Novel Fosfomycin Resistance Mechanism in Pseudomonas entomophila Due to Atypical Pho Regulon Control of GlpT

<b>Background/Objectives:</b><i>Pseudomonas entomophila</i> is a ubiquitous bacterium capable of killing insects of different orders and has become a model for host-pathogen studies and a promising tool for biological pest control. In the human pathogen <i>Pseudomonas aeruginosa</i>, spontaneous resistance to fosfomycin arises almost exclusively from mutations in the glycerol-3-phosphate transporter (GlpT), the drug's sole entry route in this species. Here, we investigated whether this specificity is conserved in <i>P. entomophila</i>, as it could provide a valuable marker system for studying mutation rates and spectra and for selection in genetic engineering. <b>Methods:</b> We isolated 16 independent spontaneous fosfomycin-resistant mutants in <i>P. entomophila</i>, and studied the genetic basis of the resistance using a combination of sequencing, phenotyping and computational approaches. <b>Results:</b> We only found two mutants without alterations in <i>glpT</i> or any of its known regulatory elements. Whole-genome sequencing revealed unique inactivating mutations in <i>phoU</i>, a key regulator of the phosphate starvation (Pho) regulon. Computational analyses identified a PhoB binding site in the <i>glpT</i> promoter, and experiments showed that <i>phoU</i> inactivation reduced <i>glpT</i> expression nearly 20-fold. While placing a sugar-phosphate transporter under the Pho regulon may seem advantageous, bioinformatic analysis shows this configuration is atypical among pseudomonads. <b>Conclusions:</b> This atypical Pho regulon control of GlpT probably reflects the peculiarities of <i>P. entomophila</i>'s habitat and lifestyle; highlighting how readily regulatory evolution can lead to the rapid divergence of resistance mechanisms, even among closely related species.