27.1: Regulation of Gene Expression in Bacteria

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Operon Organization and Transcriptional Control

• Explain the operon model of prokaryotic gene regulation: describe how structurally related genes are clustered under a single promoter to produce a polycistronic transcript, define the roles of the regulatory region (promoter and operator), and distinguish between constitutively expressed operons (housekeeping genes) and regulated operons—and explain why coordinated regulation of an entire metabolic pathway under a single promoter is energetically advantageous for a bacterium.
• Contrast repressible and inducible operons using the trp and lac operons as examples: explain how the trp operon is normally active but is repressed when tryptophan (the corepressor) binds the trp repressor protein, inducing a conformational change that allows it to bind the operator and block RNA polymerase—while the lac operon is normally repressed by the constitutively produced lac repressor but is induced when allolactose (produced from lactose) binds the repressor and prevents operator binding, allowing transcription.
• Explain the dual control of the lac operon by both the lac repressor and the catabolite activator protein (CAP): describe how glucose depletion raises cAMP levels (through EIIA phosphorylation → adenylyl cyclase activation), how cAMP-bound CAP binds upstream of the lac promoter to enhance RNA polymerase recruitment, and explain why both conditions must be simultaneously satisfied—lactose present (repressor off) AND glucose absent (CAP-cAMP on)—for maximal lac operon transcription, illustrating catabolite repression and the logic of diauxic growth.

Global and Post-Initiation Mechanisms of Gene Regulation

• Describe three mechanisms of global gene regulation in prokaryotes: explain how regulons coordinate the simultaneous control of multiple operons in response to environmental signals; how alarmones such as pppGpp mediate the stringent response (inhibiting rRNA/tRNA synthesis and redirecting resources toward stress-response genes including amino acid uptake and biosynthesis when amino acids are limiting); and how alternative σ factors allow bacteria to rapidly reprogram their entire transcriptome by replacing one σ factor with another that recognizes different promoter sequences—illustrating with sporulation σ factors in Bacillus and Clostridium.
• Explain quorum sensing as a population-level gene regulation mechanism: describe how Gram-negative bacteria use acyl homoserine lactones (AHLs) synthesized by LuxI-type synthases that diffuse across membranes and activate LuxR-type transcription factors at threshold concentrations, while Gram-positive bacteria use modified secreted peptides that bind membrane receptors triggering a two-component phosphorelay—and explain how quorum sensing regulates biofilm formation, virulence factor expression, secondary metabolite production, and motility, and why biofilms pose such significant clinical challenges (up to 1000-fold increased antimicrobial resistance, accounting for >60% of nosocomial infections).
• Describe prokaryotic attenuation and riboswitches as mechanisms that couple transcription and translation regulation: explain how trp operon attenuation works through ribosome stalling at tryptophan codons in the trpL leader sequence when tryptophan is scarce—allowing an antiterminator stem-loop (regions 2-3) to form and permitting transcription—while tryptophan abundance allows ribosome read-through and formation of the terminator stem-loop (regions 3-4), halting transcription; and explain how riboswitches in the 5′ UTR of mRNA bind small molecules to stabilize alternative RNA secondary structures that either terminate transcription or block translation (illustrating with guanine, Mg²⁺, c-di-GMP, and GlcN6P-responsive riboswitches), and why these mechanisms are exclusively prokaryotic.

Introduction

Each nucleated cell in a multicellular organism contains copies of the same DNA. Similarly, all cells in two pure bacterial cultures inoculated from the same starting colony contain the same DNA, except for changes that arise from spontaneous mutations. If each cell in a multicellular organism has the same DNA, then how is it that cells in different parts of the organism’s body exhibit different characteristics? Similarly, how is it that the same bacterial cells within two pure cultures exposed to different environmental conditions can exhibit different phenotypes? In both cases, each genetically identical cell does not "turn on" or express the same set of genes. Only a subset of proteins in a cell at a given time is expressed.

Genomic DNA contains both structural genes, which encode products that serve as cellular structures or enzymes, and regulatory genes, which encode products that regulate gene expression. The expression of a gene is a highly regulated process. Whereas regulating gene expression in multicellular organisms enables cellular differentiation, in single-celled organisms like prokaryotes, it primarily ensures that a cell’s resources are not wasted on making proteins it does not need at that time.

Elucidating the mechanisms that control gene expression is crucial to understanding human health. Malfunctions in this process in humans lead to the development of cancer and other diseases. Understanding the interaction between a pathogen's gene expression and that of its human host is crucial to understanding a particular infectious disease. Gene regulation involves a complex web of interactions within a given cell among signals from the cell’s environment, signaling molecules within the cell, and the cell’s DNA. These interactions result in the expression of specific genes and the suppression of others, depending on the circumstances.

Prokaryotes and eukaryotes share similarities in gene expression regulation; however, in eukaryotes, gene expression is more complex due to the temporal and spatial separation between transcription and translation. Thus, although most gene expression regulation in prokaryotes occurs at the transcriptional level, in eukaryotes it occurs at the transcriptional and post-transcriptional levels (after the primary transcript has been made).

In bacteria and archaea, structural proteins with related functions are typically encoded together in a genomic block known as an operon. They are transcribed together under the control of a single promoter, resulting in the formation of a polycistronic transcript, as shown in Figure \(\PageIndex{1}\). In this way, the regulation of transcription for all 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 required. For example, in E. coli, all the structural genes that encode enzymes needed to utilize lactose as an energy source are clustered together 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 demonstrate 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 \(\PageIndex{1}\): Schematic Representation of an Operon. In prokaryotes, structural genes of related functions 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. 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 encompasses the promoter and the surrounding region to which transcription factors and proteins encoded by regulatory genes can bind. Transcription factors influence RNA polymerase binding to the promoter, allowing transcription to proceed and structural genes to be transcribed. A repressor is a transcription factor that suppresses the 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 gene transcription 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 operons whose gene products are consistently required and whose expression, therefore, is not regulated. 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, other prokaryotic operons 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, eliminating the need for the cell to continue producing more of it, the expression of the operon is repressed. Conversely, inducible operons, such as the lac operon of E. coli, often encode enzymes involved in a pathway that metabolizes a specific substrate, such as lactose. These enzymes are required only when the substrate is available; thus, operon expression is typically induced only in its presence.

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, as shown in Figure \(\PageIndex{2}\). 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 binds to the operator by itself; therefore, the operon is active, and tryptophan is synthesized. However, when tryptophan accumulates in the cell, two tryptophan molecules bind to the trp repressor, changing its shape and 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 the expression of the operon. Thus, the actual product of the biosynthetic pathway controlled by the operon regulates the expression of the operon.

Figure \(\PageIndex{2}\): The Trp Operon. Parker, N., et. al. (2019) Microbiology. Openstax

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 \(\PageIndex{3}\) shows an interactive iCn3D model of the E. Coli Trp repressor-operator complex (1TRO).

Figure \(\PageIndex{3}\): E. Coli Trp repressor-operator complex (1TRO). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...tjbYwr47gVZjn8

The Trp repressor is depicted as a dimer, with one subunit shown in gray and the other in gold. The backbone of the two DNA strands is shown in spacefill magenta and cyan, except where the bases on the major groove interact with the Trp repressor. The tryptophan in each of the proton monomers is shown in spacefill with CPK colors and labeled. Noncovalent interactions (hydrogen bonds and salt bridges) between the protein and DNA are shown with dotted lines. 6 water-mediated hydrogen bonds to phosphate are not shown. Note that there are a few H bonds between the protein and base hydrogen bond donors and acceptors, suggesting that the repressor might bind specifically through geometric interactions with the backbone, along with the water-mediated hydrogen bonds.

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, as shown in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Biological Activity of the lac Operon.Figure modified from Esmaeili, A., et. al. (2015) BMC Bioinformatics 16:311

Panel (A) shows 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).

Panel (B) shows the lac repressor inhibition of the lac operon gene expression in the absence of lactose. The lac repressor binds to the operator sequence of the operon and prevents the RNA polymerase enzyme, bound to the promoter (p), from initiating transcription.

Panel (C) shows that 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 to the operator region of the operon, thereby freeing RNA polymerase and allowing transcription initiation. Expression of the lac operon genes enables the breakdown and utilization of lactose as a food source within the organism.

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

Figure \(\PageIndex{5}\): Reactions Controlled by the Expression of the Lac Operon. Andreas Piehler, Yikrazuul, and NUROtikerr Panel (A) shows the expression of the β-galactosidase enzyme, which enables the breakdown of lactose into simple sugars, glucose, and galactose for E. coli to use as a food resource. Panel (B) shows that 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. Panel (C) shows that 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.

The lac operon contains two more genes, in addition to lacZ (Fig. 4). 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.

For the lac operon to be expressed, lactose must be present. This makes sense for the cell because it would be energetically wasteful to produce the enzymes necessary for lactose processing if lactose were not available.

In the absence of lactose, the lacI gene is constitutively expressed, resulting in the production of the lac repressor protein (Fig. 28.2.3B). The lac repressor binds to the operator region of the lac operon and physically prevents RNA polymerase from transcribing the structural genes (Fig. 28.2.3B). However, when lactose is present, it is converted inside the cell to allolactose. Allolactose serves as an inducer molecule, binding to the repressor and altering its shape so that it can no longer bind to the operator DNA (Fig. 28.2.3 C). Removal of the repressor in the presence of lactose enables RNA polymerase to proceed through the operator region and initiate 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 to the lac repressor and induce the expression of the lac operon (panel C). Like Xgal, this compound has been used as a research tool for molecular cloning.

Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the lactose operon repressor and its complexes with DNA (1LBG).

Figure \(\PageIndex{6}\): Lactose operon repressor and its complexes with DNA (1LBG). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...a79MvrAHGpJk99

The resolution of the structure above was insufficient to show the amino acid side chains. Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the NMR solution structure of the dimer of LAC repressor DNA-binding domain complexed to its natural operator O1 (2KEI).

Figure \(\PageIndex{7}\): NMR solution structure of Dimer of LAC repressor DNA-binding domain complexed to its natural operator O1 (2KEI). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...p48VrPFXg6WgD9

Note the presence of white spheres representing hydrogen atoms (these do not appear in crystal structures but do in NMR structures). Color coding is the same as above. Zoom in to see specific interactions between the protein and the exposed DNA base hydrogen bond donors and acceptors. The O1 and O2 complex exhibits similar specific and nonspecific contacts, as expected, given that the lambda repressor has similar affinity for these two operator sites. In contrast, one side of the O3 complex exhibits a reduction in protein-DNA interactions, consistent with its lower affinity for the O3 operator.

The Lac Operon: Activation by Catabolite Activator Protein

Bacteria typically can use a variety of substrates as carbon sources. However, because glucose is usually preferred over other substrates, bacteria have mechanisms to ensure that alternative substrates are used only after 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 a preference for certain substrates over others through his studies of E. coli growth when cultured simultaneously in the presence of two different substrates. Such studies generated diauxic growth curves, as shown in Figure \(\PageIndex{8}\). 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 \(\PageIndex{8}\): 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. 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 an important signaling molecule involved in glucose and energy metabolism in E. coli, as shown in Figure \(\PageIndex{9}\). As a result, cAMP levels begin to rise in the cell. This signals to the cell that overall energy levels are low and that ATP is being depleted.

Figure \(\PageIndex{9}\): Conversion of ATP to cAMP. When ATP levels decrease due to the 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 crucial role in this switch from glucose to lactose utilization. When glucose is scarce, the accumulating cAMP, produced by increased adenylyl cyclase activity, binds to the catabolite activator protein (CAP), also known as the cAMP receptor protein (CRP). The complex binds to the promoter region of the lac operon, as shown in Figure \(\PageIndex{10}\). In the regulatory regions of these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter. The 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 the binding of an activating protein). When glucose levels are high, there is catabolite repression of operons encoding enzymes that metabolize alternative substrates. Because of low cAMP levels under these conditions, there is an insufficient amount of the CAP-cAMP complex to activate the transcription of these operons.

Figure \(\PageIndex{10}\): 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 alternative substrates. (b) For the lac operon to be expressed, there must be activation by cAMP-CAP as well as the removal of the lac repressor from the operator. Parker, N., et. al. (2019) Microbiology. Openstax

Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the Catabolite activator protein CAP-DNA complex with bound cAMP (2CGP).

Figure \(\PageIndex{11}\): Catabolite activator protein CAP-DNA complex with bound cAMP (2CGP). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...z6mLhBpPmPMsC6

Global Responses of Prokaryotes

In prokaryotes, several higher levels of gene regulation can 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 range of operons to respond in a coordinated manner. They do this through the production of alarmones, which are small intracellular nucleotide derivatives, such as guanosine pentaphosphate (pppGpp), as shown in Figure \(\PageIndex{12}\).

Figure \(\PageIndex{12}\): Structure of Guanosine Pentaphosphate (pppGpp). 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, inhibiting RNA synthesis when amino acid levels are low. This causes translation to decrease, and the amino acids present are therefore conserved. Furthermore, pppGpp upregulates many other genes involved in stress responses, including those for amino acid uptake from the surrounding medium 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 crucial for understanding the infection processes of many pathogens and for developing therapies to counteract them.

Quorum Sensing

Quorum sensing (QS) is an intercellular communication mechanism of bacteria used to coordinate the activities of individual cells at the population level in response to surroundings through the production and perception of diffusible signal molecules such as Acyl Homoserine Lactones or small signaling peptides, as shown in Figure \(\PageIndex{13}\). The signal synthase, signal receptor, and signal molecules are three essential elements of the basic QS circuit machinery. Genes encoding signal-generating proteins are also included among the QS target genes. This forms an autoinductive feedback loop that modulates the generation of signal molecules. Several bacterial behaviors, including the expression of virulence factors, the production of secondary metabolites, biofilm formation, motility, and luminescence, are regulated by quorum sensing (QS). Through complex regulatory networks, bacteria can express corresponding genes in proportion to their population size and behave in a coordinated manner.

Figure \(\PageIndex{13}\): Examples of Quorum Sensing Pathways. Verbeke, F., et.al. (2017) Frontiers in Neuroscience 11:183.

The left panel shows the 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 commonly diffuse and target neighboring cells within the colony to mediate a communal or population response.

The right panel illustrates that quorum-sensing peptides are synthesized by bacterial ribosomes as propeptide proteins and undergo post-translational modifications during excretion via active transport. The quorum-sensing peptides bind to membrane-associated receptors, which then autophosphorylate and activate intracellular response regulators via phosphotransfer. These phosphorylated response regulators induce increased expression of their target genes.

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, as shown in Figure \(\PageIndex{14}\). It begins with conditioning the surface by coating it with macromolecules from the surrounding aqueous environment, thereby enabling the initial reversible adhesion of microorganisms. The next step involves the formation of stronger, irreversible attachments to the surface, followed by the proliferation and aggregation of microorganisms into multicellular and multilayered clusters that actively produce an extracellular matrix. Some cells in mature biofilms continuously detach from the aggregates, providing a continuous source of planktonic bacteria that can subsequently spread and form new microcolonies.

Figure \(\PageIndex{14}\) Schematic drawing of biofilm formation. Rukavina, Z., and Vanic, Z. (2016) Pharmaceutics 8(2):18.

Biofilms are a common cause of chronic, nosocomial (originating in a hospital), and medical device-related infections because they can develop on both vital and necrotic tissue, as well as on the inert surfaces of various implanted materials. Moreover, biofilms are linked with high-level resistance to antimicrobials, frequent treatment failures, and 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. A rough estimate suggests that more than 60% of hospital-associated infections are attributable to biofilms formed on indwelling medical devices, resulting in over one million cases of infected patients annually and exceeding $1 billion in hospitalization costs per year in the USA.

Biofilm infections share several common characteristics: slow development in one or more focal points, delayed clinical manifestation, and persistence for months or years, typically with alternating periods of acute exacerbations and periods of clinical symptom absence. Although they are less aggressive than acute infections, they are more challenging to treat. There is up to a 1000-fold decrease in the susceptibility of biofilms to antimicrobial agents and disinfectants, as well as resistance to the host immune response. Thus, ways to reduce or inhibit biofilm formation are highly sought. The majority of the proposed biofilm-control methods focus 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 an electromagnetic field, ultrasound waves, photodynamic activation or specific drug delivery systems.

Alternate σ Factors

Since the σ subunit of bacterial RNA polymerase confers specificity for which promoters are transcribed, altering the σ factor used is another way for bacteria to quickly and globally change which 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 regulates the expression of the numerous genes required for sporulation in response to sporulation-stimulating signals.

Prokaryotic Attenuation and Riboswitches

Although most gene expression in prokaryotes is regulated at the level of transcription initiation, some mechanisms concurrently control both the completion of transcription and translation. 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 at the 5’ end of an mRNA as it is being transcribed determine whether transcription will complete the synthesis of this mRNA and whether it will be used for translation. Beyond the transcriptional repression mechanism already discussed, attenuation also controls the expression of the trp operon in E. coli as shown in Figure \(\PageIndex{15}\). The trp operon regulatory region contains a leader sequence, trpL, between the operator and the first structural gene, which has four RNA stretches 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, it prevents the formation of the terminator stem-loop, allowing RNA polymerase to transcribe the structural genes.

Figure \(\PageIndex{15}\): Attenuation of Transcription and Translation. Parker, N., et. al. (2019) Microbiology. Openstax

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, which enables RNA polymerase to transcribe the structural genes of the trp operon.

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, as shown in Figure \(\PageIndex{16}\). 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 \(\PageIndex{16}\): Riboswitch Form and function. Riboswitches found within prokaryotic mRNA molecules can bind to small intracellular molecules, stabilize certain RNA structures, and influence either the completion of mRNA synthesis (left) or the protein made from that mRNA (right). Parker, N., et. al. (2019) Microbiology. Openstax

Riboswitches found within prokaryotic mRNA molecules can bind to small intracellular molecules, stabilizing specific RNA structures and influencing either the completion of mRNA synthesis (left) or the protein produced from that mRNA (right).

Figure \(\PageIndex{17}\) shows interactive iCn3D models of a series of bacterial riboswitches. They are described in the legend below.

Guanine-responsive riboswitch bound to metabolite hypoxanthine (4FE5) A. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Nh1Sv7oSSyC6Z6	Divalent cation-sensing regulatory RNA (2QBZ) B. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...VbU9Vf6DUA4VE8
Cyclid-di-GMP RNA riboswitch (3IRW) C. (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...wVntbdScP3VCF8	GlmS ribozyme bound to glucosamine-6-phosphate (2Z75) D. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...w9T4K9ffK1NU27

A: Guanine-responsive riboswitch bound to metabolite hypoxanthine (4FE5) - Hypoxanthine, involved in purine metabolism, is shown bound to RNA representing the 5' untranslated region of the xanthine phosphoribosyltransferase (xbt)/ xanthine-specific purine permease (pbux) genes that lead to transcription termination.

B: The M-box in mycobacterial genes regulating Mg²⁺ transport binds divalent cations. They are transcribed under low Mg²⁺ concentrations. Salt bridges (ion-ion interactions) are shown in cyan, and pi-cation interactions in red dotted lines

C: Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger in bacteria and regulates many cellular processes, including the formation of biofilms. The riboswitch shown here is from Vibrio cholerae. The U1 small nuclear ribonucleoprotein A is shown in cyan. Figure \(\PageIndex{18}\) shows a cartoon of the actual riboswitch in the 5' untranslated region of target genes

Figure \(\PageIndex{18}\)" Consensus secondary structure and sequence conservation of Cyclic di-GMP-I riboswitch

D. This ribozyme is in the 5′ untranslated region of glucosamine-6-phosphate synthase mRNA. The protein enzyme, 2. This protein enzyme catalyzes the conversion of fructose 6-phosphate and glutamine to glucosamine 6-phosphate (GlcN6P) and glutamate. The glmS ribozyme in the 5'-untranslated region cleaves itself on binding GlcN6P. This self-cleavage is inhibited by glucose 6-phosphate (Glc6P). Hence, high levels of the synthase gene product lead to cleavage of its own mRNA. The glmS ribozyme RNA is shown in gray, and its substrate RNA is shown in cyan.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

A central question of molecular biology is how genetically identical cells can produce different phenotypes—whether in different tissues of a multicellular organism or in a single-celled bacterium responding to a changing environment. The answer lies in the precise regulation of gene expression: only specific subsets of genes are transcribed and translated under particular conditions.

In prokaryotes, gene regulation operates primarily at the level of transcription initiation. The operon—a cluster of functionally related structural genes transcribed from a single promoter as a polycistronic mRNA—provides the fundamental organizational unit of bacterial gene regulation. Jacob and Monod's landmark studies on the lac operon (Nobel Prize 1965) established the core principles: repressor proteins bind operator sequences between the promoter and structural genes to physically block RNA polymerase, while small inducer molecules can inactivate repressors, thereby permitting transcription. This framework applies to both repressible and inducible operons, which differ in their regulatory logic. Repressible operons (exemplified by the trp operon) are normally active but shut off when their biosynthetic end product—tryptophan—accumulates and binds the trp repressor, causing a conformational change that enables operator binding and transcription blockage. Inducible operons (exemplified by the lac operon) are normally repressed; allolactose generated from lactose binds the constitutively produced lac repressor, preventing its operator binding and allowing transcription of lacZ (β-galactosidase), lacY (permease), and lacA (galactoside acetyltransferase).

The lac operon is subject to an additional layer of control through catabolite repression. Because glucose is the preferred carbon source, E. coli suppresses the expression of alternative substrate-utilization genes when glucose is available. When glucose levels drop, less ATP is generated; Enzyme IIA becomes phosphorylated, activating adenylyl cyclase to convert ATP to cAMP. Elevated cAMP binds catabolite activator protein (CAP/CRP), and the CAP-cAMP complex binds upstream of the lac promoter to enhance RNA polymerase recruitment. Maximum lac operon transcription thus requires the simultaneous removal of the lac repressor (by allolactose) and activation by CAP-cAMP (signaling glucose depletion)—a logical AND gate for carbon source switching that produces the characteristic diauxic growth curve when both substrates are present.

Beyond individual operon control, bacteria coordinate gene expression at the population and genome-wide levels through several mechanisms. Regulons coordinate multiple operons in response to a single environmental signal. Alarmones such as pppGpp (guanosine pentaphosphate) mediate the stringent response to amino acid starvation, simultaneously inhibiting rRNA and tRNA synthesis while upregulating genes for amino acid biosynthesis and uptake—conserving cellular resources. Alternative σ factors allow bacteria to rapidly reprogram their entire transcriptome: when environmental conditions change, the existing σ factor is degraded and a new one is expressed that recognizes different promoter consensus sequences, redirecting RNA polymerase to an entirely different set of genes. This mechanism controls major developmental transitions such as sporulation in Bacillus and Clostridium.

Quorum sensing enables population-level gene regulation through the secretion and detection of diffusible signaling molecules. In Gram-negative bacteria, acyl homoserine lactones (AHLs) accumulate in proportion to cell density until a threshold concentration activates LuxR-type transcription factors. In Gram-positive bacteria, modified secreted peptides activate membrane-bound histidine kinase receptors that phosphorylate intracellular response regulators. Both systems form autoinduction loops that coordinate population-wide behaviors including biofilm formation, virulence factor production, secondary metabolite synthesis, and motility. Biofilms—structured communities encased in a self-produced extracellular matrix—present a major clinical challenge: they exhibit up to 1000-fold increased resistance to antimicrobials, resist immune clearance, and account for more than 60% of nosocomial infections.

Finally, attenuation and riboswitches couple transcription and translation regulation in ways possible only in prokaryotes, where the two processes are not spatially separated. In trp operon attenuation, ribosome stalling at tryptophan codons in the trpL leader sequence when tryptophan is scarce allows an antiterminator stem-loop to form, permitting RNA polymerase to continue; tryptophan abundance allows ribosome read-through and formation of a transcription terminator stem-loop. Riboswitches are structured RNA elements in the 5′ UTR that bind small intracellular metabolites directly, triggering alternative folding that either terminates transcription or sequesters the ribosome binding site to inhibit translation—as illustrated by guanine-responsive, Mg²⁺-sensing, c-di-GMP-responsive, and GlcN6P-responsive riboswitches. The self-cleaving glmS ribozyme, which destroys its own mRNA upon binding the product of the enzyme it encodes, provides an elegant example of riboswitch-mediated feedback control at the RNA level.

 

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