Proteins in the periplasmic space and outer membrane vesicles of Rhizobium etli CE3 grown in minimal medium are largely distinct and change with growth phase.

Rhizobium etli CE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and the periplasmic space were isolated from 6 and 24 h cultures and identified by proteome analysis. A total of 770 proteins were identified: 73.8 and 21.3 % of these occurred only in the periplasm and OMVs, respectively, and only 4.9 % were found in both locations. The majority of proteins found in either location were present only at 6 or 24 h: in the periplasm and OMVs, only 24 and 9 % of proteins, respectively, were present at both sampling times, indicating a time-dependent differential sorting of proteins into the two compartments. The OMVs contained proteins with physiologically varied roles, including Rhizobium adhering proteins (Rap), polysaccharidases, polysaccharide export proteins, auto-aggregation and adherence proteins, glycosyl transferases, peptidoglycan binding and cross-linking enzymes, potential cell wall-modifying enzymes, porins, multidrug efflux RND family proteins, ABC transporter proteins and heat shock proteins. As expected, proteins with known periplasmic localizations (phosphatases, phosphodiesterases, pyrophosphatases) were found only in the periplasm, along with numerous proteins involved in amino acid and carbohydrate metabolism and transport. Nearly one-quarter of the proteins present in the OMVs were also found in our previous analysis of the R. etli total exproteome of MM-grown cells, indicating that these nanoparticles are an important mechanism for protein excretion in this species.

Qualitative changes in proteins contained in outer membrane vesicles produced by Rhizobium etli grown in the presence of the nod gene inducer naringenin.

In this work, we compared the proteomic profiles of outer membrane vesicles (OMVs) isolated from Rhizobium etli CE3 grown in minimal medium (MM) with and without exogenous naringenin. One-hundred and seven proteins were present only in OMVs from naringenin-containing cultures (N-OMVs), 57 proteins were unique to OMVs from control cultures lacking naringenin (C-OMVs) and 303 proteins were present in OMVs from both culture conditions (S-OMVs). Although we found no absolute predominance of specific types of proteins in the N-, C- or S-OMV classes, there were categories of proteins that were significantly less or more common in the different OMV categories. Proteins for energy production, translation and membrane and cell wall biogenesis were overrepresented in C-OMVs relative to N-OMVs. Proteins for carbohydrate metabolism and transport and those classified as either general function prediction only, function unknown, or without functional prediction were more common in N-OMVs than C-OMVs. This indicates that naringenin increased the proportion of these proteins in the OMVs, although NodD binding sites were only slightly more common in the promoters of genes for proteins found in the N-OMVs. In addition, OMVs from naringenin-containing cultures contained nodulation factor.

The naringenin-induced exoproteome of Rhizobium etli CE3.

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.

Thiamine limitation determines the transition from aerobic to fermentative-like metabolism in Rhizobium etli CE3.

Both thiamine and biotin when added to minimal medium subcultures reversed the fermentative-like metabolism exhibited by Rhizobium etli CE3. Thiamine auxotrophs lacking thiCOGE genes were used to investigate the role of thiamine in this medium. A thiC1169::miniTn5lacZ1 thiamine auxotroph subjected to the above subcultures resulted in growth arrest, reduced pyruvate-dehydrogenase activity, and a smaller amount of poly-beta-hydroxybutyrate compared with the CE3 strain. Moreover, thiC and thiEb genes were overexpressed as result of thiamine limitation. The absence of classical thi genes suggests that thiamine is synthesized with low efficiency by an alternative pathway. Low levels of thiamine cause the CE3 strain to exhibit a fermentative-like metabolism.

Role of GOGAT in carbon and nitrogen partitioning in Rhizobium etli.

The isolation and characterization of a Rhizobium etli glutamate auxotroph, TAD12, harbouring a single Tn5 insertion, is reported. This mutant produced no detectable glutamate synthase (GOGAT) activity. The cloning and physical characterization of a 7.2 kb fragment of R. etli DNA harbouring the structural genes gltB and gltD encoding the two GOGAT subunits GltB and GltD is also reported. In comparison with the wild-type strain (CFN42), the GOGAT mutant strain utilized less succinate and glutamate and grew less with this and other amino acids as nitrogen source. R. etli assimilates ammonium by the glutamine synthetase (GS)-GOGAT pathway and a GOGAT mutant prevents the cycling of glutamine by this pathway, something that impairs nitrogen and carbon metabolism and explains the decrease in the amino-nitrogen during exponential growth, with glutamate as nitrogen source. GOGAT activity also has a role in ammonium turnover and in the synthesis of amino acids and proteins, processes that are necessary to sustain cell viability in non-growing conditions. The assimilation of ammonium is important during symbiosis and glutamate constitutes 20-40% of the total amino-nitrogen. In symbiosis, the blockage of ammonium assimilation by a GOGAT mutation significantly decreases the amino-nitrogen pool of the bacteroids and may explain why more N(2) is fixed in ammonium, excreted to the plant cell, transported to the leaves and stored in the seeds.