3 years ago

Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough.

Walian, Wooton, Zeng, Stahl
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 (DvH), a model organism for studies of sulfate-reducing bacteria, no porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of gene DVU0799, one of the most abundant proteins of the DvH 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 were reconstituted into proteoliposomes and examined for permeability against a series of test solutes. Results obtained establish DVU0799 as a pore forming protein with permeability properties similar to those observed for classical bacterial porins such as those of E. coli Taken together, these findings identify this highly abundant OM protein as the major porin of DvH. 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.Importance 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 has 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 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 improved understanding of physiological features that determine their habitat range and supporting activities.

Publisher URL: http://doi.org/10.1128/JB.00286-17

DOI: 10.1128/JB.00286-17

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