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

SIMPLE SUMMARY: The second messenger cyclic diguanylate (c-di-GMP, cdG) is a bacterial lifestyle-switch molecule, well known for its role in biofilm formation. Extracellular biofilm matrix components include diverse biopolymers such as polysaccharides, nucleic acids, proteins and lipids. The production and/or secretion of many these matrix biopolymers can be directly or indirectly regulated by cdG. We studied the extracellular proteome of a Rhizobium etli strain expressing artificially high levels of intracellular cdG. We found that, in addition to promoting the secretion of various extracellular proteins likely involved in adhesion and biofilm formation, high cdG levels also promote the export of cytoplasmic proteins (ECP) to the cell exterior. Intriguingly, most these cytoplasmic proteins have been previously described as moonlighting or multifunctional proteins in other organisms, often found extracellularly or at the bacterial cell surface. We obtained evidence that this ECP may involve an active process that would be enhanced by cdG. For a typical cytoplasmic protein, glyceraldehyde 3-phosphate dehydrogenase (Gap), we also observed that cdG increases the number of extracellular Gap proteoforms that can be separated by two-dimensional gel electrophoresis. The results suggest that cdG promotes the active exportation of cytoplasmic proteins through yet unknown mechanisms involving the post-translational modification of proteins. 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.