Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function

ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, t...

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Autores principales:	Brent W. Simpson, Tristan W. Owens, Matthew J. Orabella, Rebecca M. Davis, Janine M. May, Sunia A. Trauger, Daniel Kahne, Natividad Ruiz
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/70fd34c5f4c0484c83e80bbffb5c41d8
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spelling	oai:doaj.org-article:70fd34c5f4c0484c83e80bbffb5c41d82021-11-15T15:50:15ZIdentification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function10.1128/mBio.01729-162150-7511https://doaj.org/article/70fd34c5f4c0484c83e80bbffb5c41d82016-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01729-16https://doaj.org/toc/2150-7511ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. IMPORTANCE Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB2FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the cytoplasmic membrane prior to transport to the cell surface. How ATP hydrolysis is coupled with LPS release from the membrane is not understood. We have identified residues at the interface between the ATPase and the transmembrane domains of this heteromeric ABC complex that are important for LPS transport, some of which coordinate ATPase activity with LPS release.Brent W. SimpsonTristan W. OwensMatthew J. OrabellaRebecca M. DavisJanine M. MaySunia A. TraugerDaniel KahneNatividad RuizAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 5 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Brent W. Simpson Tristan W. Owens Matthew J. Orabella Rebecca M. Davis Janine M. May Sunia A. Trauger Daniel Kahne Natividad Ruiz Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function
description	ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. IMPORTANCE Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB2FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the cytoplasmic membrane prior to transport to the cell surface. How ATP hydrolysis is coupled with LPS release from the membrane is not understood. We have identified residues at the interface between the ATPase and the transmembrane domains of this heteromeric ABC complex that are important for LPS transport, some of which coordinate ATPase activity with LPS release.
format	article
author	Brent W. Simpson Tristan W. Owens Matthew J. Orabella Rebecca M. Davis Janine M. May Sunia A. Trauger Daniel Kahne Natividad Ruiz
author_facet	Brent W. Simpson Tristan W. Owens Matthew J. Orabella Rebecca M. Davis Janine M. May Sunia A. Trauger Daniel Kahne Natividad Ruiz
author_sort	Brent W. Simpson
title	Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function
title_short	Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function
title_full	Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function
title_fullStr	Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function
title_full_unstemmed	Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function
title_sort	identification of residues in the lipopolysaccharide abc transporter that coordinate atpase activity with extractor function
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/70fd34c5f4c0484c83e80bbffb5c41d8
work_keys_str_mv	AT brentwsimpson identificationofresiduesinthelipopolysaccharideabctransporterthatcoordinateatpaseactivitywithextractorfunction AT tristanwowens identificationofresiduesinthelipopolysaccharideabctransporterthatcoordinateatpaseactivitywithextractorfunction AT matthewjorabella identificationofresiduesinthelipopolysaccharideabctransporterthatcoordinateatpaseactivitywithextractorfunction AT rebeccamdavis identificationofresiduesinthelipopolysaccharideabctransporterthatcoordinateatpaseactivitywithextractorfunction AT janinemmay identificationofresiduesinthelipopolysaccharideabctransporterthatcoordinateatpaseactivitywithextractorfunction AT suniaatrauger identificationofresiduesinthelipopolysaccharideabctransporterthatcoordinateatpaseactivitywithextractorfunction AT danielkahne identificationofresiduesinthelipopolysaccharideabctransporterthatcoordinateatpaseactivitywithextractorfunction AT natividadruiz identificationofresiduesinthelipopolysaccharideabctransporterthatcoordinateatpaseactivitywithextractorfunction
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Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function

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