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Genetic investigation of how an ATP hydrolysis cycle is coupled to lipopolysaccharide transport

Simpson, Brent W
http://rave.ohiolink.edu/etdc/view?acc_num=osu1523988371297363

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Microbiology.
Gram-negative bacteria coat their cell surface with the glycolipid, lipopolysaccharide (LPS), that provides stringent permeability characteristics. This layer of LPS comprises the outer leaflet of the outer membrane, and prevents entry of toxic compounds, like antibiotics. After it is synthesized at the inner membrane, LPS must be extracted from the inner membrane, transported across the periplasm, and translocated across the outer membrane to the outer leaflet. This process is accomplished by seven Lpt (LPS transport) proteins which have been best studied in Escherichia coli. An unusual ATP-binding cassette (ABC) transporter, LptB2FG, powers LPS transport. The LptB dimer utilizes conserved features of ABC-motor domains to bind and hydrolyze ATP causing conformational movements. These conformational changes are then transmitted to transmembrane partners LptFG to drive LPS extraction. The objective of this dissertation is to unravel how conformational movements of ATP hydrolysis are coupled to LPS extraction in LptB2FG. Interactions between subunits of ABC transporters utilize a conserved mechanism that consists of a groove in the ATPase domains that interacts with cytoplasmic coupling helices of the transmembrane domains. The tight interactions between these conserved structures allow movements to be coupled between the ATPase and transmembrane domains and is critical for their function. In chapter 2, we identify the coupling helices of LptFG and demonstrate they interact with a groove previously identified in LptB. Using structure-function and suppressor analyses, we demonstrate that a cluster of residues in the groove of LptB and conserved Glu residues in the coupling helices of LptFG are important for coupling ATP hydrolysis and LPS extraction. In chapter 3, while selecting for suppressors of a strain with antibiotics sensitivity conferred by a mutant lptB allele, we serendipitously identified a suppressor that is only resistant to novobiocin. The suppressor mutation does not result in a change to DNA gyrase, the known antibacterial target of novobiocin, but instead is intragenic in lptB. Using genetic, biochemical, and structural approaches, we demonstrate that novobiocin can bind to LptB2FG at the interaction interface between the ATPase and transmembrane domains. Novobiocin-binding to LptB2FG increases the activity of the transporter and can rescue defects from substitutions to the coupling helices of LptFG. Thus, novobiocin alters how ATP hydrolysis is coupled to LPS transport and will be a useful tool to unravel this process. While ABC ATPases utilize many conserved motifs for binding and hydrolyzing ATP, in chapter 4, we identify that LptB has a unique domain at its C-terminus (CT) that is essential for function. Defects conferred by alterations to the CT domain of LptB can be rescued by changes to the ATP-binding motifs. Utilizing these genetic relationships and effects of novobiocin, we demonstrate that the CT domain of LptB is critical for coupling of ATP hydrolysis and LPS extraction by LptFG. These finding allows us to propose new details in the molecular mechanism of LPS extraction.
Natividad Ruiz, Ph.D. (Advisor)
315 p.
Microbiology
lipopolysaccharide transport; glycolipid; membrane biogenesis; permeability barrier; cell envelope; ABC transporter

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Citations

  • Simpson, B. W. (2018). Genetic investigation of how an ATP hydrolysis cycle is coupled to lipopolysaccharide transport [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523988371297363

    APA Style (7th edition)

  • Simpson, Brent. Genetic investigation of how an ATP hydrolysis cycle is coupled to lipopolysaccharide transport. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1523988371297363.

    MLA Style (8th edition)

  • Simpson, Brent. "Genetic investigation of how an ATP hydrolysis cycle is coupled to lipopolysaccharide transport." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523988371297363

    Chicago Manual of Style (17th edition)

osu1523988371297363
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