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A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound.

ABSTRACT: TonB-dependent receptors (TBDRs) mediate substrate-specific transport across the outer membrane, utilizing energy derived from the proton motive force transmitted from the TonB-ExbB-ExbD complex located in the inner membrane (TonB system). Although a number of TonB systems involved in the uptake of siderophores, vitamin B12 and saccharides have been identified, their involvement in the uptake and catabolism of aromatic compounds was previously unknown. Here, we show that the outer membrane transport of a biphenyl compound derived from lignin is mediated by the TonB system in a Gram-negative bacterium capable of degrading lignin-derived aromatic compounds, Sphingobium sp. strain SYK-6. Furthermore, we found that overexpression of the corresponding TBDR gene enhanced the uptake of this biphenyl compound, contributing to the improved rate of platform chemical production. Our results will provide an important basis for establishing engineered strains optimized for use in lignin valorisation.

SUBMITTER: Fujita M

PROVIDER: S-EPMC6874591 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound.

Fujita Masaya M Mori Kosuke K Hara Hirofumi H Hishiyama Shojiro S Kamimura Naofumi N Masai Eiji E

Communications biology 20191122

TonB-dependent receptors (TBDRs) mediate substrate-specific transport across the outer membrane, utilizing energy derived from the proton motive force transmitted from the TonB-ExbB-ExbD complex located in the inner membrane (TonB system). Although a number of TonB systems involved in the uptake of siderophores, vitamin B12 and saccharides have been identified, their involvement in the uptake and catabolism of aromatic compounds was previously unknown. Here, we show that the outer membrane trans ...[more]

PMID: 31799434

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Project description:We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB(+) bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepADelta3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

Project description:Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer, a Gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes hemoglobin-derived hemin as an iron source; however, the mechanism by which this bacterium acquires hemin from hemoglobin is largely unknown. Here, rhuA disruption was shown to impair iron utilization from duck hemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane hemin-binding protein, but it could not extract hemin from duck hemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A, and RhuAH149A lost hemin-binding ability, suggesting that amino acid sites at tyrosine 144 (Y144), Y177, and histidine 149 (H149) are crucial for hemin binding. Furthermore, rhuR, the gene adjacent to rhuA, encodes a TonB2-dependent hemin transporter. The function of rhuA in duck hemoglobin utilization was abolished in the rhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1 ΔrhuA rather than R. anatipestifer CH-1 ΔrhuR ΔrhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA hemin utilization locus exhibits no similarity to those of characterized hemin transport systems. Thus, this locus is a novel hemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum. IMPORTANCE In vertebrates, hemin from hemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain hemin from hemoglobin, but the mechanisms of hemin acquisition from hemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of hemin acquisition from mammalian hemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in hemin acquisition from duck hemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane hemin-binding protein, and RhuR was identified as a TonB2-dependent hemin transporter. Moreover, the function of RhuA in hemoglobin utilization is RhuR dependent and not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel hemin transportation system of Gram-negative bacteria. This study not only was important for understanding hemin uptake in R. anatipestifer but also enriched the knowledge about the hemin transportation pathway in Gram-negative bacteria.

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A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound.

Publications

A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound.

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