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. 2022 Dec 26;12(1):44.
doi: 10.3390/biology12010044.

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

María J Lorite  1 , Ariana Casas-Román  1 , Lourdes Girard  2 , Sergio Encarnación  2 , Natalia Díaz-Garrido  3 , Josefa Badía  3   4 , Laura Baldomá  3 , Daniel Pérez-Mendoza  1 , Juan Sanjuán  1
Affiliations

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

María J Lorite et al. Biology (Basel). .

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.

Keywords: adhesins; cyclic diguanylate; extracellular proteins; moonlighting proteins; protein PTM; rhizobia.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Extracellular proteome of R. etli CFN42 under high intracellular levels of c-di-GMP. (A) The increased abundance (IA, green dots) and reduced abundance (RA; red dots) culture supernatants proteins between LR101 (high levels of cdG) and LR102 (physiological levels of cdG) strains, identified by iTRAQ labelling and coupled with MS-based proteomics are represented. Identified proteins qualified as statistically Confident (q-value < 0.01 and ±1.5 fold-change) and Likely (q-value < 0.05 and ±1.5 fold-change) differentially abundant are represented as faint and strong dotted lines, respectively. (B) Predicted subcellular localization of the differentially abundant (IA and RA) proteins identified under high cdG (LR101) by 2D-GE proteomics and iTRAQ labelling coupled with quantitative MS-based proteomics.
Figure 2
Figure 2
Immunodetection of proteins EF-Tu and Gap in supernatants (SN) and cell extracts of cultures of R. etli strains LR101 and LR102. In total, 20 µg of total protein was loaded into each lane.
Figure 3
Figure 3
Subcellular localization of Gap in R. etli by immunocytochemistry and electron microscopy. In both strains, LR101 and LR102, colloidal gold particles, were localized in the cytoplasm of bacterial cells, and are associated with the inner and outer membrane in the periplasmic space and outside the cells. No gold particles were localized in vesicles (V). All scale bars = 200 nm.
Figure 4
Figure 4
Two-dimensional-GE Western Blot and Immunodetection of R. etli Gap. Immunodetection of Gap protein was performed with polyclonal antibodies against R. etli Gap. 80 µg of total proteins from supernatants (SN) or cell extracts from cultures of R. etli LR101 and LR102 were loaded, except for LR102-SN, for which 100 µg of protein was loaded. The pH gradient (3–10) applied in the first dimension is indicated.
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