Poster abstracts

Poster number 41 submitted by Andrew Wilson

Uncovering the role of an unusual transmembrane helix in the LptB2FGC ATP-binding cassette transporter

Andrew Wilson (Microbiology, The Ohio State University), Natividad Ruiz (Microbiology, The Ohio State University)

Abstract:
Gram-negative bacteria possess an inner membrane (IM) and an outer membrane (OM), which delineate the cytoplasm and periplasmic compartment. The outer leaflet of the OM is primarily formed by an essential layer of tightly packed molecules of lipopolysaccharide (LPS). LPS is synthesized in the IM, after which it must be transported to the cell surface. The cellular machinery responsible for this transport process is comprised of seven essential lipopolysaccharide transport proteins, LptA-G. The molecular “motor” of this machinery, LptB2FGC, is an ATP-binding cassette (ABC) transporter embedded in the IM. Generally, ABC transporters contain two cytoplasmic nucleotide-binding domains (LptB2) physically coupled to two transmembrane domains (LptF and LptG). However, the structure of the LptB2FGC transporter deviates from all other identified ABC transporters in that the single transmembrane α-helix from the protein LptC (TMC) is inserted into one of the contact interfaces between LptF and LptG. Interestingly, TMC is highly conserved yet it was previously reported that removing this membrane anchor from LptC yields no observable phenotypes in Escherichia coli, calling into question whether TMC has a role in LPS transport. Here, we used a genetic approach to elucidate several phenotypes for a ∆TMC mutant strain of E. coli, demonstrating a bona fide role for TMC in LPS transport. Suppressor analysis revealed that removal of TMC improves LPS transport in mutants with ATP-binding defects in LptB2. Furthermore, we show that removal of TMC is not a general suppressor of LPS transport defects, as the effect is localized to a specific step in the LPS transport cycle. Finally, we observed that removing TMC affects LptC levels, suggesting a role of TMC in the assembly of LptC into the Lpt ABC transporter.

Keywords: ABC transporter, lipopolysaccharide transport, suppressor analysis