Thank you so much for letting me share the GpsB story with you all! And there's nothing more motivating than a slice of cake right after 😉

Thank you so much!! GpsB always keeps me entertained with the many cool detours it takes :)

9/10 So, we propose GpsB acts as a peptidoglycan supply-chain sentinel: it watches cell wall synthesis through PBP2 (and PBP4, PBP2a, etc.), amplifies stress signaling through Stk1 and VraSR, and keeps MurA and MurZ protected on an as-needed basis.💪

8/10 And we find that GpsB interacts directly with VraR, and with histidine kinase VraS exclusively in the phosphomimetic state. As far as we know, this is the first direct link between GpsB and a two-component system. ΔvraR ΔgpsB and ΔvraS ΔgpsB are hypersensitive to fosfomycin.😱

7/10 murZ is transcriptionally regulated by VraRS under cell wall stress. A PmurZ-lux reporter shows murZ upregulation is badly impaired in ΔgpsB, even without antibiotic. That pointed us straight at VraRS.🫣

6/10 Fosfomycin targets MurA and MurZ (first committed step of PG synthesis). We find GpsB interacts directly with MurA, ReoM, and ReoY (MurA turnover machinery) but not MurZ (which indirectly interacts with GpsB via ReoM). Deleting reoY, which stabilizes MurA, reverses ΔgpsB fosfomycin sensitivity.

5/10 Since Stk1 is tied to drug resistance, we tested ΔgpsB against fosfomycin pressure. ΔgpsB is far more sensitive than Δstk1, and the double mutant barely survives. Complementation only works with phospho-tunable GpsB, as locking it in an “on” (T75E) state doesn’t lead to full rescue.🔄

4/10 Phosphorylation of GpsB does not impact self-interaction, but it does alter partner preference. T75E associates strongly with Ser/Thr kinase Stk1, yet much weaker with PBP2 and FtsZ. We also identified a brand-new partner in the process: PBP2a (MRSA)!😋

3/10 Upon point mutagenesis, we found 3/6 were physiologically relevant: T21, T75, and S76. Phosphomimetic substitution of just one of these three sites is enough to abolish GpsB overproduction toxicity. But why does this matter?🤔

2/10 It is known that GpsB facilitates cell division and cell wall synthesis through protein-protein interactions. But how does it prioritize them? Phosphoproteomics identified six Ser/Thr phosphosites on S. aureus GpsB, four in its IDR, hinting these could tune its interactions...