28.15: Signaling in Microorganisms

Search Fundamentals of Biochemistry

Learning Goals (ChatGPT o3-mini)

1. Differentiate Bacterial Signaling Systems:

   • Explain the three major types of bacterial signal transduction systems: one‐component systems, two‐component systems, and extracytoplasmic function (ECF) sigma factors.
   • Describe how each system transduces environmental signals across the bacterial inner membrane.

2. Understand One-Component Systems:

   • Identify the structural and functional characteristics of one-component systems (e.g., ToxR-like receptors).
   • Explain how the CadC protein functions as a pH sensor in E. coli, including its interaction with lysine and feedback inhibition by cadaverine.

3. Analyze Two-Component Systems:

   • Describe the mechanism of two-component signaling, including the roles of the membrane-integrated histidine kinase (HK) and its corresponding response regulator (RR).
   • Discuss the energetic and structural differences between phosphorylation events in histidine kinases compared to serine/threonine/tyrosine kinases.
   • Explain how multi-component phospho-relay systems work, using examples such as the NarQ-NarP system in nitrate/nitrite sensing.

4. Evaluate Second Messenger Roles in Bacteria:

   • Define the role of bacterial second messengers like cyclic di-AMP (c-di-AMP) in modulating the activity of signaling proteins (e.g., binding to the Universal Stress Protein domain in KdpD).
   • Understand how the integration of second messenger signals contributes to bacterial adaptation under stress.

5. Explore Structural Aspects of Bacterial Receptors:

   • Analyze how domain architecture (e.g., PAS, HAMP, GAF-like domains) in bacterial receptors facilitates signal detection and transduction.
   • Explain how conformational changes—such as piston-like movements and helical rotations in transmembrane regions—translate extracellular stimuli into intracellular responses.

6. Relate Signal Transduction to Physiological Outcomes:

   • Discuss how bacterial signaling systems regulate essential functions such as nutrient acquisition, stress responses, and chemotaxis/phototaxis.
   • Illustrate with examples how these systems contribute to both commensal survival and pathogenicity.

7. Integrate Knowledge of Modular Protein Structure:

   • Identify how the modularity of bacterial signaling proteins (e.g., sensor, transmembrane, and output domains) allows for versatility and specificity in response to environmental cues.
   • Predict how alterations in these domains (through mutation or ligand binding) might affect the overall signaling output.

These learning goals are designed to ensure that students not only understand the molecular details of bacterial signal transduction systems but also appreciate how these systems integrate structural, biochemical, and physiological processes to regulate bacterial behavior in diverse environments.

This section's main organization derives from Bacterial transmembrane signaling systems and their engineering for biosensing. Jung et al :25 April 2018https://doi.org/10.1098/rsob.180023. It is licensed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/). Significant content from the source has been integrated into the section.

Introduction

Bacteria constantly interact with their surroundings. They identify and actively acquire nutrient resources, sense and respond to environmental stresses, and exchange information with other cells. At the same time, commensals and pathogens adapt their lifestyles for survival in their hosts. The cytoplasmic (inner) membrane of bacterial cells separates the cytoplasm from the outer world. Therefore, all information from the outside must be transferred across this interface, which contains various sensors that carry out this function.

Bacteria use three major types of signaling systems: membrane-integrated one-component systems (for example, ToxR-like receptors), two-component systems consisting of a receptor histidine kinase and a response regulator, and extracytoplasmic function (ECF) sigma factors. These are shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Schematic presentation of the major types of transmembrane signaling systems in bacteria. One-component signaling systems, consisting of a sensor and DNA-binding domain (yellow), two-component systems with a membrane-integrated histidine kinase (HK) and a response regulator (RR) (green), and extracytoplasmic function (ECF) sigma factors (σ) that will be released from the anti-sigma factor (anti-σ) upon stimulus perception (blue).

The simplest is the one-component signaling family ToxR (named after the main virulence regulator in Vibrio cholerae). They have a periplasmic sensor domain, a single transmembrane helix, and an intracellular winged helix-turn-helix DNA-binding domain. 

In two-component systems, the membrane-integrated histidine kinase generally acts as a sensor for various stimuli and is also responsible for information transfer across the membrane. This process usually results in the autophosphorylation of the protein, and the phosphoryl group is subsequently transferred to a specific soluble response regulator, which usually acts as a transcription factor (see Figure \(\PageIndex{1}\)). The number of histidine kinase/response regulator systems varies widely between bacterial species, ranging from 30 in Escherichia coli and 36 in Bacillus subtilis to 132 in Myxococcus xanthus. In chemotactic systems, a soluble histidine kinase perceives the signal(s) conveyed by membrane-integrated chemoreceptors and transduces this information via phosphorylation/protein–proteins interaction to the flagellar motor.

The ECF sigma factors are small regulatory proteins that bind to RNA polymerase and stimulate the transcription of specific genes. Many bacteria, particularly those with more complex genomes, contain multiple ECF sigma factors, and these regulators often outnumber all other types of sigma factors. Little is known about the roles or the regulatory mechanisms employed by most ECF sigma factors. Most of them are co-expressed with one or more negative regulators. Often, these regulators include a transmembrane protein that functions as an anti-sigma factor, which binds and inhibits the cognate sigma factor.

Let's look at three examples.

One-component system: pH sensor CadC

A series of Cad proteins regulates the pH in E. Coli. CadA is a cytoplasmic decarboxylase, which converts lysine to cadaverine, while CadB is a membrane-integrated lysine/cadaverine antiporter. CadC acts as a homodimeric one-component regulator. Together, their activities lead to an increase in both internal and external pH, which favors the survival of E. coli under moderate acid stress and helps to maintain pH homeostasis. Their activities are shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): The regulation of CadC, a one-component system representative.

CadC is the regulator of the cadBA operon encoding the lysine decarboxylase CadA and the lysine/cadaverine antiporter CadB. Under non-inducing conditions, the lysine-specific transporter LysP inhibits CadC. When cells are exposed to low pH in the presence of lysine, the interaction between LysP and CadC is weakened, rendering CadC susceptible to protonation and transcriptional activation. The end-product of decarboxylation, cadaverine, binds to CadC, inactivating this receptor.

CadC is activated by two stimuli, low pH (less than 6.8) and external lysine (greater than 0.5 mM), which are perceived by different mechanisms. The periplasmic domain of CadC directly senses a decrease in pH. It has two distinct subdomains: the N-terminal subdomain comprises a mixture of β-sheets and α-helices, and the C-terminal subdomain comprises an 11-α-helix bundle. A patch of acidic amino acids (D198, D200, E461, E468, D471) detects changes in the external pH through protonation changes, altering their charges and noncovalent interactions between the subdomains/monomers. This leads to dimer formation of the periplasmic domain, triggering receptor activation.

Figure \(\PageIndex{3}\) shows an interactive iCn3D model of a Transcriptional activator CadC One Component Model - AlphaFold Model (P23890)

Figure \(\PageIndex{3}\): Transcriptional activator CadC One Component Model - AlphaFold Model (P23890) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...UfZh5LVeoaToj6

The yellow-gold represents the N-terminal CadC DNA binding domain. The green (hydrophobic) is the transmembrane helix, and the gray is the outside periplasmic domain containing the acidic side chains (D198, D200, E461, E468, D471). The N-terminal Met is magenta spacefill, and the C-terminal Ser is cyan spacefill. Again, the long intrinsically disordered region between the DNA binding domain and the transmembrane domain would have a more defined structure in the membrane-bound form and probably form additional interactions with other molecules. It presumably leads to a conformation change in the DNA-binding domain, leading to its interaction with the target DNA.

CadC senses external lysine only in interaction with the lysine-specific permease LysP. In addition, the products of lysine decarboxylation, CO₂ and cadaverine, act as feedback inhibitors on CadC. Cadaverine binds to the periplasmic domain of CadC, thereby switching off cadBA transcription.

Two Component System

Two-component histidine kinase systems have (in general) two main components

• A HK sensor protein binds a ligand in a receptor binding domain, activating the transfer of a gamma-phosphate from ATP to a His by the kinase domain. This component is often called a transmitter. The conserved His is located in the H box.
• a separate response regulator (or effector) protein containing a reactive Asp, which receives the phosphate from p-Histidine. The conserved Asp (D) is located in the D box. This activates the response regulator protein. This component is often called the receiver. It may also transfer it to another His in a phospho-relay system.

Histidine kinase/response regulator systems are the most common in bacterial signaling across the membrane. In contrast to the myriad of serine/threonine (S/T) and less abundant tyrosine (Y) kinases that dominate signaling in mammals, the histidine kinase predominates in bacterial signaling.

Before we present more details on the two-component system, let's review protein kinases in general and see what is different about histidine kinases. ATP is a donor of a gamma-phosphate in both S/T/Y and H kinases. However, their products are very different energetically. pS, pT, and pY of the O-phosphoproteome are all phosphoesters, which are not high energy compared to their hydrolysis products. (Remember, there is no such thing as a "high energy" bond.)

In contrast, pHis, a member of the N-phosphoproteome (along with pLys and pArg), is not a phosphoester but more analogous to the mixed anhydride of a carboxylic acid, as in the case of phosphorylated aspartic acid. In pHis and pAsp, an electronegative N (in pHis) and O (in pAsp) are bridging two atoms, each connected to another atom by double bonds. This type of structure, which allows for bridging resonance between the center N (in pHis) and O (in pAsp) is also high energy compared to its hydrolysis products. Hence, the phosphorylation of His by ATP is not as energetically favored as that of Ser, Thr, or Tyr since it produces another high-energy molecule (with respect to its hydrolysis products).

Since the pHis is also considered high energy compared to its hydrolysis products, it can act as a phosphate donor to another receiving group. That could be water in a simple hydrolysis reaction or, if sheltered from water in an active site of an enzyme or receptor, to a carboxylate receiver like Asp to form another high-energy mixed anhydride, which is isoenergetic with the pHis. This is the process that occurs in the two-component His kinase signaling pathways in bacteria. Figure \(\PageIndex{4}\) compares Ser, Thr, Tyr, and His kinase reaction and their products.

Figure \(\PageIndex{4}\): A comparison of Ser, Thr, Tyr, and His kinases

The more common mammalian S/T/Y kinases are shown at the top, and the histidine kinase at the bottom. Note that phosphorylation of His can occur at either nitrogen to produce either τ- or π-pHis. In the two-component system, instead of water being the receiver of the phosphate from the pHis (hydrolysis), the receiver is an Asp or another His in the same His Kinase receptor or in another receiver protein (shown in gray and its phosphorylated blue form in Figure \(\PageIndex{9}\). You can imagine the phosphate on the original pHis jumping to a receiver, which then donates it to another receiver in the signaling process in a relay process.

Proteins like the receptor His-kinase proteins in two-component systems have multiple domains with different functions. It's really helpful to present domain structure diagrams to help understand the protein's structure and activities. At the same time, the domain structures determined by various bioinformatic programs are different. Nevertheless, it is useful to see multiple representations of domain structure, especially if they are shown in conjunction with actual structures.

The first component of the two-component signaling system is the receptor His-kinase, which can be viewed as a stimulus-activated kinase (much like receptor tyrosine kinases - RTKs). The second component is the response regulator protein, typically a second protein. Each of these, in turn, has its own domain structure. For example, the periplasmic sensing domain regulates the kinase domain of the receptor His-kinase (component one). The phosphate from the p-His in the first component is transferred to an Asp in the second component. The domain structures and phospho-transfer are illustrated in Figure \(\PageIndex{5\) below.

Figure \(\PageIndex{5}\): Domain structures and phosphotransfer mechanisms in two-component systems. Silva et al. Int. J. Mol. Sci. 2021, 22(16), 9034; https://doi.org/10.3390/ijms22169034. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Panel (A) shows a prototypical two-component pathway. The transmembrane sensor HK (component one) and a cytoplasmic response regulator (RR) protein (component two) are shown. (Note: the actual protein is a dimer in the membrane.) The transmembrane segments are labeled TM₁ and TM₂. N, G₁, F, and G₂ are conserved sequence motifs in the ATP-binding domain. HK catalyzes ATP-dependent autophosphorylation of a specific conserved His residue within the HK dimerization domain. The phosphoryl group (P) is then transferred to a specific aspartate residue (D) at the conserved RR domain (component two). Phosphorylation of this domain usually triggers an associated (or downstream) effector domain, which ultimately produces a specific cellular response.

Panel(B) shows a multi-component phospho-relay system that often involves a variant of HK with an additional internal C-terminal RR domain. In these complex systems, at least two His–Asp phosphoryl transfer events occur, typically involving a His-containing phosphotransfer protein (HPT) operating as a His-phosphorylated intermediate.

In most prokaryotic systems, the RRD directly carries the response, which functions as a transcription factor. Two-component systems also exist in some eukaryotes. They often interact with other downstream signaling pathways, such as the MAPK system. However, in eukaryotic systems, the TCS are placed at the start of the pathways and establish an interface with more conventional signaling strategies such as mitogen-activated protein (MAP) kinase and cyclic nucleotide cascades

The domain structure shown in Figure \(\PageIndex{10}\) doesn't show the actual orientation of the proteins in a membrane system. Orientation is important since the sensing domain of the HK receptor must be in the environment of the stimuli. Stimuli can be encountered in the periplasmic (equivalent to extracellular) region, the transmembrane region, and the cytoplasm. Variants of the HisK receptors exist that recognize stimuli in each of these locations, as shown in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): Hannah Schramke. https://edoc.ub.uni-muenchen.de/2103...mke_Hannah.pdf

Most histidine kinases sense extracellular signals (left-hand structure). All have their cytoplasmic transmitter domains, which contain the pHis. As mentioned previously, the histidine kinases are dimeric, so when activated, they phosphorylate a His on the other monomer (transphosphorylation). In addition to the H box with the reactive histidine, they also have N, G1, F, and G2 boxes. The H box is also involved in dimerization. The transmitter domain can be further divided into two parts: the H-box is involved in dimerization and obviously in phosphotransfer. The figure also shows the CA domain (HK-type ATPase catalytic or HATPase_c), also known as the catalytic and ATP-binding (CA) domain (Figure 1.3-3)

The histidine kinase senses a variety of stimuli in its sensory domain. The stimuli can be generally grouped into organic (e.g. dicarboxylates, citrate, etc), ions (e.g., Mg²⁺, H⁺, K⁺), gaseous ligands (e.g., O₂, N₂), and physical changes (osmolarity/turgor, light, and temperature). Stimuli are "sensed" by a variety of different characterized folds. Some common sensing domains are PAS (Per-ARNT-Sim), CHASE (cyclase/histidine kinase-associated sensing extracellular), four-helix bundle (4HB), and NIT (nitrate and nitrite-sensing) classes. We will focus on one particular His kinase system, histidine kinase KdpD.

Histidine kinase KdpD and the regulation of K⁺ ion concentration

It is often difficult to identify the primary stimulus for a receptor, as exemplified by the histidine kinase KdpD, which, together with the response regulator KdpE, controls the expression of a high-affinity K⁺-uptake system in many bacteria. K⁺ is the most abundant cation in all living cells, especially in bacteria, where it is crucial for regulating cell turgor and intracellular pH and activating several enzymes. To ensure a sufficient supply of K⁺, most bacteria have more than one K⁺-uptake system. For example, E. coli has at least three such systems, the constitutively expressed systems Trk and Kup, and the inducible high-affinity K⁺-uptake system KdpFABC. The genes kdpF, kdpA, kdpB, and kdpC form an operon that codes for four inner membrane proteins. The Kdp operon is induced when E. coli is grown under K+ limitation, lacks the major K+ transporter Trk, or has an increased need for K⁺ under hyperosmotic stress. Under all these conditions, the membrane-integrated histidine kinase KdpD autophosphorylates and transfers the phosphoryl group to the cytoplasmic transcriptional (response) regulator KdpE, resulting in the induction of the kdp operon, as shown in Figure \(\PageIndex{7}\).

Amazingly, KdpD has not only kinase activity but also phosphatase activity towards phosphorylated KdpE, which switches the signaling cascade off. It is a bifunctional enzyme/receptor. A single substitution (T677A) in the C-terminal domain results in no phosphatase activity.

Figure \(\PageIndex{7}\): Schematic of the KdpD regulation system.

Hence, KdpD can be considered a bifunctional receptor acting as a kinase and a phosphatase to regulate gene expression. The bifunctional receptor histidine kinase KdpD acts as an autokinase (including phosphotransferase) and phosphatase for the response regulator KdpE. Phosphorylated KdpE activates the expression of the genes encoding the high-affinity K⁺ transporter KdpFABC. KdpD autokinase activity depends on the external K⁺ concentration, and the internal K⁺ concentration influences the phosphatase activity. K⁺ ions don't move through a channel in KdpD but through the KdpFABC.

The cartoon in Figure \(\PageIndex{7}\), as with all cartoons, can be misleading with respect to scale. Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the actual membrane domain of E. coli histidine kinase receptor KdpD (2KSF)

Figure \(\PageIndex{8}\): Membrane domain of E. coli histidine kinase receptor KdpD (2KSF)Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...yMgkv2fxn9RKo8

Four helices space the membrane. Hence, both the N-terminal and C-terminal domains are actually in the cytoplasm, not one in the periplasmic space and one in the cytoplasm as you would infer from Figure \(\PageIndex{7}\). The actual periplasmic (outside the cell) domain consists of only about six amino acids. Hence, it is most clearly represented by the middle model in Figure \(\PageIndex{6}\). Mutations of key residues in the periplasmic loop region (P466A, T469A,, L470A and V472A) drastically affect K⁺ recognition. Actually, how it "senses" periplasmic K⁺ ions is not clear.

Domain representation

We present three different domain diagrams for KdpD in Figure \(\PageIndex{9}\), not to confuse readers, but to show the utility of multiple representations they are likely to encounter in reading the literature.

Moscoso et al. Journal of Bacteriology. 198 (2016) http://dx.doi.org/10.1128 /JB.00480-15. Creative Commons Attribution 3.0 Unported license

B. Pfam

C. Dutta et al. JBC. 296, 100771 (2021). DOI:https://doi.org/10.1016/j.jbc.2021.100771. Creative Commons Attribution (CC BY 4.0)

Figure \(\PageIndex{9}\): Multiple representations of the domain structure of the histidine kinase KdpD

The four boxes btw 360 and 500 in panel A are four transmembrane helices, which would not be evident in a simple cartoon as in Figure \(\PageIndex{7}\). In panel B, the domains are shown as follows:

• Green: K+ channel His kinase sensor domain 21-230;
• Red: 4 transmembrane helices 402-508; the helices are lumped together in the red domain representation;
• Blue: GAF domain 527-644;
• Yellow and Purple combo: HK domain;
  • Yellow: HisKinase A phosphoacceptor domain 663-730 which contains the His acceptor, in effect the substrate of the kinase domain;
  • Purple: His Kinase/HSP 90 is like ATPase 773-883.

The domain structure in panel C is the most detailed and has the receiver's domain structure (response regulator). Again, the periplasmic domain, which senses K⁺, consists of only a few amino acids, 424-427 and 467-474. Neither representations A nor B show that the protein's major N-terminal and C-terminal halves are in the cytoplasm. Panel C shows more information about the domains, their function, and their orientation in the intracellular environment. There is also a sensor for intracellular K⁺ ions, which is depicted in Figure \(\PageIndex{7}\).

A central question is how histidine kinase KdpD responds to changes in K⁺ concentrations. K⁺ regulates both the kinase and phosphatase activities.

• When periplasmic (extracellular) K⁺ is > 5 mM (high), the ion appears to bind to the small extracellular loops (see Figure \(\PageIndex{8}\)), which inhibits the autokinase activity. Under the same conditions, the intracellular C-terminal tail senses K⁺ and activates the phosphatase activity, which cleaves its pHis. These combined effects inhibit high-affinity K⁺ transport.
• When periplasmic K⁺ becomes low, kinase activity is activated and the protein is autophosphorylated, ultimately leading to the activation of the gene for the high-affinity K⁺ transporter KdpFABC. As long as intracellular K⁺ levels are high, the phosphatase is active. When intracellular K⁺ levels drop sufficiently, the phosphatase becomes inhibited, further stimulating the transcription of of KdpFABC.

Hence, the histidine kinase KdpD system is regulated by both periplasmic and cytoplasmic K⁺ ions.

Yet another signal regulates the KdpD His Kinase receptor two-component signal. What has been conspicuously absent from this discussion about signaling in bacteria is the involvement of second messengers like cAMP (which activates Protein Kinase A and some membrane proteins). There does appear to be one major second messenger in bacteria - cytoplasmic di-AMP (c-di-AMP), whose structure is shown in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): The bacterial second messenger cytoplasmic di-AMP (c-di-AMP)

It binds in the N-terminal region of KdpD His Kinase receptor protein "sensor" domain region to a specific domain called the Universal stress protein (USP) domain, as shown in panels A and C of Figure \(\PageIndex{9}\). Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the Staphylococcus aureus universal stress protein (USP) domain of KdpD histidine kinase in complex with second messenger cyclic diadenosine phosphate (c-di-AMP) (7JI4)

Figure \(\PageIndex{11}\): Staphylococcus aureus universal stress protein (USP) domain of KdpD histidine kinase in complex with second messenger c-di-AMP (7JI4). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ij5DqgazLFvV5A

"Dual sensing thus emerges as a highly optimized regulation strategy. The key advantage of this strategy is that it confers on cells the ability to directly sense changes in both the supply of and demand for the limiting resource. It is, in fact, analogous to strategies that are widely used in control engineering, e.g., modern heating systems work with both exterior and interior thermometers to ensure constant room temperature."

Escherichia coli nitrate/nitrite sensor kinase NarQ

Let's examine another TCS protein, the Escherichia coli nitrate/nitrite sensor kinase NarQ, to see how the binding of a ligand to the periplasmic domain might transmit a signal so far into the cell through the plasma membrane. We won't discuss the His Kinases that lack transmembrane regions (about 1/4). The sensor domain, hence, is mostly in the periplasm, followed by the transmembrane domain (see Figure \(\PageIndex{8}\)), followed often by a cytoplasmic HAMP domain, with a four-helical parallel coiled-coil. The HAMP domain transmits the signal to the protein's downstream signaling domains like Dhp.

Nitrate/nitrite is sensed by two different two-component systems, sensing systems NarX-NarL and NarQ-NarP, which regulate anaerobic respiration. NarQ phosphorylates two different proteins, NarL and NarP, in the presence of nitrate or nitrite and dephosphorylates them without ligands. Both NarQ (and NarX) have seven domains: a four-helical periplasmic sensor domain, TM bundle, HAMP domain, so-called signaling helix (S-helix), GAF-like domain, dimerization and histidine phosphotransfer domain, and, finally, catalytic kinase domain, as shown in Figure \(\PageIndex{12}\).

Figure \(\PageIndex{12}\): Architecture of NarQ and structure of the R50S mutant. Elements belonging to the second protomer in the dimer are marked with primes. Gushchin et al. Int. J. Mol. Sci. 2020, 21, 3110; doi:10.3390/ijms21093110. Creative Commons Attribution. (CC BY) license. (http://creativecommons.org/licenses/by/4.0/).

Pane (a) shows the architecture of NarQ. Note that the functional protein is homodimeric. Approximate domain boundaries, according to InterPro [17], are TM1, residues 14–34; sensor, 39–146; TM2, 147–167; HAMP, 172–227; S-helix, 228–246; GAF-like, 247–360; DHp, 361–425; CA, 424–560.

Panel (b) shows the overall structure of the sensor-TM-HAMP fragment of the R50S mutant (which allowed crystallization). The position of Ser50 is highlighted with spheres. The backbone structure is identical to that of the WT protein.

Panel (c) shows the structure of the ligand (nitrate) -binding site in the WT protein.

Panel (d) shows the ligand-binding pocket structure in the R50S mutant. Asp133 is reoriented towards Ser50. 2F_o−F_c electron density maps are contoured at the level of 1.2 × r.m.s. Putative water molecules are shown as red spheres. Gushchin et al. Int. J. Mol. Sci. 2020, 21, 3110; doi:10.3390/ijms21093110. Creative Commons Attribution. (CC BY) license. (http://creativecommons.org/licenses/by/4.0/).

Although the sequence identity between NarQ and NarX is ~28%, their ligand binding sites—membrane-proximal parts of the sensor domain’s helices H1 called P boxes—are very well conserved: 14 out of 15 amino acids (residues 42–56 in NarQ) are identical, and the differing ones, Ile 45 in NarQ and Lys49 in NarX, are responsible for the differentiation between nitrate and nitrite.

It appears that the ligand-induced conformational change in the ligand-binding site is helical rotation, which results in diagonal scissoring of the sensor domain helices, leading to the change in the secondary structure of the sensor-TM linker and, eventually, piston-like shifts of the transmembrane α-helices.

Nitrate causes changes in the transmembrane region when the apo (nitrate-free) and holo (nitrate-bound) state structures of NarQ are compared. On nitrate binding, the induced conformation changes in NarQ have been described as a "combination of changes in the lateral arrangement of the TM helices and piston-like shifts of the helices in the direction perpendicular to the membrane plane." This results in either symmetric or asymmetric changes and scissoring of the transmembrane helices (based on two different crystal structures of the holo-form. A "piston-like" movement of the helices is observed on both holo-forms.

Results show that the binding of ligand to NarQ causes a piston-like displacement of the TM helices, which is accompanied by extensive symmetric or asymmetric rearrangements and scissoring of the TM helices. The rearrangements are different in the two presented holo-state structures, but the piston-like displacement is perfectly conserved. Thus, the latter appears to be a more robust mechanism of TM signal transduction.

Figure \(\PageIndex{13}\) shows an interactive iCn3D model of a fragment of nitrate/nitrite sensor histidine kinase NarQ (mutant R50K) in the symmetric holo state (5IJI)

Figure \(\PageIndex{13}\): Fragment of the nitrate/nitrite sensor histidine kinase NarQ (mutant R50K) in the symmetric holo state (5IJI) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...gsRXjmKBbqbis8

A homodimer (cyan and magenta) is shown with the N-terminus and C-terminus on the cytoplasmic side. Each monomer passes through the membrane with an alpha-helical domain twice. Nitrate is shown in spacefill in the extracellular domain outside of the membrane's outer leaflet (red spheres).

Figure \(\PageIndex{14}\) shows the conformational transition going from the symmetric apo state (5JEQ) magenta to the symmetric holo state (IJI) (cyan) with bound nitrate (not shown).

Figure \(\PageIndex{14}\): Conformational transition in the nitrate/nitrite sensor histidine kinase fragment going from the symmetric apo state (5JEQ) magenta to the symmetric holo state (IJI) (cyan)

Conformational changes in the HAMP domain amplify and convert the piston-like conformational changes in the transmembrane domain. In the apo form, note the splaying out of the helices at the bottom (cytoplasmic end).

Phototaxic Photoreceptors

We have discussed two-component systems that have a His-Kinase receptor transmitter protein. There are two other major types of bacterial receptors, chemoreceptors (involved in chemotaxis) and photoreceptors (involved in phototaxis). These often have similar modular domain structures. Like the His Kinase receptor, chemotaxis receptors are dimers with extracellular domains that bind the chemotactic signal. The photoreceptors appear to be active as trimers of dimers.

The basic dimeric structure contains the microbial light sensor rhodopsin, which contains the chromophore opsin, and its transducer Htr. In the halobacteria N. pharaonis (archaeal, not a bacterial cell), the proteins are sensory rhodopsin II (NpSRII) with its transducer (NpHtrII), which mediates negative phototaxis in halobacteria N. pharaonis.

Microbial rhodopsins are phototransducing proteins with a conjugated chromophore retinal, covalently attached to the protein opsin through a Schiff base (imine) linkage. The holoprotein (opsin with the attached retinal) is called rhodopsin. Retinal is derived from beta-carotene. Figure \(\PageIndex{15}\) shows the structures of animal and microbial retinals.

Figure \(\PageIndex{15}\): Structures of animal and microbial retinals

When light of the correct wavelength is absorbed, an electron in a pi molecular orbital in retinal is promoted to a pi antibonding molecular orbital, breaking a 2-electron pi bond in the structure at a certain site in the isoprenoid chain, allowing rotation around the now single bond. After the electrons return to the ground state, the final result is photoisomerization of the trans 13-14 and cis 11-12 bonds in microbial and animal retinal, respectively, to their respective cis 13-14 and trans 11-12 configuration. This conformational change in the bound retinal induces a conformational change in the protein opsin, leading to signaling.

Figure \(\PageIndex{16}\) shows an interactive iCn3D model of one monomeric of bacteria rhodopsin (1C3W) containing retinal attached through a Schiff base to Lys 216.

Figure \(\PageIndex{16}\): Bacterial rhodopsin (1C3W). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...9HzvfdFvb9kcc6

The covalently attached retinal is shown in gray spacefill. The protein opsin is a membrane protein that spans the membrane with seven helices. Hence, it is very similar to a GPCR.

In the phototaxic receptor, when light is absorbed by rhodopsin (NpSRII), the resulting conformational change in the protein causes conformational changes in the transducer protein NpHtrII associated with it, leading to signaling through a two-component signal system. (The chemotaxis response to chemical signals occurs through a similar but ligand-induced process.) Figure \(\PageIndex{17}\) shows the structure of the Archaeal photoreceptor complex.

Figure \(\PageIndex{16}\): The dimer of two NpHtrII proteins is flanked by two NpSRII proteins.

A-G, TM1, and TM2 are the transmembrane helices. The cytoplasmic part of NpHtrII consists of two HAMP domains (HAMP1 and HAMP2) connected by an α-helical linker (Inter-HAMP) and the kinase control module. Primes denote symmetry mates of the complex. Ishchenko, A., Round, E., Borshchevskiy, V. et al. New Insights on Signal Propagation by Sensory Rhodopsin II/Transducer Complex. Sci Rep 7, 41811 (2017). https://doi.org/10.1038/srep41811. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/

Figure \(\PageIndex{17}\) shows a more detailed representation of both the transmitter and receiver in the Archaeal two-component phototransduction system and how it leads to phototaxis.

Figure \(\PageIndex{17}\): Signal transduction pathway in case of the two-component phototaxis system of Natronomonas pharaonis⁵ and domain architecture of membrane chemo- and photoreceptors of TCS. Ryzhykau, Y.L., Orekhov, P.S., Rulev, M.I. et al. Molecular model of a sensor of the two-component signaling system. Sci Rep 11, 10774 (2021). https://doi.org/10.1038/s41598-021-89613-6. Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/.

Pane (A) shows that light-activated sensory rhodopsin II (NpSRII) induces conformational and/or dynamical changes in the transducer (NpHtrII), which are converted by two HAMP domains and conveyed along the 200 Å long transducer to the tip region. Activated by the transducer histidine kinase CheA (bound to the adapter protein CheW) undergoes auto-phosphorylation and further transfers the phosphate group to the response regulators CheY or CheB. CheY affects the rotational bias of the flagellar motor, while the methylesterase CheB and methyltransferase CheR control the adaptation mechanism.

Panel (B) shows cartoon representations of the chemoreceptor dimer (Tar and Tsr in complex with kinases) from E. coli and of the photosensor dimer of the complex of the sensory rhodopsin II with its cognate transducer NpHtrII and kinases from N. pharaonis.

Summary

Bacterial cells continuously interact with their environment through sophisticated signaling systems that traverse their cytoplasmic membranes. These signaling mechanisms enable bacteria to sense nutrients, detect environmental stresses, and coordinate cellular responses—critical for survival whether as free-living organisms or as commensals and pathogens within hosts.

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Major Signaling Systems

1. One-Component Systems

   • Structure & Function:
     These systems combine sensor and DNA-binding functions in a single protein. An example is the ToxR family, typified by CadC.
   • CadC Example:
     In E. coli, CadC regulates the cadBA operon, which encodes enzymes and transporters essential for pH homeostasis. CadC’s periplasmic domain directly senses low external pH (below 6.8) through acidic amino acids that undergo protonation. It also requires external lysine—interacting with LysP—to trigger transcriptional activation, while the reaction product, cadaverine, provides negative feedback by binding to CadC.

2. Two-Component Systems (TCS)

   • General Mechanism:
     These systems consist of two proteins—a membrane-bound histidine kinase (HK) and a soluble response regulator (RR). The HK senses external signals and autophosphorylates on a conserved histidine residue. The phosphoryl group is then transferred to an aspartate residue on the RR, typically activating it as a transcription factor.
   • Energy Considerations:
     Unlike mammalian serine/threonine or tyrosine kinases, histidine phosphorylation forms a high-energy mixed anhydride that facilitates phosphotransfer reactions, underpinning the robustness of these signaling cascades.
   • Key Examples:
     • KdpD System:
       Regulates high-affinity K⁺ uptake via the KdpFABC transporter in response to both extracellular and intracellular K⁺ levels. KdpD is a bifunctional enzyme—exhibiting kinase activity when external K⁺ is low and phosphatase activity when internal K⁺ is high. Its regulation is further refined by a second messenger, c-di-AMP, which binds to its universal stress protein (USP) domain.
     • NarQ System:
       Functions in nitrate/nitrite sensing to modulate anaerobic respiration. The sensor domain of NarQ, located predominantly in the periplasm, binds nitrate/nitrite, triggering a piston-like shift of the transmembrane helices. This conformational change is transmitted through a HAMP domain to affect downstream signaling, ultimately modulating the phosphorylation status of the response regulators NarL and NarP.

3. Extracytoplasmic Function (ECF) Sigma Factors

   • Overview:
     ECF sigma factors are small regulatory proteins that bind RNA polymerase to activate transcription of specific genes. Although many bacteria, especially those with larger genomes, have multiple ECF sigma factors, their precise roles and regulatory mechanisms remain less understood. They are typically regulated by co-expressed anti-sigma factors that inhibit their activity until appropriate stimuli are detected.

Additional Signal Transduction Strategies

• Phototaxis and Chemotaxis:
  Beyond chemical signals, bacteria also use light as an environmental cue.
  • Photoreceptors:
    In phototactic bacteria, such as the halobacterium Natronomonas pharaonis, sensory rhodopsin (NpSRII) binds retinal. Upon light absorption, retinal undergoes photoisomerization, inducing a conformational change in NpSRII. This signal is then relayed via its associated transducer (NpHtrII) through a two-component system, ultimately directing movement toward or away from light.
  • Chemoreceptors:
    Similarly, chemotaxis involves receptor dimers that detect chemical gradients, initiating cascades that adjust flagellar rotation to guide bacterial movement.

Integration and Regulation

Bacterial signal transduction is marked by its modularity and precision. The domain architectures of these receptor proteins—often depicted through multiple representations—reveal how different structural regions (e.g., sensor domains, transmembrane segments, and catalytic domains) are spatially organized to:

• Sense External and Internal Stimuli:
  Through specialized extracellular domains and intracellular regulatory modules.
• Facilitate Rapid Response:
  Via efficient phosphorylation relay systems, ensuring timely adaptation to environmental changes.
• Implement Feedback Control:
  By integrating signals from second messengers (like c-di-AMP) and through bifunctional proteins (such as KdpD) that modulate both activation and deactivation processes.

Overall, the chapter provides a comprehensive look at how bacteria translate external cues into coordinated cellular responses—a critical aspect of microbial physiology that underscores the adaptability and survival strategies of these organisms.

This summary emphasizes key structural features, mechanisms, and examples that illustrate the diversity and intricacy of bacterial transmembrane signaling systems, providing a robust framework for junior and senior biochemistry majors to understand both the common themes and specialized adaptations in bacterial signal transduction.