. Global changes in metabolism result, known as the "stringent response." This response causes a transient but near total inhibition of ribosome and tRNA. Stringent response occurs after nearly any nutritional restriction/shift-down (e.g., rich medium shift to minimal medium; starvation for an amino acid).

Mutant discovered in the 1950's could not reduce the rate of rRNA and tRNA synthesis. It was described as "relaxed" and the gene affected is called relA. Mutants show lags of hours when down-shifted nutritionally. Wild-type adapts often in minutes.

Mutants in the relA gene do not accumulate to nucleotides, that are derivatives of GDP and GTP. Called "Magic Spots I and II", they are 5'pppGpp3' and 5'ppGpp3'. These compounds can be synthesized in two ways.

1. RelA is (p)ppGpp synthetase I, and is ribosome associated. If the ribosome stalls with an uncharged tRNA in the A site, then pyrophosphate is transferred from ATP to GTP (or GDP) at the 3' position by RelA. This produces (p)ppGpp, which is converted completely to ppGpp by a phosphohydrolase (Gpp). (p)ppGpp is converted back to GTP or GDP by the SpoT protein, and GTP is reformed by nucleotide diphosphate kinase (Ndk).

2. A second pathway for the formation is SpoT, which is related in amino acid sequence to RelA, and is thus (p)ppGpp synthetase II. Depletion of carbon or energy source can also trigger the stringent response, which serves to conserve ATP by reducing protein synthesis. SpoT can cause both the synthesis or the degradation of (p)ppGpp. The likely scenario is that allosteric or chemical modification of SpoT cause it to behave either to synthesize or degrade (p)ppGpp. What the SpoT protein might sense in the cell is not known.

Elevation of (p)ppGpp by one of the two mechanisms leads to downstream effects on transcription of tRNA and rRNA genes, probably by a direct action of the b subunit of RNA polymerase. Perhaps anti-termination and the stringent response are related?? In any event, RNA polymerase pausing could be modified and interference with both initiation and elongation affected. Inhibition of rRNA synthesis would lead to translational repression of ribosomal protein synthesis. Virtually all processes in cells would be affected either directly or indirectly. It is known that genes encoding amino acid biosynthesis enzymes can be up-regulated, and this effect can facilitate balancing between production and consumption in a global sense. A double mutant lacking SpoT and RelA can not grow on minimal media and requires several amino acids for growth. Suppressors are easily isolated, and most map to the b or b' subunit of RNA polymerase. These observations suggest that (p)ppGpp may be required for transcription of some operons encoding amino acid biosynthesis genes. Although most required amino acids are regulated by attenuation, Trp is not required but is regulated by attenuation. '

Elevation of (p)ppGpp has significant effects on translation as well. Translational fidelity is lower when protein synthesis is very slow, and elevated levels of (p)ppGpp improves fidelity, possibly by competing with GTP and GDP for translation factors that require these nucleotides. This might improve proofreading. (p)ppGpp also blocks the binding of initiator f-Met-tRNA to the ribosome, inhibiting translation globally.

Stimulus: decrease in supply of nutrients for protein synthesis

Sensor: RelA-containing ribosome with uncharged tRNA in its A site

Signal: pppGpp and ppGpp (Magic Spots)

Regulator: Unknown. RNA polymerase???

Response: Lowered expression of stable RNAs (rRNA, tRNA), ribosomal proteins, etc. Temporary halt in DNA synthesis after replication round is completed. All biosynthesis is slowed downl-- phospholipids, LPS, proteins, RNA, etc.

Return: control of Magic Spot levels

Inorganic phosphate is a limiting nutrient in many environments, and the ability to scavenge Pi when it is only present at low concentration is very important. Alkaline phosphatase (PhoA) is an important scavenging enzyme that is inducible, secreted, and only activated once it reaches the periplasmic space. It cleaves Pi from organic phosphates. However, Pi starvation leads to the elevated production of more than 100 proteins.

Stimulus: low external concentration of Pi

Sensor: Pst transporter--PhoU

Signal: Protein phosphorylation

Transducer: PhoR

Regulator: PhoB

Output: Proteins such as PhoA

Response: Improved Pi acquisition

For mechanism, see Figures

Glutamine synthetase (GlnA) is encoded in an operon glnA-ntrB-ntrC which has several promoters. One is highly activated under NH₃ limitation. This promoter is NOT recognized by s⁷⁰ but rather is recognized by s⁵⁴ or RpoN (formerly glnF or ntrA). RpoN can bind promoters, but can not form open complexes in the absence of activators of transcription (hence, positive control elements). In this case, the activator is the phosphorylated form of NtrC, which can also be considered an ENHANCER of transcription.

The same regulation circuitry that modulates GlnA activity also modulates the transcription of the gene. A low Gln/a-KG ratio is sensed by UTase which modifies GlnB = P_II protein by uridylylation. NtrB has two activities: it can act to autophosphorylate itself and transfer those Pi groups to NtrC, and it can dephosphorylate NtrC-Pi. Modified (P_II-UMP)₃ does not interact with NtrB, which then autophosphorylates on His residues. NtrB-Pi can phosphorylate NtrC leading to activation of transcription of the glnA operon via RpoN (s⁵⁴). When the Gln/a-KG ratio is high, this is sensed by UR/UTase and UMP groups are removed from P_II. This allows P_II to interact wtih NtrB, causing it to act as a phosphatase to remove Pi groups from NtrC, inactivating transcription of the glnA operon and other operons. Other regulated operons include those for encoding enzymes to degrade organic N sources as well as transporters for various amino acids. Some additional transcriptional regulators may also be involved (e.g., Nac in Klebsiella sp.)

Stimulus: restricted supply of NH3 lead to reduction in Gln/a-KG ratio

Sensor: UR/UTase

Signal: Protein phosphorylation

Transducer: NtrB

Regulator: NtrC

Output: Increased GlnA and other proteins for N acquistion

Response: Improved efficiency of N acquistion, improved growth

Return: Feedback loops controls output from the system

For mechanism, also see Figure

Here regulation is quite different. Moderate temperature stress (30°C to 42°C shift) induces the synthesis of about 40 proteins. The critical player is RpoH = s³². At extreme temperatures (50°C), a second sigma factor (RpoE = s²⁴ = s^E) and heat-shock regulon (~13 proteins) is induced.

Operons controlled include a wide range of molecular chaperones (DnaK, DnaJ, HtpG, ClpB, GrpE, GroEL/GroES) and proteases (Lon, ClpP, ClpX, HflB). These proteins act to refold or degrade proteins in response to environmental conditions (e.g., high Temperature) that cause protein unfolding.

The key regulator is the amount of RpoH/s³² in the cell. At 30°C, there are about 10-30 molecules of RpoH per cell. After a temperature upshift, the level rises 15-fold in 5 minutes.

Present ideas: amount of free DnaK/DnaJ/GrpE determines the outcome. These proteins exert negative affects on the stability RpoH and the translation of rpoH mRNA. T-upshift would cause lower levels of free DnaK/DnaJ/GrpE, allowing RpoH levels to increase and promote transcription of heat-shock genes.

Two major regulators of Metabolism are Fnr (fumarate nitrate reduction) and Arc (aerobic respiration control). Stress regulators are SoxRS and OxyRS systems.

Fnr is a global transcription regulator with similarity to CRP; it has 4 Cys residues that have been shown to bind a 4Fe-4S cluster under some conditions and 2 [2Fe-2S] centers under other conditions. Hence, a redox-sensitive confirmational change can occur that mediates control of transcription of ~30 transcription units and >70 genes. For nitrate reductase, Fnr only potentiates expression. Nitrate must be present to relieve repression by NarX/NarL two component sensor-regulator system.

In this system, ArcB is the sensor kinase, and is a membrane-bound protein found in the cytoplasmic membrane. ArcA is the response regulator of the system and is a DNA-binding transcriptional regulator. The signal that causes autophosphorylation of ArcB and phosphotransfer to ArcA is not known, but is presumed to be either the proton motive force or a redox signal, possibly the redox state of the quinone pool. Target genes include those encoding quinol oxidase, succinate dehydrogenase, superoxide dismutase and many others involved in aerobic metabolism and energy production.

SoxRS controls about 40 proteins in E. coli that are induced in response to superoxide generating agents. These include superoxide dismutases (SodA, SodB), catalase, heme peroxidases and other protective agents. OxyRS controls about 40 proteins in response to H₂O₂. Role of OxyS not clear; OxyR is a transcriptional regulator which can be oxidized by oxygen or hydrogen peroxide to activate it.