Biology subjects

Snow, E. D.

Publications and source records attributed to Snow, E. D..

3 recordsLinked to original sources

bioRxiv2026

A TmaT-AftD interaction is required for the CmpL4 mycomembrane biogenesis pathway in Corynebacterium glutamicum.

Bacteria in the Mycobacteriales order like mycobacteria and corynebacteria surround themselves with a multilayered cell envelope. Their cytoplasmic membrane is fortified by a peptidoglycan cell wall that is decorated with branched arabinogalactan (AG) polymers. The AG glycans are further modified with mycolic acids to form an outer membrane. Biogenesis of this mycomembrane requires the transport of mycolates from their site of synthesis in the cytoplasmic membrane to the cell surface. How mycolate transport is controlled and coordinated with AG synthesis has remained unclear. Mycolate transport is mediated by the essential RND-family transporter MmpL3 in mycobacteria and a pair of related, partially redundant transporters called CmpL1 and CmpL4 in corynebacteria. The acetyltransferase TmaT has also been implicated in mycolate transport in both types of bacteria. In corynebacteria, it is required for the production of acetylated mycolates, and this modification has been proposed to promote mycolate transport via both CmpL transporters in the model organism Corynebacterium glutamicum (Cglu). Here, we reinvestigated the function of TmaT in Cglu and found that it and several factors encoded in the tmaT locus are specifically required for mycolate transport via the CmpL4 transporter pathway. Notably, one of these additional genes encodes the arabinosyltransferase AftD involved in AG biogenesis. TmaT and AftD were found to interact, and our results indicate that this interaction is required for acetylated mycolate production, mycolate transport via the CmpL4 pathway, and normal arabinan synthesis. Thus, the TmaT-AftD interaction may serve as a regulatory link connecting mycolate transport with AG biogenesis. SIGNIFICANCEMycobacteriales bacteria, including pathogens like Mycobacterium tuberculosis (Mtb), have a complex cell surface comprising an inner membrane, a cell wall modified with arabinogalactan (AG), and an outer mycomembrane made of mycolic acids linked to AG polymers. Because these surface biogenesis pathways are targeted by frontline anti-Mtb drugs, there is great interest in elucidating their underlying mechanisms. Here, we identify an interaction between factors involved in mycomembrane (TmaT) and AG biosynthesis (AftD) in the model organism Corynebacterium glutamicum. We show that this interaction is important for proper surface biogenesis and may therefore function to coordinate mycomembrane assembly with AG synthesis. This and other potential regulatory connections controlling envelope biogenesis represent attractive targets for future antibiotic development.

microbiology↗

bioRxiv2025

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

The Mycobacteriales order of bacteria includes important pathogens such as Mycobacterium tuberculosis. These organisms are surrounded by a unique cell envelope architecture that includes a two-layered cell wall composed of peptidoglycan (PG) and arabinogalactan (AG). They also build an outer membrane called the mycomembrane that is made of mycolic acids. Mycolate outer membrane proteins (MOMPs) reside within the mycomembrane and a subset are thought to form pores that allow essential nutrients to permeate the envelope. However, little is known about the structure of these proteins or the mechanism by which they are assembled. Here, we investigate MOMP assembly in Corynebacterium glutamicum (Cglu) using PorH as a model corynebacterial MOMP. PorH is encoded in an operon with the MOMP PorA, and the two small, alpha-helical proteins have been proposed to form hetero-oligomeric pores in the mycomembrane. Consistent with this proposal, AlphaFold2 predicts a high confidence structure of a hetero-oligomeric pore formed by five copies each of PorH and its partner PorA, and we show that PorA is required for the surface assembly of PorH. Using a fluorescence assay for detection of surface-exposed PorH or another MOMP called ProtX, we found that MOMP assembly occurs within zones of active PG synthesis at the cell poles. We also discovered that PorH and ProtX are linked to the cell wall. Thus, like Gram-negative bacteria, Cglu coordinates outer membrane protein assembly with PG biogenesis and uses proteins to connect the mycomembrane and the cell wall.

microbiology↗

bioRxiv2024

The mechanism of peptidoglycan O-acetylation in Gram-negative bacteria typifies bacterial MBOAT-SGNH acyltransferases

Bacterial cell envelope polymers are commonly modified with acyl groups that provide fitness advantages. Many polymer acylation pathways involve pairs of membrane-bound O-acyltransferase (MBOAT) and SGNH family proteins. As an example, the MBOAT protein PatA and the SGNH protein PatB are required in Gram-negative bacteria for peptidoglycan O-acetylation. The mechanism for how MBOAT-SGNH transferases move acyl groups from acyl-CoA donors made in the cytoplasm to extracellular polymers is unclear. Using the peptidoglycan O-acetyltransferase proteins PatAB, we explore the mechanism of MBOAT-SGNH pairs. We find that the MBOAT protein PatA catalyzes auto-acetylation of an invariant Tyr residue in its conserved C-terminal hexapeptide motif. We also show that PatB can use a synthetic hexapeptide containing an acetylated tyrosine to donate an acetyl group to a peptidoglycan mimetic. Finally, we report the structure of PatB, finding that it has structural features that shape its activity as an O-acetyltransferase and distinguish it from other SGNH esterases and hydrolases. Taken together, our results support a model for peptidoglycan acylation in which a tyrosine-containing peptide at the MBOATs C-terminus shuttles an acyl group from the MBOAT active site to the SGNH active site, where it is transferred to peptidoglycan. This model likely applies to other systems containing MBOAT-SGNH pairs, such as those that O-acetylate alginate, cellulose, and secondary cell wall polysaccharides. The use of an acyl-tyrosine intermediate for MBOAT-SGNH acyl transfer is also shared with AT3-SGNH proteins, a second major group of acyltransferases that modify cell envelope polymers.

biochemistry↗