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Andrea Vettiger @avettiger.bsky.social · 06/10/2026

Betsy then showed, that PorH and ProtX co-purify with the mAGP sacculus and interfering with AG biosynthesis weakens this interaction. Therefore, we conclude that these proteins are tightly linked to the underlying cell wall. (9/n)

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Andrea Vettiger @avettiger.bsky.social · 06/10/2026

Didi also supervised Marina, a talented Master's student, who showed that ProtX, another mycomembrane protein, is also inserted at the poles. Strikingly, once inserted, PorH & ProtX stay put, even though the mycomembrane lipids around them diffuse freely. (8/n)

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Andrea Vettiger @avettiger.bsky.social · 06/10/2026

In comes Didi, my first PhD student. Didi mastered the whole cell biology side of the project and did an amazing job at showing where and when PorH gets incorporated: The sites of active cell wall remodeling a.k.a the old and new poles! (7/n)

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Andrea Vettiger @avettiger.bsky.social · 06/10/2026

According to AlphaFold2 PorAH likely forms hetero-pentameric channel with a hydrophilic lumen and a hydrophobic outer surface. Deletion of either gene results in increased Kanamycin resistance further supporting its role as an integral channel in the mycomembrane (5/n).

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Andrea Vettiger @avettiger.bsky.social · 06/10/2026

PorH needs its partner PorA to reach the surface, and at least one of the pair must be O-mycoloylated by Cmt1 at Ser residues. Block mycoloylation on both and PorH piles up in off-pathway forms, reminiscent of assembly-defective OMPs in E. coli. Moreover, the T7SS secretion isn't required. (4/n)

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Andrea Vettiger @avettiger.bsky.social · 30/08/2025

The Vettiger Lab at #SSM2025 last week. Was good fun! Looking already forward to 2026 in Montreux 😃

Didi and Rachel with our newest hire, Marco!

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

Based on AlphaFold prediction, this PBP1b-FtsA interaction is conserved among many Enterobactericae (in red) but not beyond. However, similar intrinsically disordered domains are found on most PBP1b and may offer a way to regulate aPBP activity independent of Lpo's. (11/12)

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

In the previous movie FtsZ (yellow) gets displaced from FtsA (unlabeled) upon addition of the wt N-terminal peptide of PBP1b (magenta), but not the R6E point mutant 👇 (10/12)

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

To our surprise the peptide of PBP1b was predicted to interact with the same residue on FtsA than FtsZ. This may suggest a competitive binding for FtsZ and PBP1b to FtsA. Thanks to Roman Hajdu in @nartimsoole.bsky.social lab, we find evidence for this in supported lipid bilayer assays 🧪🔬 (9/12)

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

But how does the N-terminal peptide of PBP1b mediate septal recruitment? AlphaFold screen performed by @ernstschmid.bsky.social in Johannes Walker's lab predicted an interaction with FtsA via a salt bridge between FtsA(E303) and PBP1b(R6) 🖥️ (8/12)

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

Interestingly, septal fortification of PBP1b doesn't seem to depend on its canonical Lpo activator, LpoB. However, it is dependent on the N-terminal cytosolic peptide of PBP1b, which was omitted in previous GFP fusions resulting in uniform IM labeling pattern 🤔 (6/12)

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

This lysis phenotype is specific for cells undergoing division. When inhibiting divisome formation with SulA expression, cells lacking PBP1b lyse significantly less, and when they do, they display smaller lesions which are likely attributed to the impaired side wall repair 🛠️ (6/12)

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

Having such compromised septa leads to catastrophic lysis events under osmotically stressed conditions originating from the division site (green = released DNA) 🌊🔬(5/12)

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

And their septal cell walls show compromised integrity (more pores and less stiff) when measured by AFM ⚛️🔬 (4/12).

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

Using in situ cryo-ET (as always performed by @navarropaula.bsky.social) we show that cells lacking PBP1b fail to produce a so-called septal PG wedge (highlighted by the red arrow) ❄️🔬 (3/12)

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Andrea Vettiger @avettiger.bsky.social · 03/04/2025

aPBPs are a major cell wall synthase family and have been implemented in PG repair and fortification. Additionally, PBP1b has been proposed to play a crucial role in cell division however its precise role has remained obscure. (2/12)

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