Why do LuxR-type proteins require accessory proteins?

LuxR-type transcription factors frequently rely on PPIs to regulate receptor availability, stability, and transcriptional output. Although these receptors are classically described as AHL-responsive regulators, many LuxR-type proteins possess LBD that are unstable, aggregation-prone, or only partially functional in isolation. Accessory proteins can buffer these biophysical constraints by stabilizing specific receptor conformations, modulating dimerization, or promoting productive DNA engagement. In other cases, PPIs act in the opposite direction, restraining receptor activity to prevent premature or inappropriate activation of QS programs. Beyond receptor stabilization, PPIs provide a mechanism to control the timing, specificity, and amplitude of LuxR-dependent transcription. Negative regulators can delay activation at low-cell density (LCD), suppress self-sensing, accelerate QS shut-off following signal dilution or dispersal, or serve as nodes for integrating regulatory inputs from other systems, allowing cues beyond AHL concentration alone to permit QS activation. Global regulators can couple LuxR activity to broader physiological processes, including proteolysis and transcriptional machinery availability. Together, these interactions ensure that QS outputs reflect not only signal accumulation but also cellular state and environmental context.

PPI-based regulation is a common strategy in bacterial transcriptional regulation. Throughout bacterial regulatory networks, transcription factors are frequently controlled by non-DNA binding partner proteins, including anti-sigma factors [35], NusA/NusG-mediated control of transcriptional elongation [36], AraC-family co-regulators [37], and phosphorylation-dependent two-component systems [38]. LuxR-associated accessory proteins appear to operate under similar principles, enabling QS circuits to integrate diverse inputs without altering core receptor architecture. A growing body of evidence demonstrates that LuxR-type receptors can be modulated through direct PPIs that either enhance or inhibit receptor activity, alter promoter specificity, or redirect transcriptional outcomes. Notably, these accessory proteins generally do not bind DNA themselves, distinguishing them from canonical transcriptional activators such as sigma factors or CRP (cAMP receptor protein), which act primarily at the DNA-RNA polymerase (RNAP) interface [39]. Instead, LuxR accessory proteins act directly on the receptor, shaping its functional state before or during promoter engagement, or by influencing receptor stability and turnover.

Why some LuxR-type receptors require post-translational regulation while others appear largely ligand sufficient remains an open question. Current evidence suggests that accessory proteins compensate for intrinsic receptor instability, suppress noise and self-sensing, enforce appropriate activation thresholds, or couple QS outputs to environmental and physiological cues that are not encoded by AHL concentration alone. The systems reviewed here illustrate how LuxR-type receptor activity can be fine-tuned by diverse PPI modes of regulation rather than AHL-binding alone.

TraM establishes a paradigm for PPI-mediated LuxR inhibition

The TraR-TraM system of Agrobacterium tumefaciens provides a foundational example of LuxR negative regulation via direct PPI. In A. tumefaciens, QS regulates the conjugative transfer of the Ti plasmid, which carries virulence genes required for plant infection and is a metabolically costly process requiring tight temporal control [34,44,45]. This system is governed by a canonical LuxIR-type system: TraI synthesizes the AHL 3-oxo-octanoyl homoserine lactone (3OC8-HSL), which binds to and activates the LuxR-type transcription factor TraR and stabilizes it [1,34]. Ligand-bound TraR dimerizes and activates transcription of genes required for plasmid replication and transfer.

Initial observations suggested that TraM functions as a negative regulator of TraR, as overexpression of traM suppressed the activation of TraR-dependent genes [46]. To determine whether this inhibition was due to direct interaction, yeast two-hybrid (Y2H) assays demonstrated that TraM and TraR physically interact in vivo [46]. Subsequent in vitro binding assays and mutational analysis confirmed that the interaction involves the C-terminal DNA binding domain (DBD) of TraR [47,48]. Structural studies provided further insight into this mechanism. A crystal structure of the TraM-TraR complex revealed that TraM forms a heterotetrametric complex with TraR, binding at a surface distal to the DBD (Fig 4; top panel) [49]. This interaction induces a conformational change that allosterically prevents DNA binding and enables stepwise dissociation of TraR from DNA [49]. Functionally, this interaction prevents TraR DNA binding and, therefore, its ability to function as a transcriptional activator. As a result, TraM binding to TraR effectively blocks transcription of target genes, even in the presence of AHL.

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Fig 4. Structural basis of anti-activation in LuxR-type quorum sensing receptors.

(A) Structure of TraM-TraR complex from homologous Rhizobium sp. strain NGR234 proteins (PDB ID: 2Q0O [49]). Shown sequentially are: TraR homodimer alone (red), TraR homodimer (blue), and the full 2:2 TraM-TraR complex. TraM functions as an anti-activator by binding to TraR, inducing an allosteric conformational change in the protein and preventing DNA binding. (B) Structure of the QslA-LasR ligand-binding domain (LBD) complex from Pseudomonas aeruginosa (PDB ID: 4NG2 [61]). The complex consists of 2:1 stoichiometry, with a QslA homodimer (blue and gray) interacting with a single monomer of the LasR LBD (cyan), obstructing dimer formation and preventing LasR activation. (C) Structure of Aqs1-LasR complex from P. aeruginosa (PDB ID: 6V7W [30]). The complex consists of an Aqs1 dimer which binds to the DNA-binding domain of a LasR monomer preventing DNA binding. These co-crystal structures illustrate distinct anti-activation mechanisms: disruption of DNA binding (TraM-TraR and Aqs1-LasR), and disruption of receptor dimerization (QslA-LasR). All structure images were generated using ChimeraX.

https://doi.org/10.1371/journal.ppat.1014091.g004

Later work showed that the TraM mechanism is much more complex than initially appreciated. In the A. tumefaciens strain A6, a second homologous protein, TraM2 was discovered [50]. Although encoded at a distinct genomic locus, TraM2 also interacts with TraR and contributes to the repression of Ti plasmid conjugation, suggesting a redundant or fine-tuning role in QS regulation. Additionally, a truncated TraR-like protein encoded by A. tumefaciens octopine-type strains, TrlR, which lacks the C-terminal DNA binding domain, was found to bind to and form inactive heterodimers with TraR, further inhibiting its activity [48,51]. These findings highlight a sophisticated network of anti-activation that operates at multiple levels: direct sequestration (TraM and TraM2) and dominant-negative inhibition (TrlR), to ensure tight control over the energetically expensive process of plasmid transfer. This system set a precedent for how LuxR-type receptors can be negatively regulated by PPI, a theme that occurs in diverse QS systems across Gram-negative bacteria.

Negative regulators of LuxR-type receptors in Pseudomonas aeruginosa

QS in P. aeruginosa is subject to extensive post-translational regulation, with multiple proteins acting to restrain the LuxR-type receptors LasR and RhlR and prevent premature activation of virulence-associated genes. These negative regulators operate through distinct and, in some cases, partially overlapping post-translational strategies to collectively suppress self-sensing at LCD through direct and indirect modulation of receptor activity.

Negative regulation of CviR by VioS – an emerging candidate

Repression of bacterial QS is most often achieved indirectly, via transcriptional regulators that silence QS synthase or receptor genes [63]. Well-characterized examples include the DNA-binding repressor RsaL [64] in P. aeruginosa and the Gac two-component system in various Gram-negative species [65,66]. By comparison, direct inhibition of a LuxR-type receptors by PPI, to our knowledge, has been described in relatively few systems. One such example is found in Chromobacterium species, where the protein VioS negatively regulates the LuxR-type receptor CviR [31,67]. In Chromobacterium subtsugae (formerly C. violaceum ATCC 31532), CviR activates violacein production and other virulence-associated genes in response to its cognate AHL [68]. VioS was identified through follow-up analysis of vitR, a TetR-family transcriptional repressor identified in a transposon mutagenesis screen for altered siderophore production [31]. Transcriptome analysis of a ∆vitR mutant revealed vioS as one of the most highly upregulated genes at high cell density (HCD), placing VioS downstream of VitR in a multilayered regulatory cascade. Genetic and heterologous reporter assays indicated that VioS suppressed CviR-dependent transcription post-translationally. Overexpression of vioS attenuates CviR-dependent phenotypes, including violacein and protease production, without altering cviR transcription or AHL levels [31]. In a heterologous Escherichia coli reporter system, co-expression of VioS reduced activation of a vioA promoter-lacZ fusion by CviR more than sixfold, even in the presence of N-hexanoyl-L-homoserine lactone (C6-HSL), demonstrating that VioS is sufficient to repress CviR-mediated activation at the vioA promoter [67]. Importantly, this repressive effect was specific to the CviR system, as VioS did not repress CepR-dependent promoter activity in a Burkholderia reporter background [67]. Y2H assays further supported a direct physical interaction between VioS and CviR, consistent with a model in which VioS inhibits CviR activity through PPI [31]. However, as with several LuxR negative regulators discussed above, the precise biochemical mechanism remains unclear and warrants further investigation. Whether VioS interferes with CviR dimerization, DNA binding, or downstream engagement with transcriptional machinery has not been established. Together, these findings establish VioS as an emerging candidate of a negative regulator that directly targets CviR through direct PPI, even in the presence of AHL. Thus, VioS extends the functional spectrum of LuxR negative regulators beyond delayed activation, highlighting the versatility of post-translational control in QS networks.

PqsE establishes a paradigm as a context-dependent regulator of LuxR-type receptors

The Rhl QS system in P. aeruginosa consists of the AHL synthase RhlI, which produces N-butanoyl-L-homoserine lactone (C4-HSL), and the cognate receptor RhlR, a LuxR-type transcription factor that activates target genes in response to C4-HSL accumulation [69,70]. RhlR controls the expression of genes encoding a wide array of virulence factors, including rhamnolipids, hydrogen cyanide, and PYO, as well as functions related to biofilm formation and motility [59,70–75]. While RhlR was initially viewed as a canonical AHL-responsive transcriptional activator, it is now evident that its activity is shaped by PPIs that modulate RhlR function in a promoter- and context-dependent manner. Central among these is PqsE, a protein encoded within the pqs operon that fine-tunes RhlR-dependent transcription through both positive and negative effects depending on cellular conditions, promoter architecture, and co-factor availability. Rather than acting as a simple activator, PqsE functions as a dual regulator that modulates the amplitude, timing, and specificity of RhlR output.

PqsE was initially characterized as a dispensable thioesterase encoded within the pqs operon, involved in quinolone biosynthesis, yet its contribution to QS regulation is mechanistically separable from its enzymatic role [28,76–78]. Deletion of pqsE in laboratory strains grown planktonically, resulted in major defects in RhlR-dependent phenotypes, including loss of PYO production, and reduced elastase and hydrogen cyanide synthesis, despite intact C4-HSL and quinolone signaling [79–81]. These phenotypes closely mirror those of rhlR mutants and extend to defects in motility, biofilm formation, and in vivo pathogenesis [71,78,80]. These observations led to the long-standing hypothesis that PqsE functions as a critical co-regulator to RhlR. Supporting this hypothesis, PqsE enhances RhlR-dependent transcription in heterologous E. coli reporter systems lacking other P. aeruginosa-specific QS components, and can do so independently of PqsR or PQS production [79]. Together, these observations established PqsE as a critical regulator of RhlR.

Direct biochemical and genetic evidence subsequently demonstrated that PqsE modulates RhlR through a physical PPI. Affinity-purification experiments showed that RhlR co-purifies with PqsE in vitro [28], and site-directed mutagenesis subsequently identified a cluster of surface-exposed residues on PqsE critical for binding RhlR. Disruption of this interface abolished PqsE-dependent enhancement of RhlR-DNA binding and transcriptional output, whereas catalytic inactivation of PqsE (PqsE D73A) does not impair RhlR interaction, confirming the regulatory role of PqsE is distinct from its enzymatic activity [28,29]. Notably, a buried mutation within the PqsE active site (PqsE E182W) abolishes RhlR-binding despite being distal to the RhlR interaction interface, suggesting an allosteric coupling between the enzymatic core of PqsE and the RhlR interaction site [28]. While the physiological implications of this allostery remain unclear, it raises the intriguing possibility that conformational states of PqsE, modulated by its active site, could influence its ability to engage RhlR. In support of this regulatory relationship, structural studies showed that PqsE and RhlR form a functional tetrameric complex composed of homodimers of each protein, with PqsE potentially stabilizing a conformation of RhlR that promotes DNA binding at virulence-associated promoters [82,83].

Genome-wide analyses underscore the context-dependent nature of this interaction. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that PqsE and C4-HSL co-regulate RhlR-DNA binding as well as its ability to activate transcription at 40 genomic loci [84]. These findings demonstrate that PqsE is essential not only for enhancing the occupancy of RhlR at target promoters, but also for driving transcriptional activation of a subset of RhlR-regulated genes. Therefore, RhlR target sites may fall into several categories, some of which require both PqsE and C4-HSL, others depend on only one, and a subset appear independent of both co-factors. These findings indicate that PqsE does not uniformly enhance RhlR activity across the regulon, but instead modulates RhlR function in a promoter-specific manner. This combinatorial regulation suggests that ligand-binding and PqsE interaction cooperatively shape RhlR conformation and transcriptional output.

Physiological context further influences the relative contributions of PqsE and AHL signaling to RhlR-dependent gene expression. During planktonic growth at 37 °C, both PqsE and RhlI-synthesized C4-HSL are required for activation of the phz operons and PYO production, consistent with a model in which ligand binding and PqsE cooperatively stabilize a transcriptionally active RhlR complex [84]. In contrast, under surface-associated growth in colony biofilms, rhlI mutants retain substantial phz transcript accumulation despite the absence of canonical AHL synthesis [80], suggesting that PqsE-dependent regulation of RhlR can partially compensate for reduced AHL availability, or that alternative regulatory inputs converge on RhlR at these promoters. Temperature further influences this balance; RhlR-dependent transcription is elevated at lower temperatures even in the absence of pqsE, as observed using chromosomal phzA reporters [85], while RhlR protein levels increase at higher temperatures through post-transcriptional thermoregulation of the rhlAB-rhlR operon [86]. Together, these findings indicate that the balance between ligand- and PqsE-mediated control of RhlR is highly context-dependent and shaped by growth phase and environmental conditions.

Consistent with this view, recent studies using well-defined PqsE variants with altered RhlR-binding affinities revealed that PqsE can exert both positive and negative effects on gene expression depending on the target promoter [87]. These findings suggest that RhlR may adopt multiple active conformations, and PqsE stabilizes one such conformation, potentially the only form capable of activating certain promoters, but suboptimal or even inhibitory at others. This repressive function appears to be biologically relevant; elevated PqsE levels suppress expression of a subset of RhlR-regulated genes, including vqsR and clpP2, demonstrating that PqsE fine-tunes the RhlR regulon, rather than acting as a general amplifier of expression [88]. The regulatory complexity of the PqsE-RhlR interaction is further underscored by reciprocal feedback loops and post-translational regulation. RhlR represses pqsE expression through the pqsA promoter, with repression enhanced when the PqsE-RhlR complex is intact [79,89]. This creates a negative feedback loop: increased RhlR activity suppresses pqsE expression, thereby limiting further PqsE-mediated enhancement of RhlR [90,91]. Conversely, when RhlR or C4-HSL levels decline, pqsE expression increases, potentially reinforcing RhlR-dependent transcription at select PqsE-dependent promoters [87]. In support of this model, overexpression of pqsE leads to increased RhlR protein abundance without corresponding changes to rhlR transcripts or mRNA translation, suggesting a post-translational mechanism [92]. Separately, PqsE has been proposed to shield RhlR from proteolysis (discussed further below), suggesting a stabilizing role that may help preserve receptor abundance during QS activation [93]. This stabilizing function requires PqsE homodimerization; monomeric variants fail to bind RhlR or enhance its activity, demonstrating that PqsE dimerization is essential for complex assembly and RhlR modulation [29,93]. Given the negative feedback between PqsE and RhlR, it is likely that PqsE dimerization itself is under homeostatic regulation, serving as a point of QS-dependent control. This requirement for dimerization may serve as a regulatory checkpoint, ensuring that RhlR is stabilized and fully active only when PqsE accumulates and dimerizes, thereby restricting RhlR-dependent gene expression of virulence factors to conditions when QS thresholds have been robustly met.

Evolutionary analyses showed that PqsE is highly conserved across P. aeruginosa isolates, with strict amino acid identity at residues essential for PqsE homodimerization and RhlR-binding [93]. Importantly, while there are a limited number of species that encode a pqsE homolog, the PqsE-RhlR interaction has been observed in closely related species [92,93]. However, recent work examining PqsE from diverse P. aeruginosa isolates revealed that, despite strong sequence conservation, the contribution of PqsE to QS-regulated phenotypes varies substantially between strains. Trottier et al. showed that deletion of pqsE produces strain-dependent effects on virulence outputs, indicating that PqsE function is modulated by genetic background and regulatory context rather than being universally interchangeable across strains [94]. These findings underscore that PqsE activity cannot be inferred from sequence conservation alone and further support a model in which PqsE acts as a context-dependent modulator of RhlR-mediated transcription. In contrast, orthologs from Burkholderia species lack the structural features required for homodimerization or LuxR receptor interaction, supporting the model that this PPI evolved specifically to modulate QS in P. aeruginosa [93]. Importantly, many virulence genes in P. aeruginosa are co-regulated by both PqsE and RhlR, which makes both components indispensable for full pathogenesis. Disruption of the PqsE-RhlR interface abolishes PYO production and impairs colonization of the murine lung [71]. Accordingly, inhibitors targeting either RhlR or PqsE downregulate biofilm formation and virulence factor expression, highlighting this PPI as a promising target for anti-virulence therapies [95–99]. Together, these findings establish PqsE as a paradigm for context-dependent modulation of LuxR-type receptor activity through PPI.

BlsA-mediated context-dependent regulation of AbaR in A. baumannii

A parallel example of context-dependent LuxR modulation is found in A. baumannii, an opportunistic pathogen with a QS system composed of the AHL synthase AbaI, and the LuxR‑type receptor AbaR [100–103]. While AbaR was initially considered a canonical AHL-responsive LuxR-type receptor, subsequent work revealed an additional regulatory layer mediated by the blue light-sensing protein, BlsA. BlsA expression is highest at lower temperatures in the dark; under these conditions, BlsA interacts with multiple protein partners, including AbaR [27,104,105]. The BlsA-AbaR interaction is proposed to induce expression of the AHL synthase abaI and the subsequent production of AHL. By contrast, under blue light, BlsA does not interact with AbaR and instead induces expression of aidA, encoding a quorum-quenching lactonase, resulting in reduced QS output [27]. While these studies collectively support a model in which BlsA acts as a context-dependent modulator whose expression and interactions are tuned by environmental conditions, the precise mechanism remains unknown. Future work will be critical to determine how BlsA modulates AbaR activity at the molecular level, whether by stabilizing protein conformation, facilitating dimerization, or altering ligand responsiveness, and to establish whether BlsA is required for full AbaR function in vivo. Clarifying this mechanism will not only advance our understanding of QS regulation in A. baumannii but may also uncover new targets for anti-virulence therapeutics should the interaction increase virulence.

Interestingly, while AbaR shares modest sequence homology with RhlR, the accessory proteins BlsA and PqsE are structurally and evolutionarily unrelated (Fig 5) [93]. Despite this, both proteins modulate LuxR-type receptor activity through direct interaction, highlighting a broader evolutionary trend in which proteins with enzymatic functions appear to have evolved separate, non-enzymatic roles to regulate LuxR-type receptor activity. These findings highlight the remarkable versatility of such proteins in expanding the regulatory capacity of QS in response to environmental cues.

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Fig 5. Structural comparison of LuxR-type context-dependent regulators.

(A) Cartoon representation of a PqsE monomer (purple; PDB ID: 7KGW [28]) rotated 90° on its axis and colored by rainbow from the N-terminus to the C-terminus. Highlighted are conserved C-terminal alpha helices, α5–α9, that are unique among this class of enzymes. (B) Cartoon representation of BlsA (blue; PDB ID: 6W6Z [105]) in the ground state, rotated by 90° on its axis and colored by rainbow from the N-terminus to the C-terminus. Highlighted is the secondary structure of BlsA, which is notably different compared to PqsE. All structure images were generated using ChimeraX.

https://doi.org/10.1371/journal.ppat.1014091.g005

Proteolytic regulation of LuxR-type receptors

AAA+ proteases are ATP-dependent machines that control protein homeostasis in bacteria by unfolding and degrading targeted substrates. Members of this family, including ClpP, Lon, FtsH, and HslUV, play essential roles in degrading misfolded proteins, resolving toxic aggregates, and regulating key cellular processes through selective proteolysis [113,114]. Beyond general protein quality control, many AAA+ proteases have evolved to act as post-translational regulators of gene expression, by selectively degrading transcription factors in response to environmental or metabolic cues [113,114]. In diverse bacterial systems, transcription factor turnover by AAA+ proteases enables rapid reprogramming of gene expression. In E. coli, for example, Lon protease plays a key role in regulating the oxidative stress-response. Under stress conditions, the transcription factor SoxR is activated and induces expression of SoxS, which then activates a set of genes that help the cell detoxify reactive oxygen species [115,116]. Once the stress has passed, Lon rapidly degrades SoxS to shut down the response due to decreased soxS transcription, ensuring that stress-response genes are not expressed unnecessarily. This tightly controlled degradation underscores how AAA+ proteases can shape transcriptional programs by degrading unstable regulatory proteins. In Caulobacter crescentus, Lon controls the abundance of the DNA replication initiator DnaA [117], and the global transcription factor CtrA, coordinating cell cycle progression with environmental conditions [118]. These and other examples highlight the broad utility of AAA+ proteases in shaping transcriptional networks through the targeted degradation of regulatory proteins.

LuxR-type QS receptors may be particularly well-suited to this form of regulation. These proteins are widely understood to require their cognate AHL ligands not only for transcriptional activation but also for proper folding and protein stability [11,119]. In the absence of ligand, LuxR-type receptors often misfold, fail to dimerize, or adopt nonfunctional conformations, rendering them susceptible to proteolytic degradation [119]. This ligand-dependent folding behavior is thought to serve as a critical physiological checkpoint by limiting receptor accumulation at LCD, when AHL concentrations are insufficient to support QS activation. In doing so, bacteria avoid premature activation of QS-regulated genes. However, whether this inherent instability is actively exploited by proteolytic systems such as Lon or ClpP to shape QS output remains poorly understood. Exploring the post-translational control of LuxR-type receptors represents a promising direction for uncovering new regulatory layers in QS. The clearest example of this principle comes from the LuxR-type receptor TraR in A. tumefaciens. Apo-TraR, in the absence of its ligand 3OC8-HSL, is rapidly degraded by Lon and ClpP proteases, with a half-life of 2–3 min, and cannot fold into its functional dimeric form [119]. Only TraR synthesized in the presence of 3OC8-HSL adopts a folded, dimeric, and protease-resistant conformation. Follow-up work by Costa et al. expanded this model by identifying intrinsic degradation signals within the C-terminal DBD of TraR, including a protease recognition motif near residues 184–187 [120]. Mutation of these residues increased TraR half-life, while fusion of this domain to GFP reduced fluorescence, suggesting that these C-terminal residues function as a degron to ensure protein turnover. Moreover, this study revealed that the anti-activator TraM acts as a proteolytic adaptor, forming 2:2 complexes with TraR that prevent DNA binding and direct TraR for degradation. Importantly, evidence for TraM-dependent TraR turnover derives primarily from cell-based expression assays in which TraM levels were modulated by IPTG induction. Increasing TraM expression resulted in decreased TraR abundance, as assessed by immunoblotting; however, these experiments did not include quantitative loading controls, and altered TraR levels could also reflect indirect effects of TraM overexpression on TraR synthesis or cellular resource allocation. Notably, TraM itself is not degraded in this process and may act catalytically to promote turnover of multiple TraR molecules [120]. Therefore, while the data strongly support a model in which TraM antagonizes TraR activity and promotes its degradation, whether TraM directly stimulates proteolysis or indirectly destabilizes TraR remains to be fully resolved. Together, these findings establish TraM as a potent negative regulator of TraR and highlight regulated proteolysis as an important, though still incompletely defined, layer of LuxR-type receptor control. Together, these studies illustrate how proteolysis and ligand-dependent folding intersect to tightly regulate LuxR-type receptor abundance, and how PPIs (e.g., with TraM) modulate both activity and stability. These findings highlight proteolysis as a key regulatory layer in QS, particularly in resetting QS systems when signal levels decline. This may be a broadly conserved mechanism among LuxR-type homologs with unstable apo-forms. This “fold-to-function” model has become a conceptual framework for understanding other LuxR-type regulators.

In P. aeruginosa, early work by Takaya et al. investigated whether the ATP-dependent Lon protease plays a role in regulating LuxR-type receptors [121]. They observed that Δlon mutants accumulated elevated levels of RhlR and overproduced QS-regulated virulence factors such as PYO. However, pulse-chase labeling indicated that RhlR had a similar half-life in both wild-type and Δlon backgrounds. Furthermore, increased rhlR mRNA levels in the lon mutant pointed to indirect effects [121]. These findings led to the conclusion that Lon does not degrade RhlR directly, but instead modulates QS indirectly through degradation of the AHL synthase LasI and effects on upstream regulators like RhlI. Indeed, subsequent studies demonstrated that AHL synthases such as LasI [121] and RhlI [122–124] are direct Lon and ClpXP substrates, revealing that proteolysis plays a broader role in QS by modulating signal production and upstream regulators.

Recent findings have added new complexity by identifying a condition under which RhlR becomes a direct Lon substrate: in the absence of the accessory protein PqsE [93]. In PA14, genetic backgrounds lacking pqsE or the ability of PqsE to dimerize and interact with RhlR, showed significantly reduced RhlR protein levels. This reduction was rescued in ΔpqsEΔlon double mutants, indicating that RhlR is targeted by Lon in the absence of pqsE [93]. Importantly, the partial rescue (to WT rather than Δlon levels) suggests that additional proteases may also contribute to RhlR turnover. Notably, these experiments were performed in PA14, whereas earlier studies, including those by Takaya et al., used PAO1 [93,121]. This distinction is important, as regulatory differences between PAO1 and PA14 are well documented and may influence protease-substrate interactions [62]. These findings support a model in which PqsE stabilizes RhlR by preventing its recognition by Lon and possibly other proteases, likely through concentration-dependent interactions. However, this model remains to be fully validated, and future work will be essential to define the complete set of proteases involved, their substrate preferences, and how receptor conformation or accessory protein-binding modulates proteolytic susceptibility.

Despite these advances, many key questions remain unanswered. What structural features of LuxR-type receptors mark them for degradation? Which additional proteases, beyond Lon and ClpXP, target QS components in P. aeruginosa under various physiological conditions? How do dynamic changes in ligand occupancy or accessory protein-binding influence susceptibility to proteolysis? Future work combining genetic and biochemical studies will be essential to delineate the full proteolytic network that governs LuxR-type receptor stability.