β-Barrel proteins tether the outer membrane in many Gram-negative bacteria

Kelsi M. Sandoz; Roger A. Moore; Paul A. Beare; Ankur V. Patel; Robert E. Smith; Marshall Bern; Hyea Hwang; Connor J. Cooper; Suzette A. Priola; Jerry M. Parks; James C. Gumbart; Stéphane Mesnage; Robert A. Heinzen

doi:10.1038/s41564-020-00798-4

β-Barrel proteins tether the outer membrane in many Gram-negative bacteria

Kelsi M. Sandoz, Roger A. Moore, Paul A. Beare, Ankur V. Patel, Robert E. Smith, Marshall Bern, Hyea Hwang, Connor J. Cooper, Suzette A. Priola, Jerry M. Parks, James C. Gumbart, Stéphane Mesnage, Robert A. Heinzen

Molecular Biophysics

Research output: Contribution to journal › Article › peer-review

40 Scopus citations

Abstract

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)¹. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins^2,3. In Escherichia coli, Braun’s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability⁴; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present⁵. We used a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

Original language	English
Pages (from-to)	19-26
Number of pages	8
Journal	Nature Microbiology
Volume	6
Issue number	1
DOIs	https://doi.org/10.1038/s41564-020-00798-4
State	Published - Jan 2021

Funding

We thank M. Suzuki, A. E. Acosta Martin and M. Collins for their contribution to developing the MS method and V. Nair for his help with cryo-EM. We thank R. Kissinger for his three-dimensional modelling, with assistance from A. Athman, and A. Mora for graphics support. We thank X. De Bolle and P. Godessart for stimulating discussion and J. Zupan, P. Zambryski, D. Kelly, A. Taylor, J. Shaw, E. F. Diaz Parga, R. Wheeler, I. Boneca, M.-K. Taha and E. Hoiczyk for the bacterial cultures used for peptidoglycan extraction. The PG MS analyses were performed by the biOMICS Facility of the Faculty of Science Mass Spectrometry Centre at the University of Sheffield, UK and the Research Technologies Branch at the National Institutes of Health (NIH) in Rockville, USA. The cryo-EM work was performed at the Research Technologies Branch at the NIH in Hamilton, USA. This work was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (R.A.H. and S.A.P.), a Medical Research Council grant no. MR/S009272/1 (S.M.) and a Biotechnology and Biological Sciences Research Council grant no. BBSRC BB/N000951/1 (S.M.). R.E.S. and A.V.P. are supported by a Biotechnology and Biological Sciences Research Council studentship (doctoral training program grant no. BB/M011151/1). J.C.G. acknowledges support from the NIH under grant no. R01-GM123169. C.J.C. was supported by a National Science Foundation (NSF) Graduate Research Fellowship under grant no. 2017219379. J.M.P. was supported by the Laboratory Directed Research and Development program at Oak Ridge National Laboratory, which is managed by UT-Battelle for the US Department of Energy under contract no. DE-AC05-00OR22725. This work used resources of the Compute and Data Environment for Science at Oak Ridge National Laboratory as well as the Extreme Science and Engineering Discovery Environment (allocation no. TG-MCB130173), which is supported by an NSF grant no. ACI-1548562.

Funders	Funder number
Extreme Science and Engineering Discovery Environment	TG-MCB130173
US Department of Energy	DE-AC05-00OR22725
UT-Battelle
National Science Foundation	2017219379, ACI-1548562
National Science Foundation
National Institutes of Health	R01-GM123169
National Institutes of Health
National Institute of Allergy and Infectious Diseases
Oak Ridge National Laboratory
Division of Intramural Research, National Institute of Allergy and Infectious Diseases	ZIAAI001105, ZIA AI000931-18
Division of Intramural Research, National Institute of Allergy and Infectious Diseases
Medical Research Council	MR/S009272/1
Medical Research Council
Biotechnology and Biological Sciences Research Council	BB/N000951/1, BB/M011151/1
Biotechnology and Biological Sciences Research Council

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title = "β-Barrel proteins tether the outer membrane in many Gram-negative bacteria",

abstract = "Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun{\textquoteright}s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We used a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.",

author = "Sandoz, {Kelsi M.} and Moore, {Roger A.} and Beare, {Paul A.} and Patel, {Ankur V.} and Smith, {Robert E.} and Marshall Bern and Hyea Hwang and Cooper, {Connor J.} and Priola, {Suzette A.} and Parks, {Jerry M.} and Gumbart, {James C.} and St{\'e}phane Mesnage and Heinzen, {Robert A.}",

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AU - Moore, Roger A.

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AU - Patel, Ankur V.

AU - Smith, Robert E.

AU - Bern, Marshall

AU - Hwang, Hyea

AU - Cooper, Connor J.

AU - Priola, Suzette A.

AU - Parks, Jerry M.

AU - Gumbart, James C.

AU - Mesnage, Stéphane

AU - Heinzen, Robert A.

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β-Barrel proteins tether the outer membrane in many Gram-negative bacteria

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