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 (RND)-type effluxeffluxpumps, 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 ?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 ?acrA. Additionally, exponential-phase cultures revealed reduced expression of genes related to motility in ?acrA. The observed transcriptional changes were consistent with phenotypic assays, demonstrating that the ?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. Overall design: Three independent RNA samples, each corresponding to a pool of total RNA extracted independently from 3 biological replicates cultures of: TT01 WT grown in LB harvested in exponential phase (OD= 0.5-0.7) or stationary phase (OD=2.3-4.2) acrA mutant grown in LB harvested in exponential phase (OD= 0.5-0.7) or stationary phase (OD=2.3-4.2) mdtA mutant grown in LB harvested in exponential phase (OD= 0.5-0.7) or stationary phase (OD=2.3-4.2) Deep sequencing, performed using Illumina HiSeq 2000"