Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.
Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.
From the Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208.
Austin J
AJ
Rice
Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus Ohio 43205, and.
Alistair
A
Harrison
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Frances J D
FJ
Alvarez
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Amy L
AL
Davidson
From the Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, h-pinkett@northwestern.edu.
Heather W
HW
Pinkett
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D017136:Ion Transport; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008982:Molybdenum / Q000378:metabolism*; D009154:Mutation; D019897:Periplasm / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D026901:Membrane Transport Proteins; C044659:molybdate; D008982:Molybdenum; D000255:Adenosine Triphosphate
ABC Transporter; ATP; Electron Paramagnetic Resonance (EPR); Lipids; Membrane Transport; Nutrient Uptake