Global transcriptomics and targeted metabolite analysis
reveal the involvement of the AcrAB efflux pump in
physiological functions by exporting signaling molecules
in Photorhabdus laumondii

Linda Hadchity, Anne Lanois-Nouri, Adrien Chouchou, David Roche, Jessica Houard, Noémie Claveyroles, Alexandra Dauvé, Jacques Imbert, Maxime Gualtieri, Alain Givaudan, et al.

► To cite this version:

Linda Hadchity, Anne Lanois-Nouri, Adrien Chouchou, David Roche, Jessica Houard, et al. Global transcriptomics and targeted metabolite analysis reveal the involvement of the AcrAB efflux pump in physiological functions by exporting signaling molecules in Photorhabdus laumondii. Microbiology Spectrum, 2025, 13 (10), pp.e0110625. 10.1128/spectrum.01106-25. hal-05248666

HAL Id: hal-05248666

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Distributed under a Creative Commons Attribution 4.0 International License
# Global transcriptomics and targeted metabolite analysis reveal the involvement of the AcrAB efflux pump in physiological functions by exporting signaling molecules in *Photorhabdus laumondii*

Linda Hadchity,¹ Anne Lanois-Nouri,¹ Adrien Chouchou,² David Roche,³ Jessica Houard,⁴ Noémie Claveyroles,¹ Alexandra Dauvé,⁵ Jacques Imbert,⁵ Maxime Gualtieri,⁴ Alain Givaudan,¹ Alyssa Carré-Mlouka,¹ Ziad Abi Khattar⁶

**AUTHOR AFFILIATIONS** See affiliation list on p. 15.

**ABSTRACT** In Gram-negative bacteria, resistance-nodulation-division (RND)-type efflux pumps, particularly AcrAB-TolC, play a critical role in mediating resistance to antimicrobial agents and toxic metabolites, contributing to multidrug resistance. *Photorhabdus laumondii* is an entomopathogenic bacterium that has garnered significant interest due to its production of bioactive specialized metabolites with anti-inflammatory, antimicrobial, and scavenger deterrent properties. In previous work, we demonstrated that AcrAB confers self-resistance to stilbenes in *P. laumondii* TT01. Here, we explore the pleiotropic effects of AcrAB in this bacterium. RNA sequencing of *ΔacrA* compared to wild type revealed growth-phase-specific gene regulation, with stationary-phase cultures showing significant downregulation of genes involved in stilbene, fatty acid, and anthraquinone pigment biosynthesis, as well as genes related to cellular clumping and fimbrial pilin formation. Genes encoding putative LuxR regulators, type VI secretion systems, two-partner secretion systems, and contact-dependent growth inhibition systems were upregulated in *ΔacrA*. Additionally, exponential-phase cultures revealed reduced expression of genes related to motility in *ΔacrA*. The observed transcriptional changes were consistent with phenotypic assays, demonstrating that the *ΔacrA* mutant had altered bioluminescence and defective orange pigmentation due to disrupted anthraquinone production. These findings confirm the role of stilbenes as signaling molecules involved in gene expression, thereby shaping these phenotypes. Furthermore, we showed that AcrAB contributes to swarming and swimming motilities independently of stilbenes. Collectively, these results highlight that disrupting *acrAB* causes transcriptional and metabolic dysregulation in *P. laumondii*, likely by impeding the export of key signaling molecules such as stilbenes, which may serve as a ligand for global transcriptional regulators.

**IMPORTANCE** Recent discoveries have highlighted *Photorhabdus laumondii* as a promising source of novel anti-infective compounds, including non-ribosomal peptides and polyketides. One key player in the self-resistance of this bacterium to stilbene derivatives is the AcrAB-TolC complex, which is also a well-known contributor to multidrug resistance. Here, we demonstrate the pleiotropic effects of the AcrAB efflux pump in *P. laumondii* TT01, impacting secondary metabolite biosynthesis, motility, and bioluminescence. These effects are evident at transcriptional, metabolic, and phenotypic levels and are likely mediated by the efflux of signaling molecules such as stilbenes. These findings shed light on the multifaceted roles of efflux pumps and open avenues to better explore the complexity of resistance-nodulation-division (RND) pump-medi

**Editor** Emily Weinert, The Pennsylvania State University, University Park, Pennsylvania, USA

Address correspondence to Alain Givaudan, alain.givaudan@umontpellier.fr, Alyssa Carré-Mlouka, alyssa.carre-mlouka@umontpellier.fr, or Ziad Abi Khattar, ziad.abikhattar@balamand.edu.lb.

The authors declare no conflict of interest.

See the funding table on p. 15.

**Received** 15 April 2025

**Accepted** 4 August 2025

**Published** 8 September 2025

Copyright © 2025 Hadchity et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
ated signaling pathways in bacteria, thereby aiding in combating multidrug-resistant infections.

**KEYWORDS** *Photorhabdus*, efflux pump, pleiotropy, signaling molecules, specialized metabolites, targeted metabolite analysis, transcriptional regulation

The Gram-negative bacterium *Photorhabdus laumondii* TT01 is a bioluminescent and motile entomopathogen that forms a mutualistic relationship with soil-dwelling nematodes of the *Heterorhabditidae* family (1, 2). This bacterium-nematode complex is highly effective at infecting and killing insects by septicemia and toxemia, with death occurring within 24 to 48 h post-infection (1, 3, 4). Recently, entomopathogenic bacteria such as *Photorhabdus* have garnered considerable interest in both fundamental and applied research, owing to the extensive array of bioactive specialized metabolites they produce during the saprophytic phase within insect cadavers (5–8). The study of these compounds provides valuable insights into medicine, microbial ecology, and biochemistry (9, 10). Many of them, including stilbene derivatives (STs), carbapenem-like compounds, odilorhabdins, indole, and anthraquinones (AQs), function to inhibit bacterial and fungal contamination and/or deter scavengers in insect cadavers (6, 8, 11–14).

In *Photorhabdus*, secondary metabolite production is regulated by several common transcriptional regulators, including HexA, an ortholog of LrhA in *Escherichia coli*, which belongs to the LysR-type transcriptional regulator (LTTR) family (15, 16). This family of regulators is the most abundant in prokaryotes and features a conserved structure, with an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer binding domain (16). LTTRs are involved in sensing various chemical compounds, leading to the regulation of gene transcription through an allosteric-coupling mechanism central to biological systems (16). HexA regulates several processes in *Photorhabdus*, including the production of secondary metabolites such as AQs and STs, bioluminescence, and cellular clumping (15–21). However, the co-inducers for the LrhA family of regulators remain undefined (16).

*Photorhabdus* is the only Gram-negative bacterium known to biosynthesize stilbenes (15, 19). Stilbene biosynthesis requires both cinnamic acid, resulting from the deamination of phenylalanine catalyzed by phenylalanine ammonia-lyase encoded by the *stlA* gene, and branched-chain fatty acids (BCFA) (22, 23). The first stilbene derivative, 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS), results from the fusion of a cinnamic acid derivative and a BCFA precursor (22). IPS, clinically known as Tapinarof, is used as an anti-inflammatory agent in the treatment of psoriasis (3, 23–25). IPS has also been shown to act as an antimicrobial, insect immunosuppressant, and signaling molecule in *P. laumondii*, inhibiting the production of AQ pigments and bioluminescence, and contributing to the mutualistic interaction within the nematode (22, 23, 26).

AQs are the largest group of natural pigments, most of which have been isolated from plants and are found in various human foods such as peas, cabbage, lettuce, and beans (27). Several biological activities have been reported for AQ-pigments, including antimalarial, antitumor, antioxidant, anti-inflammatory, and antimicrobial activities (27, 28). The two *Photorhabdus* species, *P. laumondii* and *P. temperata*, are the only Gram-negative bacteria known to produce AQ-pigments via a type II polyketide-synthase system, encoded by the biosynthesis gene cluster *antABCDEFGHI* (29, 30). AntJ and CysB transcriptional regulators bind to the *antA* promoter, thereby activating AQ biosynthesis (30). Eight different AQs have been described in various *Photorhabdus* strains (29, 31). Most of these AQ molecules are derived from several O-methylations of the AQ-257 Da-derivative, involving homologous O-methyltransferase enzymes encoded in the genome of *P. laumondii* TT01 (31). AQs are responsible for the yellow to red-orange pigmentation of bacterial colonies and cultures. However, their exact function in the *Photorhabdus* life cycle remains unclear (15, 30). Nevertheless, some AQs exhibit weak antibiotic activity, leading to the suggestion that they may act as antagonistic agents against other microorganisms in the dead insect (7, 30, 32).
In the Gram-negative bacteria *E. coli*, *Salmonella*, and *P. laumondii*, the resistance-nodulation-division (RND)-type AcrAB-TolC efflux pump exports a wide range of substrates, including antibiotics, detergents, bile salts, dyes, bacterial metabolites, and other toxic compounds (33–37). AcrAB also contributes to various physiological processes, such as detoxification, biofilm formation, virulence, and motility, among others (37–40). In our previous studies, we have shown that AcrAB is not essential for virulence but contributes to resistance to antimicrobial peptides (AMPs) such as polymyxins in *P. laumondii*. However, AcrAB exports the signaling molecule IPS, and its expression occurs during the stationary growth phase of *P. laumondii* (34, 35), concomitantly with the production and accumulation of secondary metabolites, particularly AQs and STs, in the insect cadaver (41).

In this study, we investigated the role of the AcrAB efflux pump in various phenotypic traits at both the transcriptional and targeted metabolite levels, focusing on AQs. RNA sequencing (RNA-seq) analysis revealed that the *acrAB* mutation alters the expression of genes involved in the biosynthesis of AQs, STs, and fatty acids during the stationary growth phase. This mutation also negatively affected the expression of genes associated with motility during the exponential growth phase. Phenotypic analysis showed impaired motility, bioluminescence, and yellow-orange pigmentation in the *ΔacrA* mutant, whereas the *ΔstlA* mutant (deficient in stilbene production) exhibited increased bioluminescence and yellow-orange pigmentation. These results highlight the pleiotropic effects of the AcrAB efflux pump in *P. laumondii* TT01, potentially mediated in part through the export of signaling molecules such as IPS.

# RESULTS AND DISCUSSION

## Deletion of *acrA* strongly alters gene expression profile in *P. laumondii* TT01

In light of the known effects of IPS on *Photorhabdus* cellular processes (26) and its extrusion by AcrAB (34), we conducted a global transcriptomic study to explore the molecular impact of *acrAB* mutation in *P. laumondii*. The *ΔmdtA* mutant, which lacks the membrane fusion protein of the MdtABC RND-type efflux pump, was used as a negative control, as this pump does not exhibit any detrimental phenotypes in *Photorhabdus* (35, 42). DESeq2 differential analysis comparing annotated features between the transcriptomes of WT and *ΔmdtA* mutant strains revealed minimal differences, primarily attributable to the polar effect of the *mdtA* mutation in either the exponential or stationary growth phases (Fig. S1A, Table S1A and B). Only five genes were significantly differentially expressed between the WT and *ΔmdtA* strains in the exponential growth phase, while 12 genes were significantly different in the stationary phase. Among them, *mdtA*, *mdtB*, *mdtC*, and *baeS* were consistently affected in both growth phases due to the polar *mdtA* mutation (Fig. S1A, Table S1A and B). In contrast, RNA-seq analysis revealed a significant difference in the expression of 43 genes between the WT and the *ΔacrA* strains cultured to the exponential growth phase (Fig. 1A; Fig. S1B, Table S2A). Twenty-five of these genes, involved in fimbrial pilin and flagellar formation, were downregulated (Fig. 1A; Fig. S1B, Table S2A). Notably, a significant difference in the expression of 185 genes (3.6%) out of a total of 5,086 genomic objects (GO) was observed between the WT and *ΔacrA* grown to the stationary growth phase (Fig. 1A and B; Fig. S1B, Table S2B). Among these, 115 genes were significantly downregulated, many of which are involved in the biosynthesis of STs (*stlC*, *stlD*, *stlE*, *stlA*, and *plu2236* encoding a putative ST-epoxidase [13]), fatty acids (*plu2075*, *plu2217*, and *plu2218*, encoding acyl-carrier proteins), AQs (*antABCDEFGHI*, *antJ*, and *plu4892-plu4895*, encoding O-methyltransferase enzymes involved in O-methylations of the first of AQ derivative [31]), as well as genes related to bacteriophage tail fibers, cellular clumping, fimbrial pilin formation, and enterobactin biosynthesis (Fig. 1A and B; Table S2B). Conversely, 70 genes involved in T6SS (cluster 1, cluster 3, and cluster 4), T6SS-related VgrG, Tps-related proteins, CdiBA-related proteins, putative LuxR-type regulators, and other genes, such as those encoding Tcd insecticidal toxins, were upregulated in *ΔacrA* compared to WT (Fig. 1A and B; Table S2B).
<figure><img src="image_2.png" /><figcaption>**FIG 1** Identification of differentially expressed genes by RNA sequencing analysis between *P. laumondii* WT and *ΔacrA* mutant strains. *P. laumondii* WT and *ΔacrA* were cultured in LB medium to reach the exponential (OD~540~ = 0.5–0.7) and stationary (OD~540~ = 2.3–4.2) growth phases. (A) Functional classification of 82 of the 185 differentially expressed genes (|log~2~ fold change| ≥ 1, adjusted *P* value (false discovery rate [FDR] ≤ 0.05) into four groups: secondary metabolite biosynthesis, motility, quorum sensing, and toxin/secretion system biogenesis. Genes are represented by arrows labeled with their gene numbers and corresponding protein names. Downregulated genes are indicated by red arrows, upregulated genes by green arrows, and genes with similar expression levels between WT and *ΔacrA* are indicated with white arrows. (B) Clustering of 185 genes differentially expressed (|log~2~ fold change| ≥ 1, adjusted *P* value (FDR ≤ 0.05) between the WT and *ΔacrA* (EMBL accession number BX470251 and GEO accession number GSE280064), classified by cluster of orthologous groups (COG) annotation. The percentage of differentially expressed genes in each COG category is shown.</figcaption></figure>
Interestingly, a Venn diagram analysis revealed that 61 common genes (8.6%) out of 713 genes were differentially expressed between the polymyxin B-resistant subpopulation (43) and the Δ*acrA* (Fig. S2) versus WT. These genes include those encoding T6SS components, Tps-related proteins (tpsA and tpsB), CdiA effector proteins, and *pbgP4* encoding a PhoP-regulated protein involved in resistance to cationic AMPs (44). Both T6SS and CdiA toxins play crucial roles in bacterial competition, pathogenesis, and environmental stress survival (45–47). A previous study has demonstrated that the polymyxin B-resistant subpopulation is responsible for the virulence of *P. laumondii* TT01 (43). Furthermore, it has been shown that the *acrA* mutation did not significantly alter the percentage of the polymyxin B-resistant subpopulation in *P. laumondii* TT01 (35). These findings suggest that AcrAB is not involved in polymyxin B heteroresistance in *P. laumondii* TT01. Instead, the shared differentially expressed genes between Δ*acrA* and the polymyxin B-resistant subpopulation likely involve other regulatory mechanisms.

Given that AcrAB plays a key role in the transport of various compounds across the bacterial membrane (37–40, 48), its involvement in extruding IPS signals (34, 35, 42) suggests that this efflux system may significantly impact the physiology of *Photorhabdus*. The exposure of *P. laumondii* WT to relevant concentrations of IPS induces a transcriptional stringent-like response in *Photorhabdus* (26), which could be further amplified in the Δ*acrA* background, where intracellular IPS accumulates gradually. Indeed, our findings show that the differential gene expression profiles are specific to the *acrA* mutation in *P. laumondii* TT01, as no significant difference was detected between the WT and Δ*mdtA* strain lacking the RND MdtABC efflux pump (42). Reverse transcription quantitative PCR (RT-qPCR) confirmed this specificity, showing a high correlation (0.98) with the RNA-seq data (Fig. S3A and B). These stress responses likely impair the biosynthesis of secondary metabolites, namely AQ and ST derivatives, by reallocating resources from secondary metabolism to stress survival mechanisms through feedback loops.

Previous transcriptomic studies in other Enterobacterales such as *Salmonella* showed that AcrAB primarily affects central metabolic pathways, such as carbohydrate metabolism, amino acid biosynthesis, the tricarboxylic acid cycle, and fatty acid metabolism (37, 38). This differs significantly from our findings in *Photorhabdus*, highlighting the versatility of AcrAB, which may adapt its substrate profile in response to the physiological state and the environmental conditions.

## AcrAB contributes to swarming and swimming motilities of *P. laumondii* TT01 in a stilbene-independent manner

Several operons are associated with the structure and function of the flagella in *Photorhabdus*, thereby influencing the flagellum-driven motility of this bacterium (49). These operons include *motAB*, *flhBA*, *flg*, and *fli* loci in *P. laumondii* TT01 (50). Our RNA-seq analysis revealed a downregulation of several genes related to flagellar motility, including *flgBCDEFG*, *fliFGHIJKLMNO*, and *fliC*, in Δ*acrA* mutant strain grown to the exponential growth phase compared to the WT strain (Fig. 1A; Table S2A). However, no significant difference in expression levels of these genes was observed between WT and Δ*mdtA* strains (Table S1A and B). Using motility assays, we showed that Δ*acrA* exhibited reduced swarming and swimming motility compared to WT after 48 and between 18 and 24 h of incubation, respectively (Fig. 2A and B). No differences in the two types of motilities were observed between Δ*stlA* and the WT strain (Fig. 2A and B), demonstrating that motility in *P. laumondii* is IPS-independent. To test the potential involvement of RND-type efflux pumps in the export of other molecule(s), we tested the effect of phenyl-arginine-β-naphthylamide (PAβN), a competitive RND-type efflux pump inhibitor (42), on the swarming motility of *P. laumondii* strains. Our results demonstrated that PAβN strongly inhibited the swarming motility of all strains tested, including the Δ*acrA* mutant, after 48 h of incubation (Fig. 2A). Therefore, AcrAB likely contributes to *P. laumondii* motility by exporting signaling molecule(s) other than IPS, possibly by working with other efflux pumps. Our findings align with those observed in the Δ*acrB*
<figure><img src="image_3.png" /><figcaption>**FIG 2** Swarming and swimming motilities of *P. laumondii* TT01 strains. Diameters of motility halos were measured for three independent biological replicates of *P. laumondii* WT, *ΔacrA*, *ΔmdtA*, and *ΔstlA* strains grown in LB broth supplemented with (A) 0.7% Eiken agar (swarming) with or without 50 mg·L⁻¹ of PAβN or (B) 0.35% agar (swimming). The cultures were incubated at 28°C for 48 h (swarming) or 24 h (swimming). Data are presented as mean ± standard errors of the mean (SEM). Statistically significant differences between strains or treatment with PAβN are indicated as * (*P* < 0.05) or ** (*P* < 0.01) or *** (*P* < 0.001), or **** (*P* < 0.0001).</figcaption></figure>

mutant of *Salmonella*, which exhibited a less motile phenotype (51). However, they contrast with observations from a different *acrb* mutant of the same species—one with loss of efflux function—where motility-related genes, including *fli* and *flh* operons, were upregulated, resulting in a hypermotile phenotype (40). Recent studies aiming to explain the hypermotility observed in an *acrb* mutant of *E. coli* (36) have shown that endogenous metabolites, such as polyamines, can accumulate in a *Δacrb* mutant and bind to AcrR, the transcriptional repressor of *acrab*. This binding inactivates AcrR, preventing it from repressing the *flhDC* operon, which encodes the master regulator of the flagellar gene cascade, thereby promoting hypermotility (52–54). Additionally, the differences in motility behavior between *Δacrb* mutants in *E. coli* and *Salmonella* support the idea that this phenotype is species dependent. In *P. laumondii*, the reduced motility observed in the *ΔacrA* strain and in all strains treated with PAβN is likely due to the impaired export of endogenous metabolites. This suggests that the accumulation of these metabolites within the cell may adversely impact bacterial fitness and, as a consequence, repress motility gene expression through interactions with global regulators.

An alternative explanation for the transcriptomic and phenotypic changes linked to the *acrab* mutation has also been proposed in a recent study, which suggests that a mutation of *acrb* decreases proton influx across the inner membrane, leading to a perturbation of the membrane potential in *S. Typhimurium* (55). This, in turn, may activate the global ArcBA transcriptional system, causing both transcriptional and phenotypic dysregulation, which affects key physiological functions in *S. Typhimurium*, such as motility (55). Theoretically, if this mechanism is broadly conserved, it should apply to all other functional TolC- and PMF-dependent RND-type efflux pumps, including MdtABC in *P. laumondii* (35, 42). However, our RNA-seq and phenotypic analyses showed no significant changes in gene expression or motility-related phenotypes in the *ΔmdtA* compared to the WT. This contrasts with the individual deletional *ΔmdtB*, *ΔmdtC*, and *ΔtolC* mutants of *E. coli*, which exhibit hypermotility associated with upregulation of motility genes—a condition where the membrane potential hypothesis has been proposed as less probable (54). Altogether, these findings support the conclusion that the transcriptional and phenotypic dysregulations observed in the *ΔacrA* strain are specifically linked to defective AcrAB-dependent metabolite efflux in *P. laumondii*.
AcrAB contributes to bioluminescence production in a stilbene-dependent manner in *P. laumondii* TT01

Unlike many other Gram-negative bacteria, which communicate using acyl-homoserine-lactone molecules, *Photorhabdus* employs photopyrones (PPYs) for quorum sensing (QS). These pyrones are synthesized by the photopyrone synthase and detected by the PluR regulator, a member of the LuxR-type regulators that control QS (20, 56, 57). The PluR regulator directly binds to the promoter region of the *pfcABCDEF* operon, encoding *Photorhabdus* clumping factor (Pcf) to activate cellular clumping in *P. laumondii* (20, 56). Our RNA-seq analysis demonstrates a significant downregulation of *pfcAB* and *pfcD* genes involved in Pcf-mediated cellular clumping, as well as up to 25 genes encoding tail fiber or bacteriophage-related proteins in *ΔacrA* compared to WT when grown to the stationary phase (Fig. 1A and B; Table S2B). Clumping is thought to be primarily mediated by phage tail fibers, which likely function by forming bridges between bacterial cells (58). Surprisingly, our cellular clumping assays revealed only a slight reduction in clumping of the *ΔstlA* strain relative to the WT, *ΔmdtA*, and *ΔacrA* strains (Fig. S4). The global transcriptional regulator HexA has been identified as a repressor of cellular clumping in *P. laumondii* by inhibiting the expression of the *pfc* operon (20). While several studies have speculated on a potential interaction between IPS and HexA (26), direct evidence confirming IPS as a bona fide ligand of HexA remains lacking. Recently, it has been reported that various ligands, including IPS and PPYs, can compete for binding to LuxR-type regulators involved in QS in several Gram-negative bacteria, including *Photorhabdus* (59). Taken together, these results suggest that HexA and LuxR regulators may compete for ligands such as IPS, which could mask a distinct phenotype of cellular clumping between TT01 WT and *ΔacrA*. This hypothesis is further supported by our RNA-seq analysis, which shows an upregulation of six genes encoding LuxR solo regulators, while three genes in the *pfc* operon—including *pfcC*, located between *pfcAB* and *pfcD*—were downregulated in the *ΔacrA* strain compared to WT (Fig. 1A; Table S2B). QS regulates several other processes, including bioluminescence in *Photorhabdus*—the only known example of a bioluminescent terrestrial bacterium (60, 61). In *Photorhabdus*, bioluminescence is encoded by the *luxCDABE* operon, which governs luciferase-catalyzed oxidation of the luciferin substrate in the presence of oxygen, resulting in the bioluminescent reaction (61). The *ΔacrA* strain exhibited a reduction in bioluminescence intensity compared to the WT, *ΔmdtA*, and *ΔstlA* strains, with a 3 h delay compared to the other strains (Fig. 3). Interestingly, the bioluminescence intensity of *ΔstlA* was similar to that of all other strains tested up to 14 h of incubation, when it becomes higher than those of WT, *ΔmdtA*, and *ΔacrA*, particularly with a peak at 14 and 22 h of incubation (Fig. 3). No relevant difference in bioluminescence was observed between WT and *ΔmdtA* strains (Fig. 3). Interestingly, the double mutant *ΔacrA stlA* exhibits an intermediate bioluminescence phenotype between the single mutants *ΔstlA* and *ΔacrA* (Fig. 3). Although both single and double mutants have the same delay in bioluminescence onset compared to the WT, the double mutant shows higher bioluminescence than the *ΔacrA* strain—which displays the lowest signal—and is equal to the WT, though still significantly lower than the *ΔstlA* mutant (Fig. 3). This suggests a genetic interaction between *acrA* and *stlA*, possibly through compensatory or antagonistic regulatory effects, supporting our hypothesis that the bioluminescence defect in the *ΔacrA* mutant depends on stilbene production. The deletion of *stlA* in the *acrA* background likely prevents the accumulation of a repressive stilbene compound, thereby restoring bioluminescence that is reduced in *ΔacrA*. This indicates that *acrAB* acts upstream or at the level of stilbene export, modulating intracellular stilbene levels that in turn affect bioluminescence. The interaction appears compensatory rather than additive, as the combined deletion does not further reduce bioluminescence beyond the effect of *acrA* alone. Surprisingly, our RNA-seq analysis revealed no transcriptional changes in the *lux* genes in the *ΔacrA* mutant compared to WT. Hapeshi et al. (2019) demonstrated that the exogenous addition of IPS to *P. laumondii* growing exponentially causes a rapid drop in bioluminescence, while the transcript levels of the *lux* operon itself were
<figure><img src="image_4.png" /><figcaption>FIG 3 AcrAB contributes to the bioluminescence of *P. laumondii* TT01 in a stilbene-dependent manner. About 10³ of each *P. laumondii* WT, ΔacrA, ΔmdtA, ΔstlA, and ΔacrAstlA strains were inoculated into LB broth in 96-well white microtiter plates and grown for 27 h at 28°C. *Xenorhabdus nematophila* F1 and uninoculated LB broth were used as negative controls. Bioluminescence was monitored over time using an Infinite M200 microplate reader (Tecan). Data are from three independent biological and experimental replicates and are presented as means ± standard errors of the mean (SEM).</figcaption></figure>

not affected (26). This suggests that the regulation of bioluminescence in response to
stilbene compounds may occur at a post-transcriptional level or through other indirect
mechanisms. Moreover, the impaired fatty acid (FA) biosynthesis in the ΔacrA mutant
may directly impact LuxCDE-dependent production of long-chain aldehydes required for
light emission (61). Since FA metabolism contributes to both stilbene and AQ biosynthe-
sis (61), the reduced levels of stilbene (34) and AQs observed in this study support
this interpretation. Moreover, HexA is known to inhibit bioluminescence at the post-tran-
scriptional level through a small RNA-dependent mechanism (20, 60). Given that IPS,
which is exported by AcrAB, may act as a cofactor for HexA, and that the addition of
exogenous IPS rapidly reduces bioluminescence in P. laumondii, this suggests that AcrAB
plays a role in modulating bioluminescence through HexA activation. Further supporting
this model, the addition of PAβN to cultures led to a decrease in bioluminescence in
all strains tested, with the ΔacrA and ΔacrAsltA mutants showing a more pronounced
delay and reduction in bioluminescence compared to the WT, ΔmdtA, or ΔstlA strains,
particularly at 17 h of incubation (Fig. S5).

Taken together, these results suggest that AcrAB, exporting at least IPS as a signaling
molecule, likely collaborates with other efflux pumps to modulate bioluminescence
production in *P. laumondii* TT01.

AcrAB contributes to anthraquinone-mediated pigmentation in a stilbene-
dependent manner in *P. laumondii* TT01

Our RNA-seq analysis revealed significant downregulation of five genes involved in the
biosynthesis of STs, including *stlA*, *stlC*, *stlD*, and *stlE*, as well as three genes encoding
acyl carrier proteins likely involved in ST production (22) (Fig. 1A). Additionally, 10 genes
from the *antABCDEFGH*1 operon, which is implicated in AQ biosynthesis, were downregu-
lated, along with 3 genes (*plu4892*, *plu4894*, and *plu4895*) encoding enzymes involved in
AQ O-methylation (Fig. 1A; Table S2B). Our phenotypic observations indicated that AQ
production was impaired in the Δ*acrA* mutant, as evidenced by reduced pigmentation
(white-beige colonies) compared to the typical yellow pigmentation of the WT strain
on trypticase soy agar (TSA) (Fig. 4). The pigmentation defect in the ΔacrA mutant was restored upon introduction in *trans* of a WT copy of the *acrAB* operon (Fig. 4). In contrast, mutants lacking MdtABC or AcrAB-like RND-type efflux pumps (35, 42) did not exhibit pigmentation defects as compared to the WT strain (Fig. 4). Interestingly, the ΔstlA mutant exhibited hyper-yellow to red-orange pigmentation, contrasting with the typical yellow pigmentation of the WT strain (Fig. 4). Consistent with this observation, a *P. laumondii* mutant strain defective in IPS production was previously shown to develop hyper-pigmented colonies on agar media (26). Analysis of the absorbance spectra of extracts from cultures of WT, ΔacrA, ΔacrA/pBBR1-acrAB, ΔstlA, and ΔstlA/pBBR1-stlA was performed using two commercial AQ molecules—Emodin (271 Da) and Physcion (285 Da)—as positive controls (absorbance peak 400–450 nm), revealing a 2.5-fold decrease in AQ production in the ΔacrA mutant compared to both the WT and the ΔacrA mutant complemented with a WT *acrAB* operon. In contrast, the intensity of the absorbance at 400–450 nm was higher by a factor of 3.5 in the ΔstlA mutant compared to WT, suggesting increased AQ production in ΔstlA (Fig. 5A). Notably, the ΔstlA mutant strain complemented with a WT copy of the *stlA* gene displayed a decreased absorbance intensity at 400–450 nm by a factor of 6.8 compared to ΔstlA (Fig. 5A). These results are consistent with our RNA-seq analysis (Fig. 1A; Table S2B) and phenotypic observations on TSA plates (Fig. 4). Previous findings showed that *hexA* mutation activates IPS production but suppresses AQ production in *P. laumondii* (62), suggesting that in the absence of AcrAB efflux, IPS may activate *HexA* and inhibit the production of STs, while simultaneously inhibiting AQ production at the transcriptional level (26).

To confirm that the yellow to red-orange colors of *Photorhabdus* are due to AQ pigmentation, we screened several AQ derivatives produced by the WT strain. Previous reports have indicated that *Photorhabdus* primarily produces two AQ derivatives, AQ 257 Da and AQ 271 Da, while AQ 301 Da and AQ 315 Da derivatives were exclusively identified in *Photorhabdus* cultured in insect larvae (31, 56). In this study, ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of extracts from five independent cultures of the WT, ΔacrA, and ΔstlA strains in LB broth enabled us to identify four AQ derivatives naturally produced by the WT, with m/z [M+H]$^+$ values of 257.045, 271.060, 285.075, and 315.086 Da, and retention times (t<sub>R</sub>) of approximately 12.82, 12.16, 14.06, and 12.81 min, respectively (Fig. 5B; Fig. S6A through D). These were designated AQ 257 Da, AQ 271 Da, AQ 285 Da, and AQ 315 Da (Fig. 6B; Fig. S6). While the localization of AQs in *P. laumondii* has not yet been determined, we performed two sequential intracellular extraction steps. The first step recovered intracellular molecules, while the second step specifically targeted molecules attached to the bacterial surface. The analysis of the extracts obtained from the intracellular and membrane fractions of WT, ΔacrA, and ΔstlA using UHPLC-MS showed that all detected AQ-derivatives were present in the supernatant, intracellular, and bacterial surface extracts (Fig. S6). These findings are in agreement with a study showing that AQ derivatives may be present in different plant cell compartments (63), but their localization has yet to be identified in *Photorhabdus*.

<figure><img src="image_5.png" /><figcaption>**FIG 4** AcrAB contributes to anthraquinone-mediated pigmentation in *P. laumondii* TT01. Pigmentation was assessed on TSA medium after 48 h of incubation at 28°C for each strain, harboring or not the pBBR1-MCS5 or the pBBR1-acrAB plasmids. Results shown are representative of at least three independent biological experiments, each with a minimum of three technical replicates.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>FIG 5 AcrAB is involved in anthraquinone-mediated yellow to red-orange pigmentation in *P. laumondii* TT01 strains in a stilbene-dependent manner. (A) Anthraquinone absorbance spectra were determined from culture extracts of *P. laumondii* WT, Δ*acrA*, Δ*acrA/pBBR1-acrAB*, Δ*stlA*, and Δ*stlA/pBBR1-stlA* strains grown at 28°C in LB broth, supplemented with gentamicin at 15 mg·L⁻¹ where appropriate. Absorbance measurements were recorded using an Infinite M200 microplate reader (Tecan). Acetonitrile (ACN) and acetonitrile + gentamycin (ACN + Gm) were used as negative controls. Data represent averages ± standard errors of the mean (SEM) from three independent biological and experimental replicates. (B) Identification of anthraquinone derivatives was performed by extraction of targeted ion peak m/z chromatograms, analyzed by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS), from five independent extracellular and intracellular extracts of *P. laumondii* WT, Δ*acrA*, and Δ*stlA* strains grown in LB broth for 48 h at 28°C. A representative replicate of WT extracellular extracts is shown. Anthraquinone structures were adapted from reference 31 and modified from reference 29.</figcaption></figure>

Conclusion

Our study reveals an intricate interplay between the AcrAB-TolC efflux pump, specialized
metabolite production, bioluminescence, and other physiological traits in *P. laumondii*
TT01. AcrAB reduces the intracellular accumulation of toxic metabolites, including the
small IPS molecule (34). We demonstrate here that the transcriptional and phenotypic
<figure><img src="image_7.png" /><figcaption>**FIG 6** Proposed model of the AcrAB-HexA-IPS regulatory network controlling pleiotropic phenotypes in the Δ*acrA* mutant of *P. laumondii* TT01. The loss of the AcrAB efflux pump impairs export of the stilbene compound 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) (34), resulting in its intracellular accumulation. This accumulation correlates with reduced bioluminescence, despite unchanged *luxCDABE* transcript levels, supporting a mechanism of enhanced post-transcriptional repression mediated by the LysR-type regulator HexA (20). HexA also represses *stlA*, the gene required for IPS biosynthesis (23), and IPS is proposed to act as a co-effector that enhances HexA's repressive function. Accordingly, IPS and AQ pigment production are reduced in the mutant, consistent with downregulation of *stlA* and the IPS-dependent *ant* operon. Motility is also impaired in the Δ*acrA* mutant, accompanied by downregulation of motility-related genes (*fli* and *flg*), suggesting that AcrAB and/or its repressor AcrR support motility independently of IPS, possibly through regulation by other accumulating metabolites that may interact with additional, yet unidentified, regulatory proteins. Additionally, intracellular IPS or its derivatives may compete with canonical autoinducers (e.g., photopyrones) for LuxR solo binding, interfering with QS-dependent regulation (59). This competition is likely to dysregulate both the activity and expression of certain LuxR solos and their downstream targets (e.g., the *pcf* operon involved in cell clumping), thereby linking metabolite accumulation to QS interference and further contributing to the pleiotropic transcriptional and phenotypic changes observed in the Δ*acrA* mutant. This model was created using BioRender.com.</figcaption></figure>

impacts of *acrAB* mutation on *P. laumondii* align with responses to ectopic IPS, involv-
ing genes regulated by HexA (20, 62), thereby confirming the role of IPS as a signal-
ing molecule. This influences profound gene expression changes and physiological
responses in *Photorhabdus*. Notably, *hexA* expression was similar between the WT and
*ΔacrA* strains during the stationary phase (34), suggesting that the phenotypes observed
in the *ΔacrA* mutant are not due to altered *hexA* transcription but rather to enhanced
activity of HexA. This is consistent with the presence of a putative C-terminal ligand-
binding domain in HexA, suggesting that its function depends on the availability of
specific intracellular ligands or co-inducers—potentially IPS. Therefore, we propose that
*acrAB* mutation may cause an accumulation of intracellular IPS, which then acts as
a ligand, enhancing the activity of the transcriptional repressor HexA and may affect
export of as yet unidentified potential co-activator molecule(s), other than IPS, that could
be involved in bacterial behaviors such as swarming and swimming motilities (Fig. 6;
Table 1).

Overall, these findings highlight the multifaceted role of AcrAB in *P. laumondii*, extending beyond antibiotic resistance to include regulation of secondary metabolism and bacterial behavior. The regulatory connections between AcrAB, HexA, and the bacterial physiological responses underscore the complexity of bacterial regulatory networks. Future studies should elucidate the precise mechanisms by which AcrAB
<table><caption>TABLE 1 Phenotypic traits of different *P. laumondii* TT01 strains<sup>a,b</sup></caption><thead><tr><th>Phenotypic traits</th><th>TT01 WT</th><th>ΔacrA</th><th>ΔmdtA</th><th>ΔstI</th></tr></thead><tbody><tr><td>Motility</td><td>+</td><td>±</td><td>+</td><td>+</td></tr><tr><td>Cellular clumping</td><td>+</td><td>+</td><td>+</td><td>±</td></tr><tr><td>Bioluminescence</td><td>+</td><td>±</td><td>+</td><td>++</td></tr><tr><td>Pigmentation</td><td>Yellow</td><td>White beige</td><td>Yellow</td><td>Orange</td></tr></tbody></table>

<sup>a</sup>These results were observed in at least three independent biological replicates.

<sup>b</sup>±: A decrease in a phenotypic trait compared to *P. laumondii* WT; ++: an increase in a phenotypic trait compared to *P. laumondii* WT.

influences gene expression and lipidomics and explore interactions between HexA, other transcriptional regulators, and IPS. Additionally, the identification of other transcriptional regulator ligand(s) transported by RND-type efflux pumps will provide deeper insights into the regulatory architecture of Gram-negative bacteria.

# MATERIALS AND METHODS

## Bacterial strains, plasmids, and growth conditions

The bacterial strains and plasmids used in this study and their sources are listed in Table S3. *P. laumondii* TT01 (2), *Xenorhabdus nematophila* F1, *Escherichia coli* XL1-blue MRF' (Stratagene), and WM3064 (64) were routinely grown in Luria Bertani (LB) broth or on agar (Difco) or in LB broth (Sigma) as previously described (34, 35). The double mutant ΔacrAstI was constructed by disrupting stI through allelic exchange in the ΔacrA background, as previously described (34). When required, gentamicin (Gm) was added to LB broth/agar to a final concentration of 15 mg·L⁻¹ for *E. coli* and *P. laumondii* TT01 strains harboring pBBR1-MCS5-derived plasmids.

## RNA extraction

Total RNA was extracted from TT01 WT, ΔacrA, and ΔmdtA strains grown in LB broth to exponential (OD<sub>540</sub> = 0.5–0.7) and stationary (OD<sub>540</sub> = 2.3–4.2) growth phases as previously described (42). Growth and cell viability are not affected in the ΔacrA mutant under the conditions used for RNA extraction, as confirmed by our previous studies (34, 35). Nine independent biological replicates were performed per strain, and equal amounts of total RNA from three replicates of the same strain were pooled together. We thus generated three biological replicates per strain and condition that were subjected to rRNA depletion using the NEBNext rRNA Depletion Kit (Bacteria) with RNA Sample Purification Beads prior to RNA sequencing.

## RNA sequencing and analysis

The 18 RNA-seq libraries were performed and validated as described in reference 43. Clustering and sequencing were performed on a NovaSeq 6000 (Illumina) on one lane of a flow cell SP.

Demultiplexing was performed with Illumina sequencing analysis software (CASAVA 1.8.2). Data quality was assessed with FastQC (Babraham Institute) and the sequencing analysis viewer from Illumina software. Differential expression analysis was performed using the RNA-seq bioinformatics pipeline implemented in the MicroScope platform (65). In a first step, forward raw RNA-seq reads of biological replicate samples were mapped onto the *P. laumondii* TT01 genome sequence (EMBL accession number: BX470251) as the reference genome (BWA-MEM v.0.7.17) (66). To consider adapter sequences, as well as poor quality sequences, an alignment score equal to at least half of the read length was required for a hit to be retained. The samtools suite (v.1.16.1) (67) was then used to extract reliable alignments with a Mapping Quality, MAPQ ≥1, from SAM files. From the 6 to 110 million Illumina sequences obtained, between 57% and 97% of high-quality mapped reads were finally kept. The number of these reads matching the genes of the
reference genome was subsequently computed with bedtools coverageBed (v.2.30.0) (68). Finally, the R package DESeq2 (v.1.22.2) (69) with R environment (v4.1.1) (70) was used with the IfcShrink function and its default parameters to normalize read counts and test for differential expression for each experimental condition comparison. For each comparison, differential gene expression was considered significant for a computed adjusted *P* value ≤0.05 and a log₂ fold change ≥1.

The complete data set from this study has been deposited in the NCBI's Gene Expression Omnibus (GEO) database under accession number GSE280064.

## Quantitative RT-PCR analysis

RT-qPCR was performed as previously described (34), using specific primer pairs for several selected genes (Table S4). For each reaction, melting curves were analyzed, and each curve contained a single peak. Data were analyzed as the relative quantification ratio of a target gene versus the reference housekeeping gene *recA* between TT01 WT and *acrA* mutant strains using REST 2009 software as previously described (35). The *gyrB* gene was used as an internal control.

## Swarming and swimming motility assays of *P. laumondii* TT01 strains

Swarming and swimming motility assays were determined by inoculating agar plates prepared with LB broth supplemented with 0.7% Eiken agar (Gerbu) or 0.35% Bacto agar (Difco), respectively, with three independent biological replicates of bacterial strains, as previously described (71). The diameters of the halos were measured 18, 24, and 48 h after incubation at 28°C. The RND-type efflux pump inhibitor, phenyl-arginine-β-naphthylamide (PAβN, Sigma) was used at a final concentration of 50 mg·L⁻¹. Statistically significant differences between strains or treatments were determined by two-way analysis of variance followed by Fisher's LSD test, using GraphPad Prism version 10.5.0.

## Cellular clumping assays of *P. laumondii* TT01 strains

Cell clumping assays were adapted from reference 20 and tested for TT01 WT, Δ*acrA*, Δ*mdtA*, and Δ*stlA*. Three independent biological replicates of each culture were set to an optical density (OD₅₀) of 0.05 and then incubated at 28°C without shaking in LB broth until exponential (OD₅₀ = 0.28–0.4) and stationary (OD₅₀ = 1.8–4.1) growth phases. Ten microliters of each culture were dropped onto 0.5% agarose pads (SeaKem GTG) in phosphate-buffered saline (Gibco) and then subjected to microscopic observation in phase contrast (Leica).

## Bioluminescence measurements of *P. laumondii* TT01 strains

Bioluminescence kinetics in LB broth were performed in white flat-bottomed 96-well microtiter plates (Greiner) after inoculating about 10³ bacteria from an overnight culture of three independent biological replicates of bacterial strains into each microdilution well. *Xenorhabdus nematophila* F1 strain and LB broth medium were used as negative controls. Optical density (OD₅₀) of all cultures was checked, and bioluminescence was monitored for 27 h at 28°C with orbital shaking in an infinite M200 microplate reader (Tecan). Phenyl-arginine-β-naphthylamide (PAβN, Sigma) was used at a final concentration of 25 mg·L⁻¹.

## Pigmentation assays of *P. laumondii* TT01 strains

Tinctorial properties of different *P. laumondii* TT01 strains were revealed on trypticase soja agar (TSA-bioMérieux). This medium is used for the detection of the yellow-orange AQ-mediated pigmentation in *Photorhabdus* (1). Five microliters of each bacterial culture were spotted on TSA and then incubated at 28°C for 48 h for the colony pigmentation to be revealed.
## Determination of anthraquinone absorbance spectra in *P. laumondii* TT01 strains

Twenty-hour-old cultures from three independent replicates of bacterial strains grown in 5 mL LB broth (Sigma) supplemented with Gm when required were used for inoculation with 1% (vol/vol) 10 mL of LB broth (Sigma). Cultures were incubated for 48 h at 28°C with shaking. All cultures were checked for OD₅₄₀, bacterial concentration, and growth on nutrient bromothymol blue triphenyltetrazolium chloride agar (NBTA) medium as previously described (35). Subsequently, three culture replicates from the same strain were pooled together and subjected to two successive ethyl acetate extractions (vol/vol) at room temperature for 24 h for each extraction. The extracts were evaporated, using a Stuart rotary evaporator connected to a water bath at 45°C (Fisher Scientific), followed by lyophilization. The resulting dry extracts were dissolved in 3 mL acetonitrile (ACN), and each extract was analyzed in three experimental replicates using an infinite M200 microplate reader (Tecan) to determine absorbance spectra at wavelengths between 350 and 500 nm. Two anthraquinone derivatives (Sigma): Emodin (271 Da) and Physcion (285 Da) dissolved in ACN at a final concentration of 500 mg·L⁻¹ were used as positive controls. ACN with and without Gm were used as negative controls.

## Extracellular and intracellular extraction of anthraquinone pigments in *P. laumondii* TT01

Twenty-hour-old cultures from five independent replicates of bacterial strains cultured in 10 mL LB broth (Sigma) were used to inoculate at 1% (vol/vol) 100 mL of LB broth (Sigma). Cultures were incubated for 48 h at 28°C with shaking. For all cultures, OD₅₄₀, bacterial concentration, and phenotypic traits (pigmentation aspect on TSA as described above and bacterial growth on NBTA medium as reported in [35]) were checked.

The extracellular extraction of AQ pigments was performed after collecting supernatants by centrifugation of each of 50 mL cultures at 3,500 × g for 30 min. Pellets were stored at -80°C to be used later for the intracellular AQ extraction. We proceeded to two successive ethyl acetate extractions (vol/vol) at room temperature for 24 h. The extracts were evaporated, using a Stuart rotary evaporator connected to a water bath at 45°C (Fisher Scientific), followed by lyophilization.

Intracellular AQ extraction protocols were adapted from references 72, 73. For each independent replicate, the frozen bacterial pellet was resuspended with 10 mL LB broth (Sigma) and sonicated for 6 min, then cooled on ice every 1 min and 30 s. Resulting suspensions were centrifuged for 20 min at 3,500 × g to collect the supernatants (which correspond to the total intracellular content). Pellets were then resuspended in 5 mL methanol (Sigma)/water vol/vol with 1% formic acid (Sigma). All suspensions were subjected to ultrasonic treatment for 6 min and then centrifuged to collect supernatants. This final step allows for the collection of molecules that were attached to the bacterial surface. All supernatants resulting from intracellular extractions were lyophilized before being analyzed by UHPLC-MS.

## Characterization of anthraquinone pigments in *P. laumondii* TT01 by ultra-high-performance liquid chromatography-mass spectrometry

For UHPLC-MS analysis, we dissolved each extract with 2 mL of a methanol/Milli-Q water mix vol/vol. After sample preparation, 10 µL of the supernatant of each replicate was injected into a Thermo Vanquish Flex Binary UHPLC system coupled to a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic separation was performed using an AQuity Premier BEH C18 column (1.7 µm, 2.1 × 100 mm, Waters, Milford, MA, USA) and two mobile phases: mobile phase A, 0.1% formic acid in Milli-Q water, and mobile phase B, 0.1% formic acid in acetonitrile. The gradient was programmed as follows: 0-2 min, 100% A isocratic; 2-12 min, 0% to 50% B linear; 12-15 min, 50% to 100% B linear; 15-18 min, 100% B isocratic; 18-20 min, 100% to 0% B linear; followed by 2 min of re-equilibration of the
column before the next run with a total run time of 20 min. The flow rate was set at 0.4 mL/min.

The UHPLC system was coupled to a Q Exactive Hybrid Quadrupole-Orbitrap High Resolution Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The MS acquisition was performed using positive ionization with a mass resolution of 120,000 at m/z 200. The m/z range for all full scan analyses was 200–1,500. Heated electrospray ionization parameters were as follows: sheath gas flow 60 arb (arbitrary units), auxiliary gas flow 15 arb, sweep gas flow 1 arb2, spray voltage 3.5 kV, probe temperature 400°C, capillary temperature 350°C. Prior to data collection, the mass spectrometer was calibrated using Pierce negative and positive ion calibration solution (Thermo Fisher Scientific, San Jose, CA, USA). Row data were analyzed by FreeStyle 1.8 SP2 QF1 (Thermo Fisher Scientific, San Jose, CA, USA). Anthraquinones were characterized by their m/z [M+H]⁺ = 257.045, 271.060, 285.075, and 315.086 Da and tR about 12.82, 12.16, 14.06, and 12.81 min, respectively (29–32).

## ACKNOWLEDGMENTS

We thank Philippe Clair from the BioCampus facility for his expert technical assistance with real-time PCR. MGX acknowledges the financial support provided by the France Génomique National infrastructure, funded under the "Investissement d'Avenir" program managed by the "Agence Nationale pour la Recherche" (contract ANR-10-INBS-09). Additionally, support from the "Plan France Relance" managed by the ANR (Molcheco Inrae/Nosopharm contract) is gratefully acknowledged. We also thank SPE, INRAE for awarding the IB2023 Scientific Grant. This work was also funded by a research grant from the Lebanese University (2022–2023) and a PHC CEDRE project 2022 (N47846 ZE).

## AUTHOR AFFILIATIONS

¹DGIMI, INRAE, Université Montpellier, Montpellier, France

²IBMM, Université Montpellier, CNRS, ENSCM, Montpellier, France

³Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, Evry, France

⁴Nosopharm, Nîmes, France

⁵MGX-Montpellier GenomiX, c/o IGF, Montpellier, France

⁶Faculty of Medicine and Medical Sciences, University of Balamand, Tripoli, Lebanon

## AUTHOR ORCIDs

Linda Hadchity http://orcid.org/0009-0002-1589-6383

Anne Lanois-Nouri http://orcid.org/0009-0007-4075-7058

Adrien Chouchou http://orcid.org/0000-0002-2061-4670

Maxime Gualtieri http://orcid.org/0000-0001-6590-2468

Alain Givaudan http://orcid.org/0000-0002-3901-5133

Alyssa Carré-Mlouka http://orcid.org/0000-0002-6141-8115

Ziad Abi Khattar http://orcid.org/0000-0003-4386-7467

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement</td><td>IB2023</td><td>Ziad Abi Khattar</td></tr><tr><td>PHC_cedre_2022</td><td>N47846_ZE</td><td>Alain Givaudan<br>Ziad Abi Khattar</td></tr><tr><td>Agence Nationale de la Recherche</td><td>PlandeRelance_Molcheco</td><td>Alain Givaudan<br>Alyssa Carré-Mlouka</td></tr></tbody></table>
<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Université Libanaise</td><td>Grant_2022-2023</td><td>Ziad Abi Khattar</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Linda Hadchity, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft | Anne Lanois-Nouri, Data curation, Formal analysis, Methodology, Validation, Writing – review and editing | Adrien Chouchou, Data curation, Formal analysis, Methodology | David Roche, Data curation, Formal analysis, Software, Validation | Jessica Houard, Methodology | Noémie Claveyroles, Methodology | Alexandra Dauvé, Methodology | Jacques Imbert, Methodology | Maxime Gualtieri, Formal analysis, Funding acquisition | Alain Givaudan, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Validation, Writing – review and editing | Alyssa Carré-Mlouka, Funding acquisition, Methodology, Validation, Writing – review and editing | Ziad Abi Khattar, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – review and editing

## ADDITIONAL FILES

The following material is available online.

## Supplemental Material

Supplemental material (Spectrum01106-25-S0001.pdf). Fig. S1 to S6; Tables S1 to S4.

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