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

  • Hermenegildo Taboada
  • Michael F. Dunn
  • Niurka Meneses
  • Carmen Vargas-Lagunas
  • Natasha Buchs
  • Andrés Andrade-Domínguez
  • Sergio EncarnaciónEmail author
Original Paper


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.


Protein secretion Exoproteome Naringenin Nodulation factors Outer membrane vesicles Rhizobium-legume symbiosis 



We thank Dr. Carmen Quinto (Instituto de Biotecnología-UNAM) for providing R. etli strain UBP102. Part of this work was supported by CONACyT Grant 220790 and DGAPA-PAPIIT Grants IN213216 and IN207519.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

203_2019_1682_MOESM1_ESM.pdf (77 kb)
Fig. S1. Maldi-TOF mass spectrometry analysis of n-butanol extracts of OMVs and supernatant from R. etli CE3 and a CE3 nodA mutant. The strains were grown in of two liters of MM inoculated to an initial optical density of 0.5 at 540 nm OMVs were purified as described in Methods, extracted with n-butanol and the extract dried by a lyophilization. The dried residue was resuspended in distilled sterile water and analyzed by HPLC on a reverse phase C-18 column (Nova pack C18 3.9x150 mm Waters) with detection at 215 nm using CH3CN 20% as mobile phase. Peak fractions were collected lyophilized, and dissolved in methanol/water (1:1). Five µl samples were injected in the masses spectrophotometer, run was done in a LTQ XL-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) in a positive mode by DIC. Ion masse data were analyzed using the Glycomod (htpp:// data base. The ion m/z peak of 1500.9 for the Nod factor coincided with that reported previously (Cárdenas et al. 1995). (PDF 77 kb)


  1. Armengaud J, Christie-Oleza JA, Clair G et al (2012) Exoproteomics: exploring the world around biological systems. Expert Rev Proteom 9:561–575. CrossRefGoogle Scholar
  2. Bhasin M, Garg A, Raghava GPS (2005) PSLpred: prediction of subcellular localization of bacterial proteins. Bioinformatics 21:2522–2524. CrossRefGoogle Scholar
  3. Boiardi JL, Galar ML, Neijssel OM (1996) PQQ-linked extracellular glucose oxidation and chemotaxis towards this cofactor in rhizobia. FEMS Microbiol Lett 140:179–184. CrossRefGoogle Scholar
  4. Bonnington KE, Kuehn MJ (2014) Protein selection and export via outer membrane vesicles. Biochim Biophys Acta Mol Cell Res 1843:1612–1619. CrossRefGoogle Scholar
  5. Cárdenas L, Domínguez J, Quinto C et al (1995) Isolation, chemical structures and biological activity of the lipo-chitin oligosaccharide nodulation signals from Rhizobium etli. Plant Mol Biol 29:453–464. CrossRefGoogle Scholar
  6. Cevallos MA, Encarnación S, Leija A et al (1996) Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate. J Bacteriol 178:1646–1654CrossRefGoogle Scholar
  7. Chang C, Damiani I, Puppo A, Frendo P (2009) Redox changes during the Legume–Rhizobium symbiosis. Mol Plant 2:370–377. CrossRefGoogle Scholar
  8. Choi D-S, Lee J-M, Park GW et al (2007) Proteomic analysis of microvesicles derived from human colorectal cancer cells. J Proteome Res 6:4646–4655. CrossRefGoogle Scholar
  9. del Vargas MC, Encarnación S, Dávalos A et al (2003) Only one catalase, katG, is detectable in Rhizobium etli, and is encoded along with the regulator OxyR on a plasmid replicon. Microbiology 149:1165–1176. CrossRefGoogle Scholar
  10. Dombrecht B, Heusdens C, Beullens S et al (2005) Defence of Rhizobium etli bacteroids against oxidative stress involves a complexly regulated atypical 2-Cys peroxiredoxin. Mol Microbiol 55:1207–1221. CrossRefGoogle Scholar
  11. Downie JA (2010) The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol Rev 34:150–170. CrossRefGoogle Scholar
  12. Dunn MF (2015) Key roles of microsymbiont amino acid metabolism in rhizobia-legume interactions. Crit Rev Microbiol 41:411–451. CrossRefGoogle Scholar
  13. Dunn MF (2017) Rhizobial amino acid metabolism: polyamine biosynthesis and functions. In: D'Mello F (ed) The handbook of microbial metabolism of amino acids. CABI International Publishers, pp 352–370Google Scholar
  14. Dunn MF, Encarnación S, Araíza G et al (1996) Pyruvate carboxylase from Rhizobium etli: mutant characterization, nucleotide sequence, and physiological role. J Bacteriol 178:5960–5970CrossRefGoogle Scholar
  15. Elhenawy W, Debelyy MO, Feldman MF (2014) Preferential packing of acidic glycosidases and proteases into bacteroides outer membrane vesicles. MBio. Google Scholar
  16. Emerich DW, Krishnan HB (2014) Symbiosomes: temporary moonlighting organelles. Biochem J 460:1–11. CrossRefGoogle Scholar
  17. Encarnación S, Dunn M, Willms K et al (1995) Fermentative and aerobic metabolism in Rhizobium etli. J Bacteriol 177:3058–3066CrossRefGoogle Scholar
  18. Encarnación S, Guzmán Y, Dunn MF et al (2003) Proteome analysis of aerobic and fermentative metabolism in Rhizobium etli CE3. Proteomics 3:1077–1085. CrossRefGoogle Scholar
  19. Finnie C, Zorreguieta A, Hartley NM, Downie JA (1998) Characterization of Rhizobium leguminosarum exopolysaccharide glycanases that are secreted via a type I exporter and have a novel heptapeptide repeat motif. J Bacteriol 180:1691–1699Google Scholar
  20. Fujishige NA, Lum MR, De Hoff PL et al (2008) Rhizobium common nod genes are required for biofilm formation. Mol Microbiol 67:504–515. CrossRefGoogle Scholar
  21. Godlewska R, Winiewska K, Pietras Z, Jagusztyn-Krynicka EK (2009) Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis. FEMS Microbiol Lett 298:1–11. CrossRefGoogle Scholar
  22. Goedhart J, Röhrig H, Hink MA et al (1999) Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur. Biochemistry 38:10898–10907. CrossRefGoogle Scholar
  23. González V, Santamaría RI, Bustos P et al (2006) The partitioned Rhizobium etli genome: genetic and metabolic redundancy in seven interacting replicons. Proc Natl Acad Sci USA 103:3834–3839. CrossRefGoogle Scholar
  24. Górniak I, Bartoszewski R, Króliczewski J (2018) Comprehensive review of antimicrobial activities of plant flavonoids. Phytochem Rev. Google Scholar
  25. Haurat MF, Elhenawy W, Feldman MF (2015) Prokaryotic membrane vesicles: new insights on biogenesis and biological roles. Biol Chem. Google Scholar
  26. Henderson B, Martin A (2011) Bacterial virulence in the moonlight: multitasking bacterial moonlighting proteins are virulence determinants in infectious disease. Infect Immun 79:3476–3491. CrossRefGoogle Scholar
  27. Henderson B, Martin A (2013) Bacterial moonlighting proteins and bacterial virulence. Curr Top Microbiol Immunol 358:155–213. Google Scholar
  28. Hussain S, Bernstein HD (2018) The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition. J Biol Chem 293:2959–2973. CrossRefGoogle Scholar
  29. Iyer B, Rajput MS, Rajkumar S (2017) Effect of succinate on phosphate solubilization in nitrogen fixing bacteria harbouring chick pea and their effect on plant growth. Microbiol Res 202:43–50. CrossRefGoogle Scholar
  30. Krehenbrink M, Downie JA (2008) Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae. BMC Genomics 9:55. CrossRefGoogle Scholar
  31. Kuehn MJ, Kesty NC (2005) Bacterial outer membrane vesicles and the host—pathogen interaction. Genes Dev. Google Scholar
  32. Lekmeechai S, Su Y-C, Brant M et al (2018) Helicobacter pylori outer membrane vesicles protect the pathogen from reactive oxygen species of the respiratory burst. Front Microbiol 9:1837. CrossRefGoogle Scholar
  33. Liu ST, Lee LY, Tai CY et al (1992) Cloning of angene necessary for gluconic acid production and enhanced mineral phosphate solubilization in Escherichia coli HB101: nucleotide sequence and probable involvement in biosynthesis of the coenzyme pyrroloquinoline quinone. J Bacteriol 174:5814–5819CrossRefGoogle Scholar
  34. López-Gómez M, Cobos-Porras L, Prell J, Lluch C (2016) Homospermidine synthase contributes to salt tolerance in free-living Rhizobium tropici and in symbiosis with Phaseolus vulgaris. Plant Soil 404:413–425. CrossRefGoogle Scholar
  35. López-Lara IM, Geiger O (2001) The nodulation protein NodG shows the enzymatic activity of an 3-oxoacyl-acyl carrier protein reductase. Mol Plant Microbe Interact 14:349–357. CrossRefGoogle Scholar
  36. Mashburn-Warren LM, Whiteley M (2006) Special delivery: vesicle trafficking in prokaryotes. Mol Microbiol 61:839–846. CrossRefGoogle Scholar
  37. Meneses N, Mendoza-Hernández G, Encarnación S (2010) The extracellular proteome of Rhizobium etli CE3 in exponential and stationary growth phase. Proteome Sci 8:1–11. CrossRefGoogle Scholar
  38. Meneses N, Taboada H, Dunn MF et al (2017) The naringenin-induced exoproteome of Rhizobium etli CE3. Arch Microbiol 199:737–755. CrossRefGoogle Scholar
  39. Mongiardini EJ, Ausmees N, Pérez-Giménez J et al (2008) The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation. FEMS Microbiol Ecol 65:279–288. CrossRefGoogle Scholar
  40. Nelson MS, Sadowsky MJ (2015) Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes. Front Plant Sci 6:491. CrossRefGoogle Scholar
  41. Orench-Rivera N, Kuehn MJ (2016) Environmentally controlled bacterial vesicle-mediated export. Cell Microbiol 18:1525–1536. CrossRefGoogle Scholar
  42. Poole P, Ramachandran V, Terpolilli J (2018) Rhizobia: from saprophytes to endosymbionts. Nat Rev Microbiol 16:291–303. CrossRefGoogle Scholar
  43. Resendis-Antonio O, Hernández M, Salazar E et al (2011) Systems biology of bacterial nitrogen fixation: high-throughput technology and its integrative description with constraint-based modeling. BMC Syst Biol 5:120. CrossRefGoogle Scholar
  44. Reyes-Pérez A, del Vargas MC, Hernández M et al (2016) Transcriptomic analysis of the process of biofilm formation in Rhizobium etli CFN42. Arch Microbiol. Google Scholar
  45. Rinaudi LV, Giordano W (2010) An integrated view of biofilm formation in rhizobia. FEMS Microbiol Lett 304:1–11. CrossRefGoogle Scholar
  46. Roche P, Debellé F, Maillet F et al (1991) Molecular basis of symbiotic host specificity in Rhizobium meliloti: nodH and nodPQ genes encode the sulfation of lipo-oligosaccharide signals. Cell 67:1131–1143. CrossRefGoogle Scholar
  47. Roche P, Maillet F, Plazanet C et al (1996) The common nodABC genes of Rhizobium meliloti are host-range determinants. Proc Natl Acad Sci USA 93:15305–15310. CrossRefGoogle Scholar
  48. Russo DM, Williams A, Edwards A et al (2006) Proteins exported via the PrsD-PrsE type I secretion system and the acidic exopolysaccharide are involved in biofilm formation by Rhizobium leguminosarum. J Bacteriol 188:4474–4486. CrossRefGoogle Scholar
  49. Saalbach G, Erik P, Wienkoop S (2002) Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes. Proteomics 2:325–337.;2-W CrossRefGoogle Scholar
  50. Santamaría RI, Bustos P, Sepúlveda-Robles O et al (2014) Narrow-host-range bacteriophages that infect Rhizobium etli associate with distinct genomic types. Appl Environ Microbiol 80:446–454. CrossRefGoogle Scholar
  51. Schwechheimer C, Kuehn MJ (2015) Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13:605–619. CrossRefGoogle Scholar
  52. Supuran C, Capasso C (2017) An overview of the bacterial carbonic anhydrases. Metabolites 7:56. CrossRefGoogle Scholar
  53. Taboada H, Meneses N, Dunn MF et al (2018) 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. Microbiology. Google Scholar
  54. Tatusov RL, Galperin MY, Natale DA, Koonin EV (2000) The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res 28:33–36. CrossRefGoogle Scholar
  55. Tolin S, Arrigoni G, Moscatiello R et al (2013) Quantitative analysis of the naringenin-inducible proteome in Rhizobium leguminosarum by isobaric tagging and mass spectrometry. Proteomics 13:1961–1972. CrossRefGoogle Scholar
  56. Török Z, Horváth I, Goloubinoff P et al (1997) Evidence for a lipochaperonin: association of active protein-folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions. Proc Natl Acad Sci USA 94:2192–2197CrossRefGoogle Scholar
  57. Truchet G, Debellé F, Vasse J et al (1985) Identification of a Rhizobium meliloti pSym2011 region controlling the host specificity of root hair curling and nodulation. J Bacteriol 164:1200–1210Google Scholar
  58. Vázquez M, Dávalos A, de las Peñas A et al (1991) Novel organization of the common nodulation genes in Rhizobium leguminosarum bv. phaseoli strains. J Bacteriol 173:1250–1258CrossRefGoogle Scholar
  59. Wacek TJ, Brill WJ (1976) Simple, rapid assay for screening nitrogen-fixing ability in soybean1. Crop Sci 16:519. CrossRefGoogle Scholar
  60. Yokota N, Kuroda T, Matsuyama S, Tokuda H (1999) Characterization of the LolA-LolB system as the general lipoprotein localization mechanism of Escherichia coli. J Biol Chem 274:30995–30999CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Programa de Genómica Funcional de ProcariotesCentro de Ciencias Genómicas, Universidad Nacional Autónoma de MéxicoCuernavacaMéxico
  2. 2.Mass Spectrometry and Proteomics Laboratory, Department of Clinical ResearchUniversity of BernBernSwitzerland
  3. 3.Faculty of Science, Department of Chemistry and BiochemistryUniversity of BernBernSwitzerland
  4. 4.Noxgen Biotech, Centro Novatec, Palmas No. 1, Fracc. Lomas de CocoyocAtlatlahucanMéxico

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