Narlx Two-Component Signaling System as a Key Mechanism of Bacterial Adaptation

Bacteria are among the oldest and most widespread forms of life on Earth, characterised by a remarkable ability to colonise almost any conceivable environment. Their diversity and adaptability extend to extremely diverse ecosystems - from the deep ocean hydrothermal vents with temperatures above 100°C, to Antarctic glaciers, to hypersaline lakes and environments with extreme pH (Rothschild and Mancinelli 2001; Pikuta et al. 2007; Cowan et al. 2010).

To do this, they have had to specialised, among other things, in a number of mechanisms that allow them to respond to changing conditions. The systems they evolved are broadly classified according to their molecular complexity, including one-component systems (OCS), which primarily respond to internal signals and two-component systems (TCS), which can detect and respond to extracellular signals (Parkinson 1993; Stock et al. 2000; Laub and Goulian 2007).

One- and two-component systems provide a fundamental mechanism for bacteria to track and respond to the slightest changes in the surrounding environment (Mitrophanov and Groisman 2008). Among these TCS are particularly significant due to their widespread presence in bacteria, their role in regulating critical processes such as virulence, biofilm formation, and stress responses, and their potential as targets for antimicrobial therapies. The classical two-component system consists of two components - a histidine kinase (HK) that monitors external stimuli and a response regulator (RR) (Fig. 1). The former composed of a histidine kinase inside the cell and a differentiated sensing domain above the membrane. The second, on the other hand, usually being inside after receiving a signal, affects regulation of gene expression. HK switches between active and inactive states depending on the signal. In the activated state, histidine kinase transfers phosphate from ATP to histidine, its own amino acid, and then to the response regulator aspartate. TCS can respond to a number of different stimuli essential for bacterial processing. They can be activated by nutrients, pH changes, the occurrence of substances that potentially threaten the integrity of the cell, and their own misfolded proteins (Burbulys et al. 1991; Kato et al. 1999; Skerker et al. 2005).

Figure 1.

The general mechanism of action of two-component regulatory systems.

Many bacteria use nitrate and nitrite as electron acceptors in respiration in the absence of oxygen. For this metabolism to take place, the microorganism must first detect the presence of the proper conditions. Several two-component systems are responsible for detecting different forms of nitrogen. These do not occur in all bacteria in the same amount or activity. The focus of this study is to characterise NarXL, a specific two-component system associated with the detection and regulation of nitrate and nitrite metabolism, providing insights into their molecular mechanisms and their role in bacterial adaptation to anaerobic environments (Alvarez et al. 2016; Gao et al. 2019).

The NarXL system consists of the histidine kinase NarX and the response regulator NarL. The first component detects extracellular nitrate/nitrite, autophosphorylates and transfers the phosphate group to NarL, which then modulates gene expression. Using Escherichia coli as an example, their molecular complexity and evolutionary significance can be clearly traced (Cavicchioli et al. 1995). In these bacteria, NarXL systems play a fundamental role in nitrate metabolism, controlling the expression of genes responsible for nitrate and nitrite reduction under anaerobic conditions (Blattner et al. 1997; Constantinidou et al. 2006). The NarL and NarX proteins form a highly precise molecular system that enables the bacteria not only to survive but also to efficiently utilise diverse nitrogen sources under varying environmental conditions (Unden and Schirawski 1997; Stewart 2003). In E. coli, it was discovered that NarL activation is responsible for the activation of 51 operons and the inhibiting 41 of them (Constantinidou et al. 2006).

One of the main targets of the response regulator is the activation of the narKGHJI operon, which encodes a membrane-bound antiporter and nitrate reductase. The operon includes five genes. NarK belongs to the major facilitator superfamily (MFS) of transmembrane transporters and serves as a nitrate/nitrite antiporter. Further genes in the operon encode NarG (catalytic subunit), NarH (iron-sulphur cluster subunit), NarJ (chaperone) and NarI (membrane anchor), thus forming the membrane-bound respiratory nitrate reductase (Nar) (Bertero et al. 2003). This system regulates dissimilatory nitrate reduction, an energy-generating process distinct from assimilatory pathways that focus on nitrogen attachment (Unden and Schirawski 1997) (Fig. 2). Table I presents other operons regulated by NarXL system.

Figure 2.

The general mechanism of action nar nitrate reductase and nitrate/nitrite antiporter NarK.

Although the primary focus of this review is the importance of NarXL in nitrate metabolism and bacterial pathogenicity, however, studies on E. coli and Salmonella enterica have revealed the presence of an additional system, NarQP, which works alongside NarXL to detect and respond to changes in nitrate concentration. Although the two systems overlap functionally, they show differences in interaction specificity and regulatory strategies.

NarX binds to NarL more firmly than NarP, creating a high-affinity complex. The NarQ kinase, on the other hand, interacts with both NarL and NarP, allowing for a dual signal response (Chiang et al. 1997; Stewart 2003). The differences between the systems also relate to the kinetics of signal transduction. NarQ transfers a phosphate group to NarL at a rate ten times faster than NarX, facilitating rapid gene activation. However, after nitrate depletion NarX provides faster and more accurate signal termination, because it is more effective at dephosphorylating NarL. Notably, NarQ responds to both nitrate and nitrite, whereas NarX is selective for nitrate, allowing for differential regulation depending on environmental conditions. They regulate the expression of key operons such as the nitrate reductase operon (narGHJI), the fumarate reductase operon (frdABCD) and the nitrite reductase operon (nrfABCDEFG) (Rabin and Stewart 1993).

Analysis reveals significant differences in the genetic organisation of these systems. NarX genes are more closely related to nitrate reductase genes located in the narGHJI cluster, while the distribution of narQ and narP genes is characterised by greater variability (Schröder et al. 1994). The systems also differ in their DNA binding mechanisms. The phosphorylated NarL binds DNA as a stable dimer, recognising tandem inverted repeats with a sequence of 7-2-7 bp in the promoters of genes such as narGHJI and frdABCD. This structure enables cooperative binding and strong transcriptional activation of pathways crucial for energy acquisition. In contrast to NarL, the NarP protein binds DNA mainly as a monomer or briefly forming a dimer. This property limits its capacity to occupy high-affinity binding sites, narrowing NarP’s regulatory role to low-affinity targets such as the nrfABCDEFG operon encoding the NrfA periplasmic formate‐dependent nitrite reductase, which is responsible for reducing nitrite to ammonium ions involved in nitrite detoxification. Structurally, both NarQ and NarX consist of seven domains, including a periplasmic sensor domain, transmembrane region, HAMP domain, signalling helix, GAF-like domain, DHp, and CA catalytic domain. These components mediate nitrate binding, signal transduction, and response regulation across the cell membrane, spanning more than 200 Å from the extracellular ligand-binding site to the cytoplasmic catalytic core. Interestingly, NarQ contains conserved cysteine residues in its CA domain that may form a disulfide bond responsive to the cellular redox environment, potentially linking nitrate sensing to the oxidative‐stress response (Noriega et al. 2008, 2010; Godfrey et al. 2017; Gushchin et al. 2021).

Nitrate metabolism plays a key role in the regulating functioning and virulence of bacteria. For example, study by Martín-Rodríguez et al. (2020) investigated how nitrate metabolism affects the biofilm formation and pathogenicity of uropathogenic E. coli (UPEC) strains. Mutations were made in genes encoding nitrate reductases (narGHJI, narZYWV, napFDAGHBC), creating single, double and triple mutants. These mutants were then used to study the effects of nitrate reduction on biofilm formation, the expression of the biofilm master regulator CsgD, and the efficiency of bacterial colonization in a rat model of urinary tract infection (UTI). The results showed that narGHJI genes, which encode a key membrane nitrate reductase, were essential for efficient nitrate reduction under experimental conditions. Nitrate availability significantly modulated biofilm structure, particularly through its effects on the biosynthesis of curli fimbriae and cellulose. These processes were tightly regulated by CsgD, the master regulator of biofilm formation. In this study NarL was found to directly or indirectly affect the expression of csgD, leading to changes in biofilm formation. In the absence of functional nitrate reductases or under nitrate-limiting conditions, CsgD expression increased, resulting in enhanced biofilm production. In an in vivo model, mutant strains lacking nitrate-reducing ability were strongly outclassed in competitive infections with the wild-type strain, indicating that nitrate metabolism plays a role as bacterial “fitness factor” in the host environment. The results suggest that nitrate-reducing ability, although not necessary, significantly enhances UPEC adaptability in host tissues (Martín-Rodríguez et al. 2020).

Nitrate metabolism not only supports anaerobic respiration but also acts as a critical environmental signal influencing biofilm regulation and pathogenicity via two-component systems (TCS). In E. coli, the ArcAB TCS senses respiratory stress and modulates biofilm responses to sub-inhibitory antibiotics. Deletion of arcA or arcB leads to constitutively elevated biofilm levels and eliminates further stimulation by antibiotics, while nitrate supplementation suppresses this effect by relieving oxidative stress. This suppression depends on active nitrate respiration, as shown by the inability of narG mutants to respond, linking nitrate metabolism to biofilm regulation through respiratory chain activity and ArcAB signalling (Yaeger et al. 2023).

The study on Salmonella enterica serovar Typhimurium revealed that the narL and fnr genes, which are responsible for anaerobic metabolism, exhibit synergy. These two systems regulate different but overlapping metabolic pathways. Deletion of both these genes prevents the use of nitrate as an electron acceptor, leading to decrease in nitrite production by 99%. Absence of the narL gene alone results in a 73% reduction in biofilm formation under anaerobic conditions, indicating a key role for this regulator in the formation of biofilm structures, which play an important role in bacterial colonization and survival. In terms of motility, the ΔnarL mutant showed a significant reduction in swimming (by 26%) and swarming (by 61%) abilities under anaerobic conditions, which can be attributed to dysregulation of genes related to flagella activity, such as fliA. In vitro analyses in a mouse model revealed that the ΔnarL mutant has a significant decrease in replication capacity inside macrophages and epithelial cells, indicating impaired adaptability and survival in host niches. Additionally, significant reduction in the number of ΔnarL mutant bacteria was observed in organs such as the spleen and liver, indicating a limited capacity for systemic dissemination and effective colonisation. Histopathological analysis revealed less intense inflammatory processes compared to the infection caused by the wild-type strain, highlighting the significant impairment of the ΔnarL mutant’s ability to induce an inflammatory response (Priyadarsini et al. 2024). The natural inflammatory response in the host is produced during Salmonella infection drives the production of nitric oxide (NO), which is rapidly oxidised to nitrate (NO₃^-) via transient intermediates. Pathogens are able to use this inflammatory nitrate pool as an electron acceptor for anaerobic respiration, facilitating survival in hypoxic niches (Mian et al. 2013; Scales et al. 2016; Fang and Vázquez-Torres 2019).

Further expanding on this, recent research has shown that host-derived nitrate acts as a signalling molecule influencing Salmonella lifestyle transitions. Nitrate exposure downregulates the production of a major biofilm component - curli fimbriae - through repression of csgD and reduction of intracellular cyclic-di-GMP levels. However, it simultaneously promote motility via flagellar activation. This shift from a biofilm-associated to a motile phenotype, mediated through the NarXL, facilitates epithelial invasion and systemic dissemination. Inhibition of nitrate production in vivo led to increased csgD expression, confirming nitrate’s role in repressing biofilm formation during infection (Miller et al. 2022).

The NarXL mutants of Burkholderia pseudomallei, an endemic tropical bacterium is known as the etiological agent of melioidosis, show a couple of alterations, including a significant reduction in the expression of key biofilm matrix components and a significant reduction in the expression of genes responsible for the biosynthesis of secondary metabolites. These include non-ribosomally derived peptide synthetase (NRPS), polyketide synthases (PKS), and genes responsible for the biosynthesis of bacteriocins such as bactobolins, maleilactones, and syrbactins. The narX and narL mutants show key differences in their ability to replicate intracellularly. In the macrophage infection model, a significant reduction in the ability to multiply inside host cells altered gene expression patterns compared to the wild-type strain. Additionally, the disruption of immune response evasion mechanisms was observed (Mangalea and Borlee 2022).

In Pseudomonas aeruginosa the nitrate reductase system exhibits complex molecular interactions that determine its anaerobic metabolism. Notably, the narQP genes does not appear to be present in the genome of P. aeruginosa. In this bacterium, the NarL protein has a crucial function in the activation of hemeA, nirQ and dnr genes, which directly determine the metabolic transformation under anaerobic conditions (Vollack et al. 1998; Härtig et al. 1999; Schreiber et al. 2007). Further, Van Alst et al. (2007) analysed the role of the narL and narX genes, as well as the narK1K2GHJI and napEFDABC operons, in nitrate metabolism, motility and biofilm formation in P. aeruginosa. Experiments were conducted on mutants with regulatory (narL, narX) and structural (narGH, napA) genes deleted, evaluating their effects on mobility (swimming, crawling), biofilm formation and virulence in the nematode Caenorhabditis elegans infection model. The study showed that narL plays a fundamental role in regulating the balance between mobility and biofilm formation. Mutants lacking narL produced excessive amounts of rhamnolipids, which increased their crawling ability at the expense of stable biofilm formation. The narX gene was responsible for nitrate sensing; its absence led to impairing both mobility and biofilm formation. The narK1K2GHJI operon, which encodes membrane nitrate reductase, was essential for nitrate reduction and energy delivery during anaerobic circumstances. A narGH-deficient mutant not only lost the capacity to produce biofilm, but also became entirely avirulent in the nematode model, showing its critical involvement in pathogenesis (Van Alst et al. 2007).

Further, presence of assimilatory, dissimilatory nitrate reduction and complete denitrification pathway, likely provides P. aeruginosa with enhanced metabolic flexibility. Having all of this metabolic pathway is presumably advantageous for survival and competitiveness in dynamic or nutrient-limited environments.

Supporting this notion, recent findings suggest that nitrogen assimilation is not only crucial for metabolic homeostasis but also modulates host-pathogen interactions. For instance, P. aeruginosa has been shown to influence C. elegans chemotaxis and pathogenicity through the regulation of nitrogen assimilation, particularly via the production of volatile ammonia, a by-product of this pathway. The nitrogen assimilation mutants impaired both attraction and colonization of C. elegans, underscoring the significance of this pathway in infection dynamics (Marogi et al. 2024).

Moreover, a study by Kuang et al. (2021) demonstrated that inactivation of the nitrite-dependent nitric oxide (NO) biosynthesis pathway—part of the denitrification machinery—conferred increased resistance to the antibiotic cefoperazone-sulbactam in P. aeruginosa. This was associated with lower levels of NO due to impaired NADH-driven electron flow, linking metabolic shifts in nitrogen pathways to overlapping mechanisms of antibiotic resistance (Kuang et al. 2021).

A schematic comparison (Fig. 3) highlights the metabolic distinctions between E. coli, which possesses only a dissimilatory nitrate reduction pathway, and P. aeruginosa, which integrates assimilatory, dissimilatory, and complete denitrification routes—emphasising its greater ecological and pathogenic plasticity.

Figure 3.

Comparative nitrogen metabolism pathways in E. coli and P. aeruginosa.

This schematic illustrates the assimilation and dissimilation of nitrogen pathways in E. coli and P. aeruginosa. The rectangles colour-coded according to the bacteria indicate the enzymes involved in the processes. Orange represents those present in E. coli, while green indicates enzymes found in P. aeruginosa. Those shared by both organisms are shown as split-coloured boxes.

The diagram is structured around three major functional pathways: assimilatory nitrate reduction (purple), dissimilatory nitrate reduction (pink), and denitrification (blue). Substrates and products are shown as blue circles, and directional arrows denote the enzymatic flow from extracellular nitrate or nitrite towards terminal products, such as ammonia (NH₃) or dinitrogen gas (N₂).This schematic was reconstructed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps as a primary reference (Kanehisa et al. 2023), with organism-specific nitrogen metabolism data curated for E. coli and P. aeruginosa.

Studies on BCG (Bacillus Calmette-Guérin) strain of Mycobacterium bovis, which is used as a TB vaccine, shed light on the processes of metabolic adaptation in anaerobic conditions. Deleting the narG gene drastically reduces the bacterium’s capacity to thrive in important target organs. In mouse studies, a mutant lacking the narG gene was shown to exhibit significantly reduced colonization of the lungs, liver and kidneys. Of particular interest, the effect of lacking the nitrate reductase enzyme is highly selective - minimal changes in bacterial adaptive potential were observed in the spleen tissue (Fritz et al. 2002). Interestingly, while the BCG genome encodes the narXL two-component regulatory system involved in nitrate sensing and response, its components are dispersed across the genome, suggesting potential regulatory decoupling or modularity. Moreover, the genome lacks the narQP system altogether (Gomes et al. 2011).

Unlike many other bacteria, Staphylococcus aureus lacks the binary systems NarXL and NarQP. However, these bacteria make up for this with a network of alternative regulatory systems that can contribute to the regulation of nitrate reductase activity. One component of this network is the two-component system SrrAB. It responds to both nitrosative stress and hypoxia by modulating genes involved in electron transport, anaerobic metabolism. Although SrrAB does not directly replace NarXL or NarQP, it can influence pathways involved in nitrate respiration by indirectly detecting electron transport chain activity and redox status (Kinkel et al. 2013). Recent detailed studies indicate that S. aureus has as many as 17 different two-component systems, many of which share similar regulatory pathways, making it more complicated to control genes related to virulence and metabolism, including those for nitrate and nitrite processing (Ahator et al. 2024). This creates a more complex regulatory network than in organisms with dedicated nitrate systems and highlights the adaptive plasticity of the S. aureus regulatory architecture.

Studies on S. aureus, particularly in the context of methicillin-resistant strains (MRSA), have revealed an important role for the nitrate reductase NarGHJI in virulence regulation. The NarGHJI operon affects the expression of virulence genes such as RNAIII, agrBDCA, hla, psmα and psmβ, and its inactivation leads to their down-regulation and a reduction in haemolytic activity. Observations on mouse models and Galleria mellonella confirmed that narG-deficient strains show significantly reduced virulence. Expression of narGHJI is the highest in the early and mid-log phases, suggesting a major role for these phases in the regulation of agr, a global regulator of virulence genes. RNA-seq analysis showed that inactivation of narGHJI results in an increase in the expression of 63 genes and a decrease in 89, including vraX, which encodes a protein involved in pathogenesis. NarGHJI can modulate virulence in an agr-dependent and independent manner, indicating a complex regulatory mechanism that requires further study. The results highlight the novel role of NarGHJI in the control of molecular determinants of virulence and provide a potential basis for developing strategies to combat S. aureus infections (Li et al. 2023).

The NarXL system is a key signalling mechanism for the survival and establishment of bacterial virulence in the host. Its role in bacterial virulence, particularly in the context of biofilm formation and the potential for urinary tract infection, makes it an extremely promising therapeutic target, especially for uropathogenic bacteria.

In the context of a growing global antibiotic threat, two-component systems (TCS) such as NarXL offer an alternative approach to combating bacterial infections. The World Health Organization (WHO) indicates that antibiotic resistance is one of the ten most serious global public health threats, with up to 10 million deaths predicted annually by 2050 and economic losses estimated at around $100 trillion (Jonas et al. 2017; Murray et al. 2022).

Studies to date point to a remarkable variety of bacterial resistance mechanisms that can be activated by two-component systems. These include but are not limited to modification of the cell surface, decreased drug uptake or increased drug removal, activation of antibiotic-degrading enzymes, and biofilm production (Beier and Gross 2006; Alvarez and Georgellis 2023). Further research on NarXL requires a deeper understanding of its mechanisms of action and the development of highly specific inhibitors. It will also be crucial to conduct thorough analyses to avoid potential side effects associated with possible interaction with host cells.

Identifying small-molecule inhibitors of the NarXL system would allow precise modulation of pathogenic bacterial behaviour, such as biofilm formation and expression of virulence factors. This type of approach could not only be used as an adjuvant therapy in combination with traditional antibiotics but could also minimise side effects of the treatment, such as destruction of the gut microbiota. In addition, the high structural similarity of signalling circuit elements in different bacterial species offers the possibility of developing broad-spectrum medicine (Barrett et al. 1998; Worthington et al. 2013).

A potential solution may be to search databases of chemical molecules using bioinformatics methods and molecular modelling. In this study authors used the ZINC22 database of 37 billion commercially available compounds (including 4.5 billion in 3D docking-ready form) to identify potential inhibitors of the NarL protein in Mycobacterium tuberculosis. After virtual screening, the researchers selected ZINC63191404 a nitrobenzeneaminopiperidine derivative. It was shown that it can potentially bind to the NarL phosphorylation site more stable than acetylphosphate, natural phosphate donor. In silico molecular dynamics analysis suggested that the selected compound forms stable hydrogen bonds with the protein and inhibits its activity at nanomolar concentrations. Despite encouraging theoretical results, the compound efficacy has not yet been confirmed experimentally. The authors emphasized the need for further in vitro and in vivo studies to assess its potential as an antimicrobial agent (Shivakumar et al. 2014; Tingle et al. 2023).

Two-component systems (TCS) are fundamental adaptive mechanisms in bacteria and are emerging as promising therapeutic targets due to their key roles in bacterial survival, virulence and antibiotic resistance. One of the process regulated by TCS is nitrate/nitrite metabolism, where NarXL proteins play important role.

Research on the NarXL system has significantly advanced our understanding of its molecular mechanisms. While additional interdisciplinary studies are needed to fully understand its functions, current evidence indicates that the NarXL system plays an essential role in regulating nitrate and nitrite metabolism, as well as mediating adaptive processes including biofilm formation and virulence factor expression. Therefore, these characteristics, combined with their specificity for bacterial cells, render the NarXL system a promising target for innovative therapeutic strategies, especially against uropathogenic bacteria.