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Title: Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough

Abstract

Due in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a β-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, suchmore » as those of Escherichia coli. Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes. Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core β-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.« less

Authors:
 [1];  [2];  [3]; ORCiD logo [1]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
  2. Univ. of California, Berkeley, CA (United States). Department of Molecular and Cell Biology
  3. Univ. of Washington, Seattle, WA (United States). Department of Civil and Environmental Engineering
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1456963
Grant/Contract Number:  
[AC02-05CH11231]
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Bacteriology
Additional Journal Information:
[ Journal Volume: 199; Journal Issue: 23; Related Information: © 2017 American Society for Microbiology.]; Journal ID: ISSN 0021-9193
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; membrane channel proteins; outer membrane proteins; porins

Citation Formats

Zeng, Lucy, Wooton, Etsuko, Stahl, David A., and Walian, Peter J. Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough. United States: N. p., 2017. Web. doi:10.1128/JB.00286-17.
Zeng, Lucy, Wooton, Etsuko, Stahl, David A., & Walian, Peter J. Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough. United States. doi:10.1128/JB.00286-17.
Zeng, Lucy, Wooton, Etsuko, Stahl, David A., and Walian, Peter J. Tue . "Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough". United States. doi:10.1128/JB.00286-17. https://www.osti.gov/servlets/purl/1456963.
@article{osti_1456963,
title = {Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough},
author = {Zeng, Lucy and Wooton, Etsuko and Stahl, David A. and Walian, Peter J.},
abstractNote = {Due in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a β-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, such as those of Escherichia coli. Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes. Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core β-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.},
doi = {10.1128/JB.00286-17},
journal = {Journal of Bacteriology},
number = [23],
volume = [199],
place = {United States},
year = {2017},
month = {9}
}

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