Impact of c-di-GMP on the Extracellular Proteome of Rhizobium etli

Abstract

Extracellular matrix components of bacterial biofilms include biopolymers such as polysaccharides, nucleic acids and proteins. Similar to polysaccharides, the secretion of adhesins and other matrix proteins can be regulated by the second messenger cyclic diguanylate (cdG). We have performed quantitative proteomics to determine the extracellular protein contents of a Rhizobium etli strain expressing high cdG intracellular levels. cdG promoted the exportation of proteins that likely participate in adhesion and biofilm formation: the rhizobial adhesion protein RapA and two previously undescribed likely adhesins, along with flagellins. Unexpectedly, cdG also promoted the selective exportation of cytoplasmic proteins. Nearly 50% of these cytoplasmic proteins have been previously described as moonlighting or candidate moonlighting proteins in other organisms, often found extracellularly. Western blot assays confirmed cdG-promoted export of two of these cytoplasmic proteins, the translation elongation factor (EF-Tu) and glyceraldehyde 3-phosphate dehydrogenase (Gap). Transmission Electron Microscopy immunolabeling located the Gap protein in the cytoplasm but was also associated with cell membranes and extracellularly, indicative of an active process of exportation that would be enhanced by cdG. We also obtained evidence that cdG increases the number of extracellular Gap proteoforms, suggesting a link between cdG, the post-translational modification and the export of cytoplasmic proteins.

Cyclic di‐GMP signaling—Where did you come from and where will you go?

“…This common physiological trait of cyclic di-GMP signaling, unique among ubiquitous second messenger signaling molecules, is surprising as the cyclic di-GMP binding sites of receptors can be highly diverse with cyclic di-GMP binding in multiple conformations (Chou & Galperin, 2016). Equally can the components of the extracellular matrix, which include conserved moonlighting ribosomal and glycolytic proteins such as the ribosomal protein elongation factor EF-Tu and glyceraldehyde-3-phosphate dehydrogenase, respectively, be chemically, structurally, and evolutionary unrelated (Lorite et al, 2022). Furthermore, the environmental conditions under which distinct biofilms are formed can vary substantially even within a single isolate (Cole et al, 2014).…”

“…Interference with other associated fundamental traits such as the cell cycle, recombination, and DNA repair and alteration in cell morphology seems to require specific cyclic di-GMP turnover proteins (Fernandez et al, 2018;Gupta et al, 2016;Hwang & Harshey, 2023;Kaczmarczyk et al, 2020;Manikandan et al, 2018). This diversity of biofilm traits and their flexibility to respond to different environmental conditions partly answers the conundrum of the | 567 RÖMLING abundant presence of numerous cyclic di-GMP turnover proteins encoded by bacterial genomes (Amikam & Galperin, 2006;López-Ochoa et al, 2017;Lorite et al, 2022;Roelofs et al, 2015;Römling et al, 2005). Cell lysis and release of DNA moonlighting as extracellular eDNA biofilm matrix component is stimulated at low cyclic di-GMP and cyclic di-AMP levels though (DeFrancesco et al, 2017;Ueda & Wood, 2010).…”