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

Abstract

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 770 proteins were identified: 73.8 and 21.3 % of these proteins 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, autoaggregation 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.\n\nIMPORTANCEThe reduction of atmospheric nitrogen to ammonia by rhizobia symbiotically associated with legumes is of major importance in sustainable agricultural. Rhizobia excrete a variety of symbiotically important proteins using canonical secretion systems. In this work, we show that Rhizobium etli grown in culture also excretes proteins in membrane-enclosed structures called outer membrane vesicles (OMVs). This study reports OMV production by rhizobia. Proteins identified in the OMVs included Rhizobium adhering (Rap) and autoaggregation proteins, polysaccharidases, RTX toxins, porins and multidrug efflux proteins. Some of these proteins have important roles in the R. etli-common bean symbiosis, and their packaging into OMVs could deliver them to the environment in a concentrated yet diffusible form protected from degradation. The work described here provides a basis for future studies on the function of rhizobial OMVs in free life and symbiosis.