Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

Davinia Salvachúa; Allison Z. Werner; Isabel Pardo; Martyna Michalska; Brenna A. Black; Bryon S. Donohoe; Stefan J. Haugen; Rui Katahira; Sandra Notonier; Kelsey J. Ramirez; Antonella Amore; Samuel O. Purvine; Erika M. Zink; Paul E. Abraham; Richard J. Giannone; Suresh Poudel; Philip D. Laible; Robert L. Hettich; Gregg T. Beckham

doi:10.1073/pnas.1921073117

New Research In

Edited by Alexis T. Bell, University of California, Berkeley, CA, and approved February 27, 2020 (received for review December 1, 2019)

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Significance

The valorization of the plant polymer lignin is critical to enable the bioeconomy, but the heterogeneity of lignin presents a barrier to its use. Natural microbial conversion processes funnel aromatic compound mixtures to single products and thus, have emerged as a means to overcome lignin heterogeneity. Accordingly, understanding the mechanisms that bacteria use to convert lignin degradation products is of importance for their eventual industrial application. Here, we demonstrate that a promising bacterial chassis for lignin-relevant synthetic biology, Pseudomonas putida, secretes outer membrane vesicles that turn over aromatic compounds extracellularly. From this work, we propose a mechanism for extracellular nutrient acquisition from aromatic compounds by soil bacteria, which holds promise for improving the efficiency of microbial lignin conversion.

Abstract

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

Footnotes

↵¹D.S., A.Z.W., I.P., and M.M. contributed equally to this work.
↵²To whom correspondence may be addressed. Email: gregg.beckham{at}nrel.gov or hettichrl{at}ornl.gov.

Author contributions: D.S., A.Z.W., I.P., M.M., B.S.D., P.E.A., R.J.G., P.D.L., R.L.H., and G.T.B. designed research; D.S., A.Z.W., I.P., M.M., B.A.B., B.S.D., S.J.H., R.K., S.N., K.J.R., A.A., S.O.P., E.M.Z., P.E.A., R.J.G., and S.P. performed research; D.S., A.Z.W., I.P., M.M., B.S.D., R.K., K.J.R., A.A., S.O.P., P.E.A., R.J.G., S.P., P.D.L., R.L.H., and G.T.B. analyzed data; and D.S., A.Z.W., I.P., and G.T.B. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Data deposition: The proteomics data from Pseudomonas putida, Rhodoccocus jostii, and Amycolatopsis sp. have been deposited in the MassIVE database (accession no. MSV000084524) and the ProteomeXchange database (accession no. PXD016114). The proteomics data from P. putida OMV and VFS are available at the MassIVE database (accession no. MSV000084506).
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921073117/-/DCSupplemental.