Identification and Characterization of a Novel Fosfomycin Bacillithiol Transferase FosB8 in Paenibacillus illinoisensis

Objective To characterize a novel fosfomycin resistance gene, fosB8 , elucidate its enzymatic parameters, and analyze the genetic environment of fosB8 in the clinical Paenibacillus illinoisensis strain WP77, as has been done with other fosfomycin resistance genes such as fosA7 in Salmonella and fosB in Staphylococcus aureus . Method Species identification was conducted using the VITEK 6 system and Laser Desorption Ionization-Time of Flight Mass Spectrometry. The minimum inhibitory concentrations (MICs) were then determined by the agar dilution method, adhering to the guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI), ensuring standardized and reliable results. The genetic profile of strain WP77, as revealed by whole-genome sequencing, indicates a genome length of 6.64 Mbp with a single chromosome and a GC content of 47.2%. The analysis identified key chemotaxis genes such as mcp , che , and mot , and antibiotic resistance genes including drr and emr , conferring resistance to various antibiotics. fosB8 was characterized through conjugation experiments, molecular cloning, bioinformatics analysis, and enzymatic kinetic assays. Results P. illinoisensis WP77 exhibited resistance to fosfomycin (512 mg/L). Finally, A novel functional fosfomycin resistance gene, designated fosB8, was identified in WP77. The gene encodes a putative fosfomycin bacillithiol transferase that exhibits 43% to 62% amino acid identity with known metallothiol transferases. Expression of fosB8 in a recombinant strain resulted in an 256-fold increase in the fosfomycin MIC. FosB8 exhibited a catalytic efficiency (kcat/Km) of (7.9 ± 0.8) × 10³ M⁻¹ s⁻¹ toward fosfomycin. Genetic environment analyses indicated that fosB8 is probably intrinsic ingenus Paenibacillus . Conclusion The study for the first time reported a novel fosfomycin bacillithiol transferase FosB8. Notably, FosB8 was confirmed to play an essential role in the resistancemechanism exhibited by Paenibacillus illinoisensis against fosfomycin. FosB8 represents the eighth characterized example of a FosB enzyme in the existing literature.