# 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

Hermenegildo Taboada, Niurka Meneses, Michael Dunn, Carmen Vargas-Lagunas, Natasha Buchs, Jaime Castro-Mondragon, Manfred Heller, Sergio Encarnación

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Hermenegildo Taboada, Niurka Meneses, Michael Dunn, Carmen Vargas-Lagunas, Natasha Buchs, et al. 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, 2018, 10.1099/mic.0.000720. [https://doi.org/10.1099/mic.0.000720](https://doi.org/10.1099/mic.0.000720). [hal-01981959](https://hal HAL Id:)

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# 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

Hermenegildo Taboada,¹† Niurka Meneses,¹,²,³† Michael F. Dunn,¹† Carmen Vargas-Lagunas,¹ Natasha Buchs,²
Jaime A. Castro-Mondragón,⁴ Manfred Heller² and Sergio Encarnación¹,.*

## 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 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.

## INTRODUCTION

Bacterial protein secretion is a vital function involving the transport of proteins from the cytoplasm to other cellular locations, the environment or to eukaryotic host cells [1]. Of the proteins synthesized by *Escherichia coli* on cytoplasmic ribosomes, about 22% are inserted into the inner membrane (IM) while 15% are targeted to periplasmic, outer membrane (OM) and extracellular locations [2].

The IM is a phospholipid bilayer that surrounds the cytoplasm. The OM is comprised of an inner leaflet containing phospholipids and lipoproteins and an outer leaflet comprised mostly of lipopolysaccharide (LPS) and also containing proteins such as porins [3]. The periplasmic space of Gram-negative bacteria is delineated by the IM and OM, with a thin peptidoglycan layer attached to both membranes

by membrane-anchored proteins. The periplasm of *E. coli*, for example, contains hundreds of proteins including transporters, chaperones, detoxification proteins, proteases and nucleases [4, 5]. About a dozen specialized export systems for bacterial protein secretion have been described [1]. Gram-negative bacteria also excrete proteins and other substances in outer membrane vesicles (OMVs). Phospholipid accumulation in the OM triggers the formation of these spherical structures, which are composed of a membrane bilayer derived from the bacterial OM [6]. The amount of OMVs produced by a given bacterium varies in response to environmental conditions including growth phase, nutrient sources, iron and oxygen availability, abiotic stress, presence of host cells and during biofilm formation [7]. Depending on the species and growth conditions, OMVs may enclose cytoplasmic, periplasmic and transport proteins, as well as

Received 28 May 2018; Accepted 25 August 2018; Published 25 October 2018

**Author affiliations:** ¹Programa de Genómica Funcional de Procariotes, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos C. P. 62210, México; ²Mass Spectrometry and Proteomics Laboratory, Department of Clinical Research, University of Bern, 3010 Bern, Switzerland; ³Faculty of Science, Department of Chemistry and Biochemistry, University of Bern, 3010 Bern, Switzerland; ⁴Aix Marseille University, INSERM, TAGC, Theory and Approaches of Genomic Complexity, UMR_S 1090, Marseille, France.

*Correspondence: Sergio Encarnación, encarnac@ccg.unam.mx

**Keywords:** protein secretion; outer membrane vesicles; periplasm; exoproteome; *rhizobium*-legume interactions.

**Abbreviations:** IM, inner membrane; MM, minimal medium; OM, outer membrane; OMV, outer membrane vesicle.
†These authors contributed equally to this work.

Three supplementary tables and two supplementary figures are available with the online version of this article.
DNA, RNA and outer membrane-derived components such as LPS and phospholipids. The inclusion of proteins in the OMVs is not random but appears to be determined by specific sorting mechanisms [2–5, 8]. Suggested roles for OMVs include invasion, adherence, virulence, antibiotic resistance, modulation of the host immune response, biofilm formation, intra- and interspecies molecule delivery, nutrient acquisition and signalling [2, 8–12].

Rhizobia are Gram-negative bacteria that reduce atmospheric nitrogen to ammonia in symbiotic association with leguminous plants. The excretion of specific proteins and polysaccharides by rhizobia is an essential component of this process [13]. The alpha-proteobacterium *Rhizobium etli* CE3 establishes a nitrogen-fixing symbiosis with *Pha-seolus vulgaris* (common bean). We have shown that *R. etli* CE3 secretes many proteins during exponential and stationary phase growth in minimal medium cultures [14] and suggested that some of the secreted proteins might be exported in OMVs, although these nanoparticles have not been reported in rhizobia [15]. Mashburn-Warren and Whiteley have hypothesized that hydrophobic rhizobial nodulation (Nod) factors could be packaged in OMVs for delivery to the plant root, where they induce plant responses required for nodulation [10]. OMVs produced by symbiotic rhizobia have not, however, been studied experimentally.

Relatively few studies have been done on OMVs produced by plant-associated bacteria, where these protein and molecule-bearing structures could enhance the benefits obtained by the prokaryote in mutualistic or pathogenic interactions [16, 17]. Our major aim in this work was to identify proteins present in purified *R. etli* OMVs obtained from cells grown in culture. Because periplasmic proteins could be (perhaps nonspecifically) incorporated into the OMVs, we also identified proteins in the periplasm of cells grown under the same conditions. 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* [14] are excreted in OMVs.

# METHODS

## Bacterial strains and growth conditions

*Rhizobium etli* strain CE3 was maintained in 15% glycerol stocks prepared from PY-rich medium cultures containing 200 and 20 µg ml⁻¹ of streptomycin and nalidixic acid, respectively. Minimal medium (MM) contained 10 mM each of succinate and ammonium chloride as carbon and nitrogen sources, respectively [14]. *Bacillus subtilis* 168 was obtained from E. Martínez (Centro de Ciencias Genómicas-UNAM, Cuernavaca, México) and maintained on PY-rich medium [14].

## Isolation of periplasmic proteins

A modification of the hypo-osmotic shock technique used with *R. leguminosarum* by Krehenbrink *et al.* [18] was used to isolate *R. etli* periplasmic proteins. To do this, bacterial cells obtained by centrifugation (6000 g) were washed twice with cold 1.0 M NaCl, resuspended in cold sucrose buffer [20% (w/v) sucrose, 1 mM EDTA and 10 µl of protease inhibitor cocktail (#13743200, Roche)] per 10 ml and incubated for 5 min at room temperature. The samples were centrifuged at 6000 g for 10 min at 4 °C, and the pellets were resuspended in cold distilled water for 5 min at room temperature followed by centrifugation as above for 30 min. The supernatants were transferred to fresh tubes and precipitated with 2.5 volumes of cold acetone followed by overnight incubation at –20 °C. The samples were centrifuged at 8000 g for 45 min, and the pellets were washed with 80% acetone and resuspended in urea solubilization buffer (7 M urea, 2% (v/v) CHAPS, 1 mM DTT) and stored at –20 °C until required for analysis.

## Isolation of OMV proteins

Cultures of *R. etli* were grown in 2 L of MM under conditions described previously [14] for 6 and 24 h, reaching optical densities (ODs) of 0.2 and 0.6 at 540 nm (Beckman Coulter DU 800 spectrophotometer), respectively, or approximately 20 and 110 µg ml⁻¹ of protein, respectively, with a standard deviation less than 10% between the two replicates [19]. To isolate OMVs, the cultures were centrifuged at 6000 g for 10 min at 4 °C. The supernatant was filtered through a 0.22 µm filter and concentrated to dryness by lyophilization. Lyophilized samples were resuspended in 1 mM Tris-HCl (pH 8.0) buffer and ultra-centrifuged in transparent tubes (Beckman 14×89 mm), at 100 000 g for 2 h at 4 °C in a SW41 swinging-bucket rotor (Beckman) [20]. The pellet was resuspended in 500 µl of 1 mM Tris-HCl (pH 8.0 buffer), and the proteins were extracted with phenol and kept at –20 °C until required [15].

## Transmission electron microscopy

Samples from different purification stages of the OMV and periplasm isolations were resuspended in 20 mM Tris-HCl (pH 8). Five microlitres of purified OMV samples were applied to 400-mesh copper grids, then 2% acid phosphotungstic was added followed by incubation for 1 min at room temperature. Grids were observed in a JEM1011 (JEOL, Japan) at 100 kilowatts of acceleration voltage.

## SDS-PAGE and protein digestion

The protein concentration of OMVs and periplasm samples was determined by the Bradford method [21]. Gel electrophoresis was carried out on 12.5% resolving gels loaded with 20 µg of total protein per lane. Gels were stained with Coomassie blue and 5 mm-wide gel slices were excised, transferred to micro-centrifuge tubes and covered with 20 µl of 20% ethanol. Gel slices were sequentially washed with 100 mM Tris-HCl (pH 8) and 50% aqueous acetonitrile, then reduced with 50 mM DTT in 50 mM Tris-HCl (pH 8) for 30 min at 37°C. Samples were alkylated with 50 mM
iodoacetamide in 50 mM Tris-HCl (pH 8) for 30 min at 37 °C (in darkness), and incubated for 5 h at 37 °C with trypsin (10 ng µl⁻¹, sequencing grade, Promega, Switzerland) in 20 mM Tris-HCl (pH 8). The tryptic fragments were extracted with 20 µl of 20 % (v/v) formic acid. Two independent experiments to obtain OMV and periplasmic fractions were performed, with 85 % of the exoproteins identified being found in both experiments. Only these proteins are included in the data reported here.

## Mass spectrometry

Peptide sequencing was performed on a LTQ XL-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen; Germany) equipped with a Rheos Allegro nano-flow system with AFM flow splitting (Flux Instruments, Reinach; Switzerland) and a nano-electrospray ion source operated at a voltage of 1.6 kV. Peptide separation was performed on a magic C18 nano column (5 µm, 100 Å, 0.075×70 mm) using a flow rate of 400 nl min⁻¹ and a linear gradient (60 min) from 5 to 40 % acetonitrile in H₂O containing 0.1 % formic acid. Data acquisition was in data-dependent mode on the top five peaks with an exclusion for 15 s. Survey full-scan MS spectra were from 300 to 1800 m/z, with resolution R=60 000 at 400 m/z, and fragmentation was achieved by collision-induced dissociation with helium gas in a LTQ XL-Orbitrap mass spectrometer.

## Protein identification and prediction of subcellular localization

Mascot generic files (mgf) were created by means of a pearl script using Hardklor software, v1.25 (M. Hoopmann and M. MacCoss, University of Washington). MS/MS data (mgf files) were submitted to EasyProt (version 2.3) for a search against the SwissProt database (Rhizobium_Homo_Tryp_-ForRev (20100415) in two rounds. First-round parameters were: parent error tolerance 20 ppm, normal cleavage mode with one missed cleavage, permitted amino acid modifications (fixed CysCAM, variable Oxidation_M), minimal peptide z-score 5, maximum *p*-value 0.01 and AC score of 5. Second round parameters were: parent error tolerance 20 ppm, half-cleaved mode with four missed cleavages, permitted amino acid modifications (variable CysCAM, variable Deamid, variable phos, variable Oxidation_M, variable pyrr) minimal peptide z-score 5, maximum *p*-value 0.01. Protein identifications were accepted only with an AC score of 10, i.e. when two different peptide sequences could be matched. The PSLpred program [22] was used to predict subcellular localization of the proteins. We determined potential protein-protein interactions among the *R. etli* exoproteome using the ProLinks server (http://prl.mbi.ucla.edu/prlbeta/) [23].

## Cytotoxic OMV activity

OMVs purified from 24 h *etli* CE3 were extracted with ethyl acetate. *B. subtilis* 168 was cultured overnight in PY at 30 °C, and cells were washed twice with sterile distilled water and diluted with sterile water to an optical density of 0.05 at 540 nm. Two hundred microlitres of *B. subtilis* cells were

plated on Petri dishes of PY supplemented with 0.1 mM CaCl₂. Whatman filter paper circles (0.5 cm diameter) were placed on the plate and impregnated with 5 µl test samples. The plates were incubated for 48 h at 30 °C before determining the presence of zones of growth inhibition surrounding the paper discs.

# RESULTS AND DISCUSSION

## General characteristics of the *R. etli* periplasmic and OMV fractions

We used proteome analysis to identify proteins in the periplasmic and OMV fractions prepared from *R. etli* cultures grown in MM for 6 and 24 h (OD of 0.2 and 0.6, respectively, at 540 nm; see Methods) (Table S1, available in the online version of this article). Only proteins found in two experimental replicates are included in the dataset (see Methods). The suitability of hypo-osmotic shock protocols similar to that used here to isolate periplasmic proteins has been demonstrated in several rhizobia [18, 24]. The presence of cytoplasmic proteins in periplasmic protein preparations is commonly reported in the literature (see below), and could be an artefact resulting from cell lysis [24]. In *Pseudomonas aeruginosa* periplasm (obtained by spheroplasting), 39 and 19 % of 395 proteins identified were predicted to be cytoplasmic and periplasmic, respectively [25]. The periplasmic proteomes of *Pseudoalteromonas haloplanktis* [26] and *Xanthomonas campestris* pv. *campestris* [27], both obtained by hypo-osmotic shock, contained many cytoplasmic enzymes for carbohydrate and amino acid metabolism, among others. In contrast, over 94 % of the 140 proteins obtained by osmotic shock and identified in the periplasms of *E. coli* strains BL21(DE3) and MG1655 were predicted to be periplasmic [28].

We used the PSLPred program to predict the subcellular localization of *R. etli* proteins. While the localization predictions made with this program are over 90 % accurate for proteins from Gram-negative bacteria [22], we noted that several proteins had an unexpected predicted localization. For example, the ribosomal proteins S12 and L31 were predicted to be periplasmic rather than cytoplasmic. Analysis of the *R. etli* exoproteome using an alternative protein localization prediction program, LocTree3 [29], gave significantly different results in comparison to PSLpred. In the case of the ribosomal proteins described above, LocTree3 predicted that S12 is in fact cytoplasmic, but that L31 was secreted.

Based on PSLpred, the predicted cellular localizations of the 568 proteins found only in *R. etli* periplasm (Table S2) were 57 % cytoplasmic, 25 % periplasmic, 14.9 % IM, 1.8 % extracellular and 1.4 % OM. Importantly, the eight highest-abundance proteins found in the total proteome of *R. etli* CE3 grown in MM [30] under conditions similar to those used in the present study were absent from both the periplasmic and OMV fractions (Table S1). This result argues against significant contamination of the periplasmic and OMV fractions by proteins resulting from cell lysis. Among
the proteins identified in the *R. etli* OMVs and periplasm, all those classed as phosphatases, phosphodiesterases or pyrophosphatases were found only in the periplasm (Table S1), consistent with the biochemically determined localization of these enzymes in other rhizobia [24, 31]. In addition, electron microscopic examination of the cell preparations obtained after hypo-osmotic shock showed that the IMs were still intact (results not shown).

The *R. etli* OMV fraction was obtained by differential centrifugation of culture filtrates. Transmission electron microscopic examination of the OMVs purified from 6 and 24 h cultures showed that the vesicles were spherical and had diameters of 40 to 100 nm, within the size range expected for OMVs [16, 32]. No pili, bacteria, flagella or membrane debris were detected (Fig. 1). SDS-PAGE analysis showed that the OMV protein patterns differed significantly from those of whole-cell extracts (Fig. S1). Although artefactual protein contamination of the OMVs cannot be completely excluded [33], these results are consistent with the proposal that specific protein-sorting mechanisms are important in determining the protein content of bacterial OMVs [3, 10, 34].

For proteins present only in the OMVs (Table 1), 39 and 34% were predicted to be periplasmic and cytoplasmic, respectively, followed by 14% IM, 8% OM and 4% extracel-lular. Although the presence of cytoplasmic and IM proteins as *bona fide* components of bacterial OMVs is controversial [3, 34], they have been found in similar proportions in OMVs from several species [32, 35–38]. In comparison to the periplasm, the OMVs contain 5.7 times the number of OM proteins, consistent with the enrichment of these pro-teins in bacterial OMVs [34]. For the 38 proteins found in both periplasmic and OMV fractions (Table S3), the pre-dicted localizations were biased towards periplasmic pro-teins (53%), followed by cytoplasmic (29%), IM (16%) and OM (3%).

Previously, we identified 383 extracellular proteins in *R. etli* MM culture filtrates (14), which would include OMVs. Ninety of the proteins that occurred exclusively in OMVs or in both periplasm and OMVs (Tables 1 and S3) were also found in the previously determined exoproteome [14]. Thus, nearly one-quarter of the exoproteins identi-fied 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 reveal their presence or absence in a sample.

The 770 proteins identified in the *R. etli* periplasmic and OMV fractions at 6 and/or 24 h (Table S1) represent 12.8% of the 6022 predicted ORFs encoded in its genome. Only 14.2% of these proteins are plasmid-encoded, representing less than half of the 32% of the *R. etli* proteome that is extra-chromosomally encoded. There was no significant dif-ference in the relative proportion of plasmid-encoded

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** OMVs from *Rhizobium etli* CE3 from 24 h. Panel A. Electron micrograph of a culture of strain CE3 showing OMVs (arrows). Panel B. Electron micrograph of purified OMVs.</figcaption></figure>

proteins in the periplasm-only, OMV-only, and in both the periplasm and OMV categories.

Of the 770 proteins identified, 568 and 164 (74 and 21%) occurred exclusively in the periplasm (Table S1) and OMV (Table 1) fractions, respectively. Remarkably, only 4.9% of the total proteins were found in both fractions (Table S3),
<caption>**Table 1.** Proteins occurring in *R. etli* OMVs but absent from the periplasm</caption>

The presence of the protein at 6 and/or 24 h is indicated by a shaded box.

<table><thead><tr><th>Protein(s) and accession number(s)*</th><th>Loc.†</th><th>COG‡</th><th>6 h</th><th>24 h</th></tr></thead><tbody><tr><td>Autoaggregation protein RHE_RS11065</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>Autoaggregation protein RHE_RS12180</td><td>Cyt</td><td>-</td><td></td><td></td></tr><tr><td>Autoaggregation protein RHE_RS23910</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>Hypothetical protein RHE_RS20105</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>Hypothetical protein RHE_RS26095</td><td>IM</td><td>-</td><td></td><td></td></tr><tr><td>Hypothetical proteins RHE_RS04195, RHE_RS04515</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>Hypothetical proteins RHE_RS05200, RHE_RS11420, RHE_RS16465, RHE_RS21050</td><td>Cyt</td><td>-</td><td></td><td></td></tr><tr><td>Hypothetical proteins RHE_RS05795, RHE_RS07780, RHE_RS08450, RHE_RS13310, RHE_RS13575</td><td>Cyt</td><td>-</td><td></td><td></td></tr><tr><td>Hypothetical proteins RHE_RS06290, RHE_RS17695, RHE_RS18165, RHE_RS24115, RHE_RS24445, RHE_RS24870</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>Hypothetical proteins RHE_RS08890, RHE_RS11225</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>Polysaccharidase RHE_RS03110</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>Polysaccharidase RHE_RS13340</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>Porin RHE_RS18285</td><td>OM</td><td>-</td><td></td><td></td></tr><tr><td>Porins RHE_RS06895, RHE_RS12455</td><td>OM</td><td>-</td><td></td><td></td></tr><tr><td>Pseudo RHE_RS31185, RHE_RS31705</td><td>Ext</td><td>-</td><td></td><td></td></tr><tr><td>Right-handed parallel beta-helix repeat-containing protein RHE_RS13345</td><td>Per</td><td>-</td><td></td><td></td></tr><tr><td>RTX toxin RHE_RS09670</td><td>Ext</td><td>-</td><td></td><td></td></tr><tr><td>SPOR domain-containing protein RHE_RS09300</td><td>IM</td><td>-</td><td></td><td></td></tr><tr><td>ATP synthase subunit alpha RHE_RS19810</td><td>IM</td><td>C</td><td></td><td></td></tr><tr><td>ATP synthase subunit B 1 RHE_RS04365</td><td>Cyt</td><td>C</td><td></td><td></td></tr><tr><td>C4-dicarboxylate transporter RHE_RS15195</td><td>IM</td><td>C</td><td></td><td></td></tr><tr><td>Cytochrome b RHE_RS15535</td><td>IM</td><td>C</td><td></td><td></td></tr><tr><td>Cytochrome c family protein RHE_RS13405</td><td>Per</td><td>C</td><td></td><td></td></tr><tr><td>Cytochrome c oxidase subunit II CoxB RHE_RS04795</td><td>IM</td><td>C</td><td></td><td></td></tr><tr><td>Cytochrome c1 family protein RHE_RS15530</td><td>Per</td><td>C</td><td></td><td></td></tr><tr><td>Dihydrolipoyl dehydrogenase RHE_RS19855</td><td>Cyt</td><td>C</td><td></td><td></td></tr><tr><td>F<sub>0</sub>F<sub>1</sub> ATP synthase subunit gamma RHE_RS19805</td><td>IM</td><td>C</td><td></td><td></td></tr><tr><td>NADH dehydrogenase subunit E RHE_RS08225</td><td>Cyt</td><td>C</td><td></td><td></td></tr><tr><td>NADH-quinone oxidoreductase subunit B 1 RHE_RS08200</td><td>Cyt</td><td>C</td><td></td><td></td></tr><tr><td>NADH-quinone oxidoreductase subunit C RHE_RS08205</td><td>Cyt</td><td>C</td><td></td><td></td></tr><tr><td>NADH-quinone oxidoreductase subunit D 1 RHE_RS08215</td><td>Cyt</td><td>C</td><td></td><td></td></tr><tr><td>NADH-quinone oxidoreductase subunit G RHE_RS08240</td><td>Per</td><td>C</td><td></td><td></td></tr><tr><td>NADH-quinone oxidoreductase subunit I 1 RHE_RS08250</td><td>Cyt</td><td>C</td><td></td><td></td></tr><tr><td>NADH-ubiquinone oxidoreductase RHE_RS09650</td><td>Per</td><td>C</td><td></td><td></td></tr><tr><td>NADH:ubiquinone oxidoreductase subunit NDUFA12 RHE_RS09530</td><td>Per</td><td>C</td><td></td><td></td></tr><tr><td>NADP-dependent malic enzyme RHE_RS01970</td><td>IM</td><td>C</td><td></td><td></td></tr><tr><td>Pyruvate dehydrogenase complex E1 component subunit beta RHE_RS09875</td><td>Cyt</td><td>C</td><td></td><td></td></tr><tr><td>Septum formation inhibitor Maf RHE_RS02960</td><td>IM</td><td>D</td><td></td><td></td></tr><tr><td>ABC transporter substrate-binding protein RHE_RS15485</td><td>IM</td><td>E</td><td></td><td></td></tr><tr><td>ABC transporter substrate-binding proteins RHE_RS10970, RHE_RS22990</td><td>Per</td><td>E</td><td></td><td></td></tr><tr><td>Alanine dehydrogenase Ald RHE_RS09050</td><td>Cyt</td><td>E</td><td></td><td></td></tr><tr><td>Amino acid ABC transporter substrate-binding protein RHE_RS07475</td><td>Per</td><td>E</td><td></td><td></td></tr><tr><td>Argininosuccinate synthase RHE_RS20070</td><td>Cyt</td><td>E</td><td></td><td></td></tr><tr><td>Asparagine synthetase B AsnB RHE_RS03835</td><td>IM</td><td>E</td><td></td><td></td></tr><tr><td>Cysteine synthase A CysK RHE_RS01645</td><td>Cyt</td><td>E</td><td></td><td></td></tr><tr><td>Glycine dehydrogenase GcvP RHE_RS11470</td><td>Cyt</td><td>E</td><td></td><td></td></tr><tr><td>NAD-glutamate dehydrogenase RHE_RS20990</td><td>Per</td><td>E</td><td></td><td></td></tr><tr><td>Periplasmic alpha-galactoside-binding protein RHE_RS24485</td><td>Per</td><td>E</td><td></td><td></td></tr><tr><td>Adenylosuccinate lyase RHE_CH02273</td><td>Cyt</td><td>F</td><td></td><td></td></tr><tr><td>Dihydroorotase RHE_RS08400</td><td>Cyt</td><td>F</td><td></td><td></td></tr><tr><td>Multifunctional 2',3'-cyclic-nucleotide 2'-phosphodi-esterase/5'-nucleotidase/3'-nucleotidase-5' RHE_RS18170</td><td>Per</td><td>F</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 1.** cont.</caption><thead><tr><th>Protein(s) and accession number(s)*</th><th>Loc.†</th><th>COG‡</th><th>6 h</th><th>24 h</th></tr></thead><tbody><tr><td>Phosphoribosylaminoimidazolesuccinocarboxamide synthase RHE_RS11645</td><td>Cyt</td><td>F</td><td></td><td></td></tr><tr><td>Ribose-phosphate pyrophosphokinase RHE_RS15465</td><td>IM</td><td>F</td><td></td><td></td></tr><tr><td>ABC transporter permease RHE_RS16190</td><td>IM</td><td>G</td><td></td><td></td></tr><tr><td>ABC transporter substrate-binding protein RHE_RS02490</td><td>Per</td><td>G</td><td></td><td></td></tr><tr><td>Arabinose ABC transporter substrate-binding protein RHE_RS18895</td><td>Per</td><td>G</td><td></td><td></td></tr><tr><td>Carbohydrate ABC transporter substrate-binding protein RHE_RS08805</td><td>Per</td><td>G</td><td></td><td></td></tr><tr><td>Carbohydrate ABC transporter substrate-binding protein RHE_RS10590</td><td>Per</td><td>G</td><td></td><td></td></tr><tr><td>Sugar ABC transporter ATP-binding protein RHE_RS24975</td><td>Per</td><td>G</td><td></td><td></td></tr><tr><td>Sugar ABC transporter substrate-binding protein RHE_RS22625</td><td>Per</td><td>G</td><td></td><td></td></tr><tr><td>Sugar ABC transporter substrate-binding protein RHE_RS26655</td><td>IM</td><td>G</td><td></td><td></td></tr><tr><td>Porphobilinogen synthase RHE_RS07675</td><td>Cyt</td><td>H</td><td></td><td></td></tr><tr><td>Riboflavin synthase RHE_RS07710</td><td>Cyt</td><td>H</td><td></td><td></td></tr><tr><td>Acetyl-CoA carboxylase carboxyl transferase subunit alpha RHE_RS19575</td><td>Cyt</td><td>I</td><td></td><td></td></tr><tr><td>Beta-ketoacyl-ACP reductase RHE_RS20550</td><td>Cyt</td><td>I</td><td></td><td></td></tr><tr><td>Enoyl-[acyl-carrier-protein] reductase RHE_RS04730</td><td>Cyt</td><td>I</td><td></td><td></td></tr><tr><td>Transporter RHE_RS09265</td><td>OM</td><td>I</td><td></td><td></td></tr><tr><td>30S ribosomal protein S12 RHE_RS08550</td><td>Per</td><td>J</td><td></td><td></td></tr><tr><td>50S ribosomal protein L1 RHE_RS08520</td><td>Per</td><td>J</td><td></td><td></td></tr><tr><td>50S ribosomal protein L15 RHE_RS08670</td><td>Cyt</td><td>J</td><td></td><td></td></tr><tr><td>50S ribosomal proteins L22 RHE_RS08600 L24 RHE_RS08630, L30 RHE_RS08665</td><td>Cyt</td><td>J</td><td></td><td></td></tr><tr><td>50S ribosomal protein L31 RHE_RS17920</td><td>Per</td><td>J</td><td></td><td></td></tr><tr><td>Ribonuclease PH RHE_RS01835</td><td>Cyt</td><td>J</td><td></td><td></td></tr><tr><td>Translation initiation factor IF-1 InfA RHE_RS02950</td><td>IM</td><td>J</td><td></td><td></td></tr><tr><td>Transcription elongation factor GreA RHE_RS15185</td><td>Cyt</td><td>K</td><td></td><td></td></tr><tr><td>DNA gyrase subunit A RHE_RS10805</td><td>IM</td><td>L</td><td></td><td></td></tr><tr><td>Integration host factor subunit alpha RHE_RS07825</td><td>Cyt</td><td>L</td><td></td><td></td></tr><tr><td>Chromosome partitioning protein ParA RHE_RS16540</td><td>IM</td><td>M</td><td></td><td></td></tr><tr><td>Complex I NDUFA9 subunit family protein RHE_RS01580</td><td>Cyt</td><td>M</td><td></td><td></td></tr><tr><td>Curlin RHE_RS24865</td><td>Per</td><td>M</td><td></td><td></td></tr><tr><td>d-alanyl-d-alanine carboxypeptidase RHE_RS11305</td><td>Per</td><td>M</td><td></td><td></td></tr><tr><td>Efflux RND transporter periplasmic adaptor subunit RHE_RS18860</td><td>Cyt</td><td>M</td><td></td><td></td></tr><tr><td>Exopolysaccharide glucosyl ketal-pyruvate-transferase RHE_RS16450</td><td>Cyt</td><td>M</td><td></td><td></td></tr><tr><td>GDP-fucose synthetase RHE_RS03860</td><td>IM</td><td>M</td><td></td><td></td></tr><tr><td>Glycosyl transferase RHE_RS16485</td><td>Cyt</td><td>M</td><td></td><td></td></tr><tr><td>MexE family multidrug efflux RND transporter periplasmic adaptor subunits RHE_RS17125, RHE_RS17180 (MexE2)</td><td>IM</td><td>M</td><td></td><td></td></tr><tr><td>Nodulation protein NodT RHE_RS17445</td><td>OM</td><td>M</td><td></td><td></td></tr><tr><td>Organic solvent tolerance protein OstA RHE_RS07410</td><td>Per</td><td>M</td><td></td><td></td></tr><tr><td>Outer membrane protein assembly factor BamA RHE_RS09805</td><td>OM</td><td>M</td><td></td><td></td></tr><tr><td>Peptidoglycan-binding protein RHE_RS09350</td><td>OM</td><td>M</td><td></td><td></td></tr><tr><td>Porin RHE_RS0409</td><td>OM</td><td>M</td><td></td><td></td></tr><tr><td>Sugar ABC transporter substrate-binding protein RHE_RS07970</td><td>Per</td><td>M</td><td></td><td></td></tr><tr><td>Sugar ABC transporter substrate-binding protein RHE_RS16550</td><td>OM</td><td>M</td><td></td><td></td></tr><tr><td>Flagellar basal body rod modification protein FlgD RHE_RS03460</td><td>Per</td><td>N</td><td></td><td></td></tr><tr><td>Flagellar basal body rod proteins FlgF RHE_RS03315, FlgG RHE_RS03345</td><td>Per</td><td>N</td><td></td><td></td></tr><tr><td>Flagellar hook protein FlgE RHE_RS03435</td><td>Per</td><td>N</td><td></td><td></td></tr><tr><td>Flagellar hook-associated protein FlgK RHE_RS03440</td><td>Per</td><td>N</td><td></td><td></td></tr><tr><td>Flagellar hook-associated protein FlgL RHE_RS03445</td><td>Per</td><td>N</td><td></td><td></td></tr><tr><td>Flagellin C protein RHE_RS14380</td><td>Ext</td><td>N</td><td></td><td></td></tr><tr><td>Flagellin RHE_RS03400</td><td>Ext</td><td>N</td><td></td><td></td></tr><tr><td>GlcNAc transferase RHE_RS18450</td><td>OM</td><td>N</td><td></td><td></td></tr><tr><td>Heat-shock protein Hsp20 RHE_RS01855</td><td>Cyt</td><td>O</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 1.** cont.</caption><thead><tr><th>Protein(s) and accession number(s)*</th><th>Loc.†</th><th>COG‡</th><th>6 h</th><th>24 h</th></tr></thead><tbody><tr><td>Metallopeptidase RHE_RS03660</td><td>Ext</td><td>O</td><td></td><td></td></tr><tr><td>Metalloprotease RHE_RS08470</td><td>Cyt</td><td>O</td><td></td><td></td></tr><tr><td>Molecular chaperone SurA RHE_RS07405</td><td>Per</td><td>O</td><td></td><td></td></tr><tr><td>Peptidylprolyl isomerase RHE_RS11115</td><td>Per</td><td>O</td><td></td><td></td></tr><tr><td>Protease modulators RHE_RS14300 (HflC), RHE_RS14305 (HflK)</td><td>Per</td><td>O</td><td></td><td></td></tr><tr><td>Carbonic anhydrase RHE_RS30365</td><td>Cyt</td><td>P</td><td></td><td></td></tr><tr><td>Copper oxidase RHE_RS12890</td><td>Per</td><td>P</td><td></td><td></td></tr><tr><td>Fe<sup>3+</sup> ABC transporter substrate-binding protein RHE_RS13955</td><td>Per</td><td>P</td><td></td><td></td></tr><tr><td>Ferrichrome ABC transporter substrate-binding protein RHE_RS13730</td><td>Per</td><td>P</td><td></td><td></td></tr><tr><td>Hemin ABC transporter substrate-binding protein RHE_RS16725</td><td>Per</td><td>P</td><td></td><td></td></tr><tr><td>ABC transporter RHE_RS11985</td><td>Per</td><td>R</td><td></td><td></td></tr><tr><td>Acyltransferase RHE_RS30975</td><td>Cyt</td><td>R</td><td></td><td></td></tr><tr><td>Membrane protein RHE_RS24075</td><td>Cyt</td><td>R</td><td></td><td></td></tr><tr><td>Outer membrane protein assembly factor BamD RHE_RS14515</td><td>Cyt</td><td>R</td><td></td><td></td></tr><tr><td>RNA-binding protein Hfq RHE_RS09975</td><td>Cyt</td><td>R</td><td></td><td></td></tr><tr><td>Zinc/cadmium-binding protein RHE_RS27255</td><td>Per</td><td>R</td><td></td><td></td></tr><tr><td>DUF992 domain-containing protein RHE_RS22070</td><td>Per</td><td>S</td><td></td><td></td></tr><tr><td>Hypothetical protein RHE_RS00750</td><td>Per</td><td>S</td><td></td><td></td></tr><tr><td>Hypothetical protein RHE_RS06795</td><td>Cyt</td><td>S</td><td></td><td></td></tr><tr><td>Hypothetical protein RHE_RS09255</td><td>IM</td><td>S</td><td></td><td></td></tr><tr><td>Hypothetical protein RHE_RS12895</td><td>Per</td><td>S</td><td></td><td></td></tr><tr><td>L,D-transpeptidase RHE_RS00275</td><td>Per</td><td>S</td><td></td><td></td></tr><tr><td>L,D-transpeptidase RHE_RS04095</td><td>OM</td><td>S</td><td></td><td></td></tr><tr><td>L,D-transpeptidase RHE_RS06695</td><td>Cyt</td><td>S</td><td></td><td></td></tr><tr><td>Membrane protein RHE_RS19980</td><td>Ext</td><td>S</td><td></td><td></td></tr><tr><td>Peptidoglycan-binding protein LysM RHE_RS07230</td><td>Cyt</td><td>S</td><td></td><td></td></tr><tr><td>Polyhydroxyalkanoate synthesis repressor PhaR RHE_RS20540</td><td>Cyt</td><td>S</td><td></td><td></td></tr><tr><td>Restriction endonuclease RHE_RS01175</td><td>IM</td><td>S</td><td></td><td></td></tr><tr><td>Ribosome maturation factor RimP RHE_RS00610</td><td>Cyt</td><td>S</td><td></td><td></td></tr><tr><td>Secretion protein RHE_RS02740</td><td>Cyt</td><td>S</td><td></td><td></td></tr><tr><td>Inosine-5-monophosphate dehydrogenase RHE_RS11270</td><td>Cyt</td><td>T</td><td></td><td></td></tr><tr><td>Conjugal transfer protein TrbB RHE_RS21970</td><td>Cyt</td><td>U</td><td></td><td></td></tr><tr><td>Conjugal transfer proteins RHE_RS21935 (TrbF), RHE_RS21930 (TrbG), RHE_RS21920 (TrbI)</td><td>Per</td><td>U</td><td></td><td></td></tr><tr><td>Hypothetical protein RHE_RS01020</td><td>OM</td><td>U</td><td></td><td></td></tr><tr><td>Hypothetical protein RHE_RS25690</td><td>Per</td><td>U</td><td></td><td></td></tr><tr><td>Preprotein translocase subunit YajC RHE_RS09360</td><td>OM</td><td>U</td><td></td><td></td></tr><tr><td>Protein TolR RHE_RS17705</td><td>IM</td><td>U</td><td></td><td></td></tr><tr><td>VirB4 family type IV secretion/conjugal transfer ATPase RHE_RS21955</td><td>Per</td><td>U</td><td></td><td></td></tr></tbody></table>

*Protein names and accessions from GenBank. Where multiple proteins have the same COG, predicted localization and temporal distribution in periplasm and/or OMV, these are listed as a group.

†Predicted cellular localization based on *in silico* analysis. Cyt, cytoplasmic; Ext, extracellular; IM, inner membrane; Per, periplasmic; OM, outer membrane.

‡COGs (Clusters of Orthologous Groups) represent the following functional groups: minus sign, without COG; C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, co-enzyme metabolism; I, lipid metabolism; J, translation; K, transcription; L, replication and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover, chaperone functions; P, inorganic ion transport and metabolism; R, general functional prediction only; S, function unknown; T, signal transduction; U, intracellular trafficking and secretion.

which argues against the random inclusion of periplasmic proteins in the OMVs during their formation and supports the idea that specific protein-sorting mechanisms are at least partly responsible for determining OMV protein content [34].

The number and identity of periplasmic and OMV proteins produced by bacteria change with culture age and growth conditions [4, 34, 37]. In *R. etli*, we found significant differences in the identities of the proteins present in the periplasm and OMVs at 6 versus 24 h (Table S1). In the
periplasm-only fraction (Table S2), 31 and 45% of the proteins were present only at 6 and 24 h, respectively, and 24% were present at both 6 and 24 h. For the proteins found exclusively in the OMVs (Table 1), 49 and 42% were present only at 6 and 24 h, respectively, and 9% were present at both times. The largely distinct protein profiles for OMVs from log and stationary phase cultures, with relatively few proteins present at both sampling times, indicate a time-dependent differential packaging of proteins into the OMVs. For example, several Cluster Orthologous Groups (COGs) (C, D, I, J, L, O, T and U) comprising proteins found only in OMVs contain a majority of proteins that are present at 6 but not 24 h. What accounts for the disappearance of these proteins between 6 and 24 h? Possibly, these proteins are selectively degraded within the OMVs, or are released from them, as the culture ages. It has been proposed that different sub-populations of OMVs with a distinctive protein content could exist in the same bacterial culture, but this has hardly been addressed experimentally [39].

Proteins without a dedicated transport mechanism might enter the exoproteome by interacting with one or more other proteins that are specifically excreted. We determined potential protein-protein interactions in the *R. etli* proteome using the ProLinks server (http://prl.mbi.ucla.edu/prlbeta/) [23]. While highly probable (P=1.0) interactions were predicted to occur between certain members of the total proteome, none were found among the proteins identified in the periplasm and/or OMVs, even at the lowest probability setting (P=0.4).

## Functional distribution of periplasmic and OMV proteins

An important reason for identifying proteins in the *R. etli* periplasm was to determine whether the OMVs also contained a significant number of these proteins. As mentioned previously, less than 5% of the proteins identified were shared between the two locations. The identity of periplasmic proteins has perhaps been best established in *E. coli*, which contains a wide functional diversity of proteins among the hundreds that are present (4). The *R. etli* periplasmic proteins are also diverse in functional categories (Fig. 2a). The general functional prediction-only COG (R) had the greatest number of proteins (13% of the total), and proteins involved in amino acid and carbohydrate metabolism and transport were also highly represented. With the exception of the COGs for periplasmic proteins having very few or no members (cell cycle control, replication, motility, secondary metabolism and trafficking/secretion), at least 10 proteins were present in the other COGs and had a relatively even (Fig. 2a) numerical distribution among them.

For proteins present in OMVs but absent in the periplasm, those without an assigned COG were the most abundantly represented category, accounting for about 21% of the total (Table 1 and Fig. 2b). About 59% of OMV-exclusive proteins in this COG were present only at 24 h. In comparison, only 5.1% of those present in the periplasm but absent in

OMVs were in this category (Fig. 2a). Note that the majority (61–70%) of proteins without COG present only in the periplasm or only in OMVs were hypothetical proteins. The remainder of the OMV-exclusive proteins were more or less evenly distributed among many of the remaining COGs, with 6 h samples usually having the greatest number of proteins. Over half of the COGs lacked representatives from 6 and/or 24 h, with several COGs (e.g. cell cycle control, coenzyme metabolism, transcription) having a markedly lower number of total proteins (Fig. 2b). These comparisons highlight the fact that distinct temporal and numerical patterns in periplasm and OMV proteins occur during the growth of *R. etli* in culture. The exoproteins in our dataset (Table S1) were not biased towards being the products of *R. etli* genes expressed as part of specific regulons [40–42] or under conditions of biofilm formation [43].

## OMV-localized proteins of physiological interest

Here we mention some of the OMV proteins with potentially important physiological roles. Based on their analysis of proteins reported in a variety of OMV proteomes, Lee *et al.* [44] found that many of the proteins belonged to a relatively limited number of protein functional families, including porins, murein hydrolases, multidrug efflux pumps, ABC transporters, proteases/chaperones, adhesins/invasins and cytoplasmic proteins. Many of the *R. etli* OMV proteins described below fit into one of these categories.

Based on what is known of orthologous proteins in other rhizobia, the Rhizobium-adhering proteins (Rap) and polysaccharidases described below are probably secreted by the *R. etli* PrsDE type I secretion system (T1SS) [45, 46]. The three auto-aggregation/adherence proteins (accessions RHE_RS11065, RHE_RS12180 and RHE_RS23910; Table 1) found in OMVs but not in the periplasm belong to the Rap family and include the sole plasmid-encoded Rap paralogue and two of the four chromosomally encoded Raps found in *R. etli*. These calcium-binding, cell surface-localized proteins are found exclusively in *Rhizobium leguminosarum* biovars and in *R. etli* [47]. They are important in rhizobial autoaggregation in both species, in *R. leguminosarum* bv. trifolii RapA1 influence binding to host cell roots and, when overexpressed, nodulation competitiveness on clover [47–49]. Curlin (RHE_RS24865) forms curli fimbriae, surface proteins that are common in bacteria and that in Enterobacteria are important for attachment to host cells and for biofilm formation [50]. Whether the *R. etli* Rap proteins and curlin in OMVs act as adhesion bridges to surfaces (12) is a topic for future research.

In *R. leguminosarum* bv. *viciae*, the PlyA, B and C polysaccharidases degrade and reduce the molecular mass of *R. leguminosarum* exopolysaccharides (EPS) and also attack carboxymethylcellulose, an analogue of plant cell wall cellulose [45, 51]. A number of similar polysaccharidases are unique to the *R. etli* OMV fraction, namely the right-handed helix repeat-containing pectin-lyase-like PlyB orthologue RHE_RS13345, the contiguously encoded PlyC orthologue RHE_RS13340 and polysaccharidase RHE_RS03110. In
<figure><img src="image_5.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Distribution of periplasm-only (a) and OMV-only proteins (b) by functional category and presence at 6 h, 24 h, or at both 6 and 24 h. COG categories are as described in Table 1.</figcaption></figure>

R. leguminosarum, polysaccharidases PlyA and PlyB have been well characterized while PlyC, which shares a high sequence identity with PlyB has not. PlyA and PlyB are not required for effective symbiosis between R. leguminosarum and pea or vetch [51], but mutants in either polysaccharidase form significantly less biofilm than the parent strain [52]. In R. leguminosarum PlyA and PlyB are secreted by the TISS and diffuse away from the producing cells, but are inactive until they interact with EPS on the surface of cells in the vicinity. Both enzymes are able to cleave nascent but not mature EPS chains, and are not activated by partially purified R. leguminosarum EPS [53]. If the R. leguminosarum polysaccharidases are present in OMVs like their orthologues in R. etli (Table 1), this might facilitate their

delivery to R. leguminosarum cells or host plant roots. That a protein exported by a TISS can be present in OMVs was demonstrated for the *E. coli* α-haemolysin [54].

The R. etli OMVs contained the RTX toxin haemolysin-type calcium-binding protein RHE_RS09670. In R. leguminosarum bv. viciae, an exported protein of the same family, designated NodO, has been shown to bind calcium and may be important for the attachment of bacteria to roots [55]. Other OMV-localized proteins that determine cell surface characteristics include the glucosyl ketal-pyruvate-transferase RHE_RS16450 (a PssM orthologue), the glycosyl transferase RHE_RS16485, peptidoglycan-binding protein LysM and the organic solvent tolerance protein OstA. The last of these has varied roles in membrane synthesis in different
bacteria and is an essential protein in *E. coli* [56]. The L,D-
transpeptidases RHE_RS00275, RHE_RS04095 and
RHE_RS06695 likely catalyse alternative peptidoglycan
cross-linking reactions. In *E. coli*, the alternative cell wall
cross-links introduced by these enzymes are essential for
resistance to certain antibiotics [57]. Antibiotic resistance in
rhizobia is potentially important for their competition with
antibiotic-producing soil organisms [58]. Other cell wall-
modifying enzymes identified in OMVs include the
peptidoglycan-binding protein RHE_RS09350 and the D-
alanyl-D-alanine carboxypeptidase RHE_RS11305.

Many proteins involved in transport or excretion were iden-
tified in the OMVs. Three chromosomally encoded outer
membrane Rhizobium outer membrane protein A (RopA)
orthologues were found in the OMV fraction. The function
of these porins in rhizobia is largely undefined, but it was
determined that the RopA1, RopA2 and RopA3 proteins
found in R. etli OMVs are not involved in copper transport
like the plasmid-encoded RopAe [59], which was not pres-
ent in the exoproteome. In Sinorhizobium meliloti RopA1 is
a major phage-binding site and, presumably due to other, as
yet undefined physiological roles, is essential for cell viabil-
ity [60, 61]. Certain bacteria produce OMVs containing
phage-binding proteins as decoy targets for the virus [62,
63]. In R. leguminosarum bv. viciae, RopA1 and RopA2 are
secreted, along with polysaccharidases PlyA1 and PlyA2, by
the TISS [52]. Other secretion-related OMV proteins
include the preprotein translocase subunit YajC
RHE_RS09360, the ExbD/TolR biopolymer transport family
protein RHE_RS17705 and the VirB4 family type IV secre-
tion/conjugal transfer ATPase RHE_RS21955. Two proteins
annotated as sugar ABC transporter substrate-binding pro-
teins (RHE_RS07970 and RHE_RS16550) have sequence
similarities to proteins involved in polysaccharide export. In
the 24 h OMVs we identified two IM-localized MexE family
multidrug efflux RND (resistance nodulation cell division)
transporter periplasmic adaptor subunits, MexE1 and
MexE2. These are expected to form part of the HlyD (Type
I) multi-drug efflux system. A related protein,
RHE_RS18860, encodes an efflux RND transporter periplas-
mic adaptor subunit. RND efflux pumps contribute to nod-
ulation competitiveness and antimicrobial compound
resistance in S. meliloti [64]. Mex efflux pumps were present
in OMVs from Pseudomonas species [37]. BamA
(RHE_RS09805) is the OM component of the β-barrel
assembly machinery (BAM) responsible for the insertion of
virtually all OM proteins in Gram-negative bacteria [65].
BamD, the outer membrane protein assembly factor that
forms part of the Bam complex, was also present in OMVs.
Although the reconstituted E. coli Bam complex is able to
insert proteins into artificial membrane vesicles [66], it is
not known whether Bam complex components can do the
same in natural OMVs. Porin RHE_RS04090 also has a
sequence indicative of an OM beta-barrel protein. Three
porins (RHE_RS18285, RHE_RS06895 and RHE_RS12455),
all predicted to be OM proteins of the porin family common
in alphaproteobacteria, were present in the OMVs.

Nodulation protein NodT RHE_RS17445 is a chromosom-
ally encoded OM lipoprotein that is not involved in Nod
factor synthesis or transport. A functional nodT is essential
for the viability of R. etli CE3, where NodT is proposed to
play a role in chromosome segregation or maintaining OM
stability rather than as an export pump [67]. The dicarboxy-
late transporter RHE_RS15195 (DctA) is expected to be a
symbiotically essential gene in R. etli, since rhizobial
mutants defective in dicarboxylate transport are unable to
fix nitrogen [68]. Three other ABC transporter substrate-
binding proteins similar to UgpB are probably involved in
glycerol 3-phosphate transport (RHE_RS08805,
RHE_RS24975 and RHE_RS10590). Polyamines are
involved in growth and stress resistance in rhizobia [69],
RHE_RS10970 resembles a lysine/arginine/ornithine-
binding periplasmic protein that could transport polyamine
precursor amino acids into the cell, and RHE_RS22990 is
likely to be a spermidine/putrescine-binding periplasmic
protein (PotD). Certain bacteria package signal molecules
for quorum sensing in OMVs [10, 11, 70]. Transporter
RHE_RS09265 is a FadL orthologue: in rhizobia, these are
important for the uptake of quorum sensing system long-
chain N-acyl-homoserine lactones. These FadL orthologues
are probably involved in transporting long-chain acyl-
homoserine lactones across the OM [71], but the presence
of a FadL transporter in OMVs could provide for their
uptake into vesicles, which could deliver them, perhaps in a
concentrated dose, to recipient cells.

Proteins involved in energy production and conversion rep-
resent more than 11 % of the OMV-exclusive proteins, over
twofold more than among the periplasm-only proteins. For
oxidative phosphorylation, numerous components of
NADH dehydrogenase, cytochrome c, NADH-quinone oxi-
doreductase and ATP synthase were found principally in
the OMVs, although not all of the proteins required to
completely assemble these complexes was present.

Among the cytoplasmic proteins present only in OMVs, we
found Tme, the NADP⁺-specific malic enzyme that in
S. meliloti appears to serve as a secondary pathway for pyru-
vate synthesis during growth on succinate [72]. Porphobili-
nogen synthase is required for the synthesis of tetrapyrrole
pigments such as porphyrin and vitamin B₁₂. Ribonuclease
PH is involved in tRNA processing. Ribosomal protein sub-
units accounted for 2.6 and 4.2 %, respectively, of the R. etli
periplasm- and OM-exclusive proteins, and have been
found in OMVs from E. coli and Neisseria meningitides
[73, 74].

OMV-localized chaperones include the cytoplasmic heat-
shock protein Hsp20 and the periplasmic peptidyl-prolyl
isomerases SurA and RHE_RS11115, the protease modula-
tors HflC and HflK and the RNA chaperone Hfq. The latter
protein has diverse functions in riboregulation in rhizobia
[75].

Finally, because certain of the OMV-localized proteins such
as peptidoglycan-modifying enzymes may have
antimicrobial activity, we assayed for the ability of purified *R. etli* OMVs to inhibit the growth of *Bacillus subtilis*, a soil bacterium that co-exists with *R. etli* and is able to sporulate and resist multiple environmental conditions [76] (Fig. S2). In plate assays, we found that purified OMVs and, especially, ethyl acetate extracts of OMVs, inhibited the growth of *B. subtilis*. It is possible that peptidoglycan-modifying enzymes cause the cytotoxicity of OMVs. No inhibition was observed in assays with *R. etli* MM culture or culture supernatant, or with ethyl acetate.

In summary, we show here that *R. etli* produces OMVs with significant temporal differences in their protein content during growth in culture. The proteins in the OMVs are largely distinct from those of the periplasmic space and include many proteins of physiological interest, including some with known symbiotic roles. The excretion of these proteins in OMVs could give a survival or metabolic advantage to free-living or symbiotically associated *R. etli* cells, and provides an exciting topic of research that we are currently exploring.

**Funding information**

Part of this work was supported by CONACYT grant 220790 and DGAPA-PAPIIT grant IN213216.

**Acknowledgements**

We thank Esperanza Martínez (CCG-UNAM) for supplying *B. subtilis* 168.

**Conflicts of interest**

The authors declare that there are no conflicts of interest.

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