28.2: Regulation of Gene Expression in Bacteria

<h2prokaryotetxn">28.2.1 Prokaryotic Gene Regulation

In bacteria and archaea, structural proteins with related functions are usually encoded together within the genome in a block called an operon and are transcribed together under the control of a single promoter, resulting in the formation of a polycistronic transcript (Figure 28.2.1). In this way, regulation of the transcription of all of the structural genes encoding the enzymes that catalyze the many steps in a single biochemical pathway can be controlled simultaneously, because they will either all be needed at the same time, or none will be needed. For example, in E. coli, all of the structural genes that encode enzymes needed to use lactose as an energy source are encoded next to each other in the lactose (or lac) operon under the control of a single promoter, the lac promoter. French scientists François Jacob (1920–2013) and Jacques Monod at the Pasteur Institute were the first to show the organization of bacterial genes into operons, through their studies on the lac operon of E. coli. For this work, they won the Nobel Prize in Physiology or Medicine in 1965.

Figure 28.2.1 Schematic Representation of an Operon. In prokaryotes, structural genes of related function are often organized together on the genome and transcribed together under the control of a single promoter. The operon’s regulatory region includes both the promoter and the operator. If a repressor binds to the operator, then the structural genes will not be transcribed. Alternatively, activators may bind to the regulatory region, enhancing transcription.

Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

Each operon includes DNA sequences that influence its own transcription; these are located in a region called the regulatory region. The regulatory region includes the promoter and the region surrounding the promoter, to which transcription factors, proteins encoded by regulatory genes, can bind. Transcription factors influence the binding of RNA polymerase to the promoter and allow its progression to transcribe structural genes. A repressor is a transcription factor that suppresses transcription of a gene in response to an external stimulus by binding to a DNA sequence within the regulatory region called the operator, which is located between the RNA polymerase binding site of the promoter and the transcriptional start site of the first structural gene. Repressor binding physically blocks RNA polymerase from transcribing structural genes. Conversely, an activator is a transcription factor that increases the transcription of a gene in response to an external stimulus by facilitating RNA polymerase binding to the promoter. An inducer, a third type of regulatory molecule, is a small molecule that either activates or represses transcription by interacting with a repressor or an activator.

In prokaryotes, there are examples of operons whose gene products are required rather consistently and whose expression, therefore, is unregulated. Such operons are constitutively expressed, meaning they are transcribed and translated continuously to provide the cell with constant intermediate levels of the protein products. Such genes encode enzymes involved in housekeeping functions required for cellular maintenance, including DNA replication, repair, and expression, as well as enzymes involved in core metabolism. In contrast, there are other prokaryotic operons that are expressed only when needed and are regulated by repressors, activators, and inducers.

Prokaryotic operons are commonly controlled by the binding of repressors to operator regions, thereby preventing the transcription of the structural genes. Such operons are classified as either repressible operons or inducible operons. Repressible operons, like the tryptophan (trp) operon, typically contain genes encoding enzymes required for a biosynthetic pathway. As long as the product of the pathway, like tryptophan, continues to be required by the cell, a repressible operon will continue to be expressed. However, when the product of the biosynthetic pathway begins to accumulate in the cell, removing the need for the cell to continue to make more, the expression of the operon is repressed. Conversely, inducible operons, like the lac operon of E. coli, often contain genes encoding enzymes in a pathway involved in the metabolism of a specific substrate like lactose. These enzymes are only required when that substrate is available, thus expression of the operons is typically induced only in the presence of the substrate.

The trp Operon: A Repressible Operon

E. coli can synthesize tryptophan using enzymes that are encoded by five structural genes located next to each other in the trp operon (Figure 28.2.2). When environmental tryptophan is low, the operon is turned on. This means that transcription is initiated, the genes are expressed, and tryptophan is synthesized. However, if tryptophan is present in the environment, the trp operon is turned off. Transcription does not occur and tryptophan is not synthesized.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. However, when tryptophan accumulates in the cell, two tryptophan molecules bind to the trp repressor molecule, which changes its shape, allowing it to bind to the trp operator. This binding of the active form of the trp repressor to the operator blocks RNA polymerase from transcribing the structural genes, stopping expression of the operon. Thus, the actual product of the biosynthetic pathway controlled by the operon regulates the expression of the operon.

Figure 28.2.2 The Trp Operon. The five structural genes needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is absent, the repressor protein does not bind to the operator, and the genes are transcribed. When tryptophan is plentiful, tryptophan binds the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan biosynthesis genes.

Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

The Lac Operon: An Inducible Operon

The lac operon is an example of an inducible operon that is also subject to activation in the absence of glucose. The lac operon encodes three structural genes, lacZ, lacY, and lacA, necessary to acquire and process the disaccharide lactose from the environment (Fig 28.2.3A).

Figure 28.2.3 Biological Activity of the lac Operon. (A) Schematic representation of the lac operon in E. coli. The lac operon has three structural genes, lacZ, lacY, and lacA that encode for β-galactosidase, permease, and galactoside acetyltransferase, respectively. The promoter (p) and operator (o) sequences that control the expression of the operon are shown. Upstream of the lac operon is the lac repressor gene, lacI, controlled by the lacI promoter (p). (B) Shows the lac repressor inhibition of the lac operon gene expression in the absence of lactose. The lac repressor binds with the operator sequence of the operon and prevents the RNA polymerase enzyme which is bound to the promoter (p) from initiating transcription. (C) In the presence of lactose, some of the lactose is converted into allolactose, which binds and inhibits the activity of the lac repressor. The lac repressor-allolactose complex cannot bind with the operator region of the operon, freeing the RNA polymerase and causing the initiation of transcription. Expression of the lac operon genes enables the breakdown and utilization of lactose as a food source within the organism.

Figure modified from: Esmaeili, A., et. al. (2015) BMC Bioinformatics 16:311

The lacZ gene encodes the β-galactosidase (β-gal) enzyme responsible for the hydrolysis of lactose into simple sugars glucose and galactose (Fig. 28.2.4A). The β-gal enzyme can also mediate the breakdown of the alternate substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal) (Fig. 28.2.4B). The breakdown product, 5-bromo-4-chloro-3-hydroxyindole – 1, spontaneously dimerizes to form the intensely blue blue product, 5,5′-dibromo-4,4′-dichloro-indigo – 2. Thus, Xgal has been a valuable research tool, not only in the study of the enzymatic activity of β-gal, but also in the development of the commonly used blue-white DNA cloning system that utilizes the β-gal enzyme as a marker in molecular cloning experiments.

The lac operon contains two more genes, in addition to lacZ (Fig. 28.2.3A). The lacY gene encodes a permease that increases the uptake of lactose into the cell and lacA encodes a galactoside acetyltransferase (GAT) enzyme. The exact function of GAT during lactose metabolism has not been conclusively elucidated but acetylation is thought to play a role in the transport of the modified sugars.

Figure 28.2.4 Reactions Controlled by the Expression of the Lac Operon. (A) Expression of the β-galactosidase enzyme enables the breakdown of lactose into the simple sugars, glucose and galactose for E. coli to use as a food resource. (B) The β-galactosidase enzyme also mediates the breakdown of the non-native substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal). Breakdown product (1) 5-bromo-4-chloro-3-hydroxyindole quickly dimerizes into the intensely blue product (2) 5,5′-dibromo-4,4′-dichloro-indigo making it a useful tool for molecular biology. (C) β-D-1-thiogalactopyranoside (IPTG) can serve as a non-native inducer of the lac operon. It mimics the structure of lactose and binds with the Lac Repressor.

Figure modified from: Andreas Piehler, Yikrazuul, and NUROtiker

For the lac operon to be expressed, lactose must be present. This makes sense for the cell because it would be energetically wasteful to create the enzymes to process lactose if lactose was not available.

In the absence of lactose, the lacI gene is constituitively expressed, expressing the lac repressor protein (Fig. 28.2.3 B). The lac repressor binds with an operator region of the lac operon and physically prevents RNA polymerase from transcribing the structural genes (Fig. 28.2.3 B). However, when lactose is present, the lactose inside the cell is converted to allolactose. Allolactose serves as an inducer molecule, binding to the repressor and changing its shape so that it is no longer able to bind to the operator DNA (Fig. 28.2.3 C). Removal of the repressor in the presence of lactose allows RNA polymerase to move through the operator region and begin transcription of the lac structural genes. In addition to lactose, laboratory experiments have revealed that the non-natural compound Isopropyl β-D-1-thiogalactopyranoside (IPTG) can also bind with the lac repressor and cause the expression of lac operon (Figure 28.2.4 C). Similar to Xgal, this compound has also been used as a research tool for molecular cloning.

The Lac Operon: Activation by Catabolite Activator Protein

Bacteria typically have the ability to use a variety of substrates as carbon sources. However, because glucose is usually preferable to other substrates, bacteria have mechanisms to ensure that alternative substrates are only used when glucose has been depleted. Additionally, bacteria have mechanisms to ensure that the genes encoding enzymes for using alternative substrates are expressed only when the alternative substrate is available. In the 1940s, Jacques Monod was the first to demonstrate the preference for certain substrates over others through his studies of E. coli’s growth when cultured in the presence of two different substrates simultaneously. Such studies generated diauxic growth curves, like the one shown in Figure 28.2.5. Although the preferred substrate glucose is used first, E. coli grows quickly and the enzymes for lactose metabolism are absent. However, once glucose levels are depleted, growth rates slow, inducing the expression of the enzymes needed for the metabolism of the second substrate, lactose. Notice how the growth rate in lactose is slower, as indicated by the lower steepness of the growth curve.

Figure 28.2.5. Utilization of Glucose in E. Coli. When grown in the presence of two substrates, E. coli uses the preferred substrate (in this case glucose) until it is depleted. Then, enzymes needed for the metabolism of the second substrate are expressed and growth resumes, although at a slower rate.

Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

The ability to switch from glucose use to another substrate like lactose is a consequence of the activity of an enzyme called Enzyme IIA (EIIA). When glucose levels drop, cells produce less ATP from catabolism and EIIA becomes phosphorylated. Phosphorylated EIIA activates adenylyl cyclase, an enzyme that converts some of the remaining ATP to cyclic AMP (cAMP), a cyclic derivative of AMP and important signaling molecule involved in glucose and energy metabolism in E. coli (Fig. 28.2.6). As a result, cAMP levels begin to rise in the cell. This is an indicator to the cell, that overall energy levels are low and that ATP is being depleted.

Figure 28.2.6. Conversion of ATP to cAMP. When ATP levels decrease due to depletion of glucose, some remaining ATP is converted to cAMP by adenylyl cyclase. Thus, increased cAMP levels signal glucose depletion.

Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

The lac operon also plays a role in this switch from using glucose to using lactose. When glucose is scarce, the accumulating cAMP caused by increased adenylyl cyclase activity binds to catabolite activator protein (CAP), also known as cAMP receptor protein (CRP). The complex binds to the promoter region of the lac operon (Figure 28.2.7). In the regulatory regions of these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter. Binding of the CAP-cAMP complex to this site increases the binding ability of RNA polymerase to the promoter region to initiate the transcription of the structural genes. Thus, in the case of the lac operon, for transcription to occur, lactose must be present (removing the lac repressor protein) and glucose levels must be depleted (allowing binding of an activating protein). When glucose levels are high, there is catabolite repression of operons encoding enzymes for the metabolism of alternative substrates. Because of low cAMP levels under these conditions, there is an insufficient amount of the CAP-cAMP complex to activate transcription of these operons.

Figure 28.2.7 Effect of CAP on the Lac Operon. (a) In the presence of cAMP, CAP binds to the promoters of operons, like the lac operon, that encode genes for enzymes for the use of alternate substrates. (b) For the lac operon to be expressed, there must be activation by cAMP-CAP as well as removal of the lac repressor from the operator.

Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

Global Responses of Prokaryotes

In prokaryotes, there are also several higher levels of gene regulation that have the ability to control the transcription of many related operons simultaneously in response to an environmental signal. A group of operons all controlled simultaneously is called a regulon.

Alarmones

When sensing impending stress, prokaryotes alter the expression of a wide variety of operons to respond in coordination. They do this through the production of alarmones, which are small intracellular nucleotide derivatives, such as guanosine pentaphosphate (pppGpp) (Fig. 28.2.8).

Figure 28.2.8 Structure of Guanosine Pentaphosphate (pppGpp)

Figure from: Yikrazuul

Alarmones change which genes are expressed and stimulate the expression of specific stress-response genes. For example, pppGpp signaling is involved in the stringent response in bacteria, causing the inhibition of RNA synthesis when there is a shortage of amino acids present. This causes translation to decrease and the amino acids present are therefore conserved. Furthermore, pppGpp causes the up-regulation of many other genes involved in stress response such as the genes for amino acid uptake (from surrounding media) and biosynthesis.

The use of alarmones to alter gene expression in response to stress appears to be important in pathogenic bacteria, as well. On encountering host defense mechanisms and other harsh conditions during infection, many operons encoding virulence genes are upregulated in response to alarmone signaling. Knowledge of these responses is key to being able to fully understand the infection process of many pathogens and to the development of therapies to counter this process.

Quorum Sensing

Quorum sensing (QS) is an intercellular communication mechanism of bacteria used to coordinate the activities of individual cells in population level in response to surroundings through production and perception of diffusible signal molecules such as Acyl Homoserine Lactones or small singaling peptides (Fig. 28.2.9). The signal synthase, signal receptor, and signal molecules are three essential elements of the basic QS circuit machinery (Fig. 28.2.9). Genes encoding signal generating proteins are also included among the QS target genes. This forms an autoinduction feedback loop to modulate generation of signal molecules. Several bacterial behaviors including virulence factors expression, secondary metabolites production, biofilm formation, motility, and luminescence are regulated by QS. Through complex regulatory networks bacteria are capable of expressing corresponding genes according to their own population size and of behaving in a coordinated manner.

Figure 28.2.9 Examples of Quorum Sensing Pathways. (Left panel) Typical Gram-negative quorum sensing mechanism. Acyl homoserine lactone molecules, synthesized by LuxI, passively pass the bacterial cell membrane and when a sufficient concentration is reached (threshold level) activate the intracellular LuxR which subsequently activates target gene expression in a coordinated way. Note that a single cell is shown for simplicity. However, acyl homoserine lactones will commonly diffuse and target neighboring cells within the colony to mediate a communal or population response within the bacterial colony. (Right panel) Quorum sensing peptides are synthesized by the bacterial ribosomes as pro-peptidic proteins and undergo posttranslational modifications during excretion by active transport. The quorum sensing peptides bind membrane associated receptors which get autophosphorylated and activate intracellular response regulators via phosphor-transfer. These phosphorylated response regulators induce increased target gene expression.

Figure from: Verbeke, F., et.al. (2017) Frontiers in Neuroscience 11:183.

For example, some microbial species, such as Staphylococcus aureus, can encase their community within a self-produced matrix of hydrated extracellular polymeric substances that include polysaccharides, proteins, nucleic acids, and lipid molecules. These encasements are known as biofilms. The formation of the biofilm on solid surfaces is a step-wise process comprising several stages (Fig. 28.2.10). It starts with the conditioning of the surface through the coating with macromolecules from the aqueous surrounding, which enables initial reversible adhesion of microorganisms. The next step is a formation of stronger, irreversible attachments to the surface, followed by the proliferation and aggregation of microorganisms into multicellular and multilayered clusters, which actively produce extracellular matrix. Some cells in the mature biofilms continuously detach and separate from the aggregates, representing a continuous source of planktonic bacteria that can subsequently spread and form new microcolonies.

Figure 28.2.10 Schematic drawing of biofilm formation.

Figure from: Rukavina, Z., and Vanic, Z. (2016) Pharmaceutics 8(2):18.

Biofilms are a common cause of chronic, nosocomial and medical device-related infections, due to the fact that they can develop either on vital or necrotic tissue as well as on the inert surfaces of different implanted materials. Moreover, biofilms are linked with high-level resistance to antimicrobials, frequent treatment failures, increased morbidity and mortality. As a consequence, biofilm infections and accompanying diseases have become a major health concern and a serious challenge for both modern medicine and pharmacy. The rough estimation shows that more than 60% of hospital-associated infections are attributable to the biofilms formed on indwelling medical devices, which result in more than one million cases of infected patients annually and more than $1 billion of hospitalization costs per year in the USA.

Biofilm infections share some common characteristics: slow development in one or more hot-spots, delayed clinical manifestation, persistency for months or years, usually with interchanging periods of acute exacerbations and absence of clinical symptoms. Even though they are less aggressive than acute infections, their treatment is challenging to a greater extent. The main reason for the aforesaid is up to 1000-fold decrease in susceptibility of biofilms to antimicrobial agents and disinfectants as well as resistance to host immune response. Thus, ways to reduce or inhibit biofilm formation are highly sought. The majority of the proposed biofilm-control methods focuses on: (i) prevention and minimization of biofilm formation by selection and surface modifications of anti-adhesive materials; (ii) debridement techniques including ultrasound and surgical procedures; (iii) disruption of biofilm QS-signaling system; or (iv) achieving proper drug penetration and delivery to formed biofilms by the use of electromagnetic field, ultrasound waves, photodynamic activation or specific drug delivery systems.

Alternate σ Factors

Since the σ subunit of bacterial RNA polymerase confers specificity as to which promoters should be transcribed, altering the σ factor used is another way for bacteria to quickly and globally change what regulons are transcribed at a given time. The σ factor recognizes sequences within a bacterial promoter, so different σ factors will each recognize slightly different promoter sequences. In this way, when the cell senses specific environmental conditions, it may respond by changing which σ factor it expresses, degrading the old one and producing a new one to transcribe the operons encoding genes whose products will be useful under the new environmental condition. For example, in sporulating bacteria of the genera Bacillus and Clostridium (which include many pathogens), a group of σ factors controls the expression of the many genes needed for sporulation in response to sporulation-stimulating signals.

Prokaryotic Attenuation and Riboswitches

Although most gene expression is regulated at the level of transcription initiation in prokaryotes, there are also mechanisms to control both the completion of transcription, as well as translation, concurrently. Since their discovery, these mechanisms have been shown to control the completion of transcription and translation of many prokaryotic operons. Because these mechanisms link the regulation of transcription and translation directly, they are specific to prokaryotes, because these processes are physically separated in eukaryotes.

One such regulatory system is attenuation, whereby secondary stem-loop structures formed within the 5’ end of an mRNA being transcribed determine if transcription to complete the synthesis of this mRNA will occur and if this mRNA will be used for translation. Beyond the transcriptional repression mechanism already discussed, attenuation also controls expression of the trp operon in E. coli (Fig. 28.2.11). The trp operon regulatory region contains a leader sequence called trpL between the operator and the first structural gene, which has four stretches of RNA that can base pair with each other in different combinations. When a terminator stem-loop forms, transcription terminates, releasing RNA polymerase from the mRNA. However, when an antiterminator stem-loop forms, this prevents the formation of the terminator stem-loop, so RNA polymerase can transcribe the structural genes.

Figure 28.2.11. Attenuation of Transcription and Translation. When tryptophan is plentiful, translation of the short leader peptide encoded by trpL proceeds, the terminator loop between regions 3 and 4 forms, and transcription terminates. When tryptophan levels are depleted, translation of the short leader peptide stalls at region 1, allowing regions 2 and 3 to form an antiterminator loop, and RNA polymerase can transcribe the structural genes of the trp operon.

Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

A related mechanism of concurrent regulation of transcription and translation in prokaryotes is the use of a riboswitch, a small region of noncoding RNA found within the 5’ end of some prokaryotic mRNA molecules (Figure 28.2.12). A riboswitch may bind to a small intracellular molecule to stabilize certain secondary structures of the mRNA molecule. The binding of the small molecule determines which stem-loop structure forms, thus influencing the completion of mRNA synthesis and protein synthesis.

Figure 28.2.12. Riboswitch Form and Function. Riboswitches found within prokaryotic mRNA molecules can bind to small intracellular molecules, stabilizing certain RNA structures, influencing either the completion of the synthesis of the mRNA molecule itself (left) or the protein made using that mRNA (right).

Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

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