The naringenin-induced exoproteome of Rhizobium etli CE3

Abstract

Flavonoids excreted by legume roots induce the expression of symbiotically essential nodulation (nod) genes in rhizobia, as well as that of specific protein export systems. In the bean microsymbiont Rhizobium etli CE3, nod genes are induced by the flavonoid naringenin. In this study, we identified 693 proteins in the exoproteome of strain CE3 grown in minimal medium with or without naringenin, with 101 and 100 exoproteins being exclusive to these conditions, respectively. Four hundred ninety-two (71%) of the extracellular proteins were found in both cultures. Of the total exoproteins identified, nearly 35% were also present in the intracellular proteome of R. etli bacteroids, 27% had N-terminal signal sequences and a significant number had previously demonstrated or possible novel roles in symbiosis, including bacterial cell surface modification, adhesins, proteins classified as MAMPs (microbe-associated molecular patterns), such as flagellin and EF-Tu, and several normally cytoplasmic proteins as Ndk and glycolytic enzymes, which are known to have extracellular "moonlighting" roles in bacteria that interact with eukaryotic cells. It is noteworthy that the transmembrane ß (1,2) glucan biosynthesis protein NdvB, an essential symbiotic protein in rhizobia, was found in the R. etli naringenin-induced exoproteome. In addition, potential binding sites for two nod-gene transcriptional regulators (NodD) occurred somewhat more frequently in the promoters of genes encoding naringenin-induced exoproteins in comparison to those ofexoproteins found in the control condition.

Proteins in the periplasmic space and outer membrane vesicles ofRhizobium etliCE3 grown in minimal medium are largely distinct and change with growth phase

“…A major finding was that only a small fraction of the periplasmic proteins were also present in OMVs, which suggests that they are not randomly incorporated into the latter during vesicle formation. Our data also indicate that nearly one-quarter of the previously identified exoproteins produced by R. etli (15) are excreted in OMVs.…”

“…Thus, nearly one-quarter of the exoproteins identified in our previous study were apparently excreted in OMVs. It should be noted that the mass spectrometric methods used for protein identification in this and our previous (14, 15) work do not allow the quantitation of proteins, but only reveals their presence or absence in a sample.…”

“…Lyophilized samples were resuspended in 1 mM Tris-HCl (pH 8.0) buffer and ultracentrifuged in transparent tubes (Beckman 14 X 89 mm), at 100,000 x g for 2 h at 4°C in a SW41 swinging bucket rotor (Beckman). The pellet was resuspended in 500 μl of 1 mM Tris-HCl (pH 8.0 buffer), the proteins were extracted with phenol and kept at -20°C until use (15).…”

Multidisciplinary approaches for studying rhizobium–legume symbioses

“…Additionally, comparative proteomic studies have been used in the identification of putative symbiotic genes (Gomes et al 2012b). Several processes of more direct relevance to SNF have also been studied using proteomics (Table 3), such as the proteomic response to flavonoids (Guerreiro et al 1997;Chen et al 2000b;Hempel et al 2009;da Silva Batista and Hungria 2012;Arrigoni et al 2013;Tolin et al 2013;Gao et al 2015;Meneses et al 2017); and to micro-aerobic or anaerobic conditions (Dainese-Hatt et al 1999). The secretome of rhizobia, with a particular focus on the proteins exported by type III secretion systems, has also been investigated (Saad et al 2005;Rodrigues et al 2007;Hempel et al 2009;Okazaki et al 2009).…”

Transcriptomic Studies of the Effect of nod Gene-Inducing Molecules in Rhizobia: Different Weapons, One Purpose

“…Results obtained by Ramachandran et al [ 42 ] described a common core of rhizosphere-induced genes of Rhizobium leguminosarum biovar viciae 3841. Despite the fact that most of these genes code for proteins with unknown functions (66%), the up-regulation of well-known genes indicates that several physiological processes are enhanced when this rhizobial species colonizes plant roots, such as acid organic metabolism ( dctA and pckA genes, implied in C4-dicarboxylate transport and Phosphoenolyruvate (PEP)-carboxykinase production, respectively), molecular transport ( rmrA , coding for a multi-drug resistance family efflux pump) [ 54 ] and osmotic adaptation ( ndvA , whose product is responsible for the export of cyclic β-1-2-glucans to the bacterial periplasm) [ 55 ]. The activation of this set of genes appears to be the way R. leguminosarum deals with adverse conditions in the plant rhizosphere.…”