The mycomembrane proteins PorH and ProtX are inserted at polar growth zones and linked to the cell wall

Area of Science:

• Microbiology
• Bacterial Cell Envelope Structure
• Protein Assembly Mechanisms

Background:

• Mycobacteriales possess a unique cell envelope with peptidoglycan, arabinogalactan, and a mycomembrane made of mycolic acids.
• Mycolate outer membrane proteins (MOMPs) are embedded in the mycomembrane, with some forming pores for nutrient permeation.
• The structure and assembly mechanisms of MOMPs remain largely uncharacterized.

Purpose of the Study:

• To investigate the assembly mechanism of Mycolate Outer Membrane Proteins (MOMPs) in the model organism *Corynebacterium glutamicum* (Cglu).
• To elucidate the structural basis and assembly process of MOMPs, using PorH as a model.
• To understand the relationship between MOMP assembly and cell wall biogenesis.

Main Methods:

• Utilized AlphaFold2 to predict the structure of hetero-oligomeric pores formed by MOMPs PorH and PorA.
• Employed a fluorescence assay to detect surface-exposed MOMPs (PorH and ProtX).
• Investigated the requirement of PorA for PorH surface assembly and the localization of MOMP assembly.

Main Results:

• AlphaFold2 predicted a high-confidence structure of a hetero-oligomeric pore composed of PorH and PorA.
• PorA was found to be essential for the surface assembly of PorH.
• MOMP assembly was localized to zones of active peptidoglycan (PG) synthesis at the cell poles.
• PorH and ProtX were shown to be linked to the cell wall.

Conclusions:

• MOMP assembly in *Cglu* is coordinated with peptidoglycan biogenesis, occurring at the cell poles.
• Proteins are utilized to connect the mycomembrane to the cell wall, similar to mechanisms in Gram-negative bacteria.
• These findings provide insights into the assembly of essential outer membrane proteins in Mycobacteriales.