AUTHOR=Bobková Šárka , Plechatá Michaela , Kovaľ Tomáš , Šanderová Hana , Blažková Simona , Akçin Rüveyda , Kameník Zdeněk , Dohnálek Jan , Krásný Libor , Wiedermannová Jana 
  
TITLE=The bacterial phosphotransferase system-mediated rifampicin phosphorylation: ancestral links to rifampicin-inactivating enzyme
  
JOURNAL=Frontiers in Microbiology
  
VOLUME=Volume 17 - 2026
  
YEAR=2026
  
URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2026.1789656
  
DOI=10.3389/fmicb.2026.1789656
  
ISSN=1664-302X
  
ABSTRACT=IntroductionThe bacterial phosphotransferase system (PTS) transports and phosphorylates sugars. Some PTS proteins share structural motifs with rifampicin phosphotransferases (RPHs), which inactivate rifampicin by phosphorylation. This homology suggests that the PTS may represent an evolutionary ancestor of the multi domain RPHs, though direct biochemical evidence has been lacking.MethodsBacillus subtilis strains lacking genes encoding PTS proteins were evaluated in growth assays in the absence/presence of rifampicin; liquid chromatography-mass spectrometry was used to monitor the ability of B. subtilis PTS proteins to phosphorylate rifampicin; thermophoresis was employed to characterize protein–rifampicin interactions.ResultsDeletion of B. subtilis ptsH, ptsI genes (encoding PTS proteins: HPr and EI) or rphT (encoding RphT-B. subtilis RPH) impaired growth in the presence of rifampicin. In vitro, the PTS complex (HPr, EI, MtlF, and PckA) phosphorylated rifampicin, with EI alone sufficient for this activity. However, no rifampicin phosphorylation by EI was detected in vivo. Heterologous expression of rphT then strongly increased rifampicin resistance, while ptsH/ptsI expression did not.ConclusionThis study shows that part of the PTS, protein EI, can phosphorylate rifampicin, supporting its evolutionary link to RPHs. We also establish that RphT, a putative rifampicin phosphotransferase misannotated as phosphoenolpyruvate synthase (Pps), is a bona fide rifampicin-modifying enzyme in B. subtilis. Finally, we demonstrate that derepressing RphT or its horizontal transfer confers high-level resistance to rifampicin.