Chouchou, A. | bioRxiv Science

bioRxiv2025

Pleiotropic role of PAX cyclolipopeptides in the Xenorhabdus bacterium mutualistically associated with entomopathogenic nematodes

Xenorhabdus is an entomopathogenic bacterium involved in a mutualistic relationship with Steinernema nematodes. Xenorhabdus produces a multitude of specialized metabolites by Non-Ribosomal Peptide Synthetases (NRPSs) pathways to mediate bacterial-nematode- insect interactions. PAX cyclolipopeptides are a family of NRP-type molecules whose ecological role remains poorly understood. In this study, the pleiotropic role of PAX peptides in the life cycle of Xenorhabdus nematophila has been investigated. By mass spectrometry analysis, we first demonstrated that PAX peptides were detected from the pathogenic stage up to the necrotrophic stage. We discovered that the bromothymol blue adsorption phenotype historically used to discriminate Xenorhabdus variants was associated with the presence of PAX peptides. We found that PAX peptides were positively involved in biofilm formation and negatively involved in swimming motility. PAX peptides were also shown to promote in vivo the production of infective Steinernema juveniles, suggesting their involvement in the mutualistic relationship between Xenorhabdus and its nematode partner. Finally, we showed that the paxTABC cluster as well as PAX peptide production was conserved across the whole Xenorhabdus genus except in Xenorhabdus poinarii and Xenorhabdus ishibashii. This work has revealed multiple new ecological roles for NRP-type peptides. ImportanceXenorhabdus bacteria are models of particular interest for their mutualistic relationship with Steinernema nematodes and their ability to produce a wide range of natural NRP-type bioactive metabolites. These compounds are mostly studied for their medical or industrial applications, but their ecological role is poorly understood. This study provides a dynamic characterization of PAX cyclolipopeptide presence during Xenorhabdus nematophila life cycle, as well as confirmation of their production by 7 different strains within the Xenorhabdus genus. We revealed new multiple functions for PAX peptides in biofilm formation, swimming motility and juvenile nematode production. A deeper understanding of how PAX peptides interact with the nematode host would provide a better insight into the role of these cyclolipopeptides in bacterial-nematode mutualism.

microbiology↗

bioRxiv2025

Global Transcriptomics and Targeted Metabolite Analysis Reveal the Involvement of the AcrAB Efflux Pump in Physiological Functions by Exporting Signaling Molecules in Photorhabdus laumondii

In Gram-negative bacteria, resistance-nodulation-division-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 deterrents 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 {Delta}acrA compared to wild-type revealed growth-phase-specific gene regulation, with stationary-phase cultures showing significant downregulation of genes involved in stilbenes, 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 {Delta}acrA. Additionally, exponential-phase cultures revealed reduced expression of genes related to motility in {Delta}acrA. The observed transcriptional changes were consistent with phenotypic assays, demonstrating that the {Delta}acrA 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. ImportanceRecent 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 RND pump-mediated signaling pathways in bacteria, thereby aiding combat multidrug resistant infections.

microbiology↗