Микроаэробный маленький белок от теплового шока способствует ризобию лимуминозараму/Писуму симбиозу и взаимодействует с широким диапазоном бактерироидных белков.

INTRODUCTION

Nitrogen-fixing Rhizobium -legume symbiosis is the result of a molecular dialogue established among both partners that leads to the formation of the root nodules containing the symbiosomes, intracellular organelle-like structures in which bacteria are differentiated into bacteroids, the N 2 -fixing form of rhizobia ( 1 , 2 ). Bacteroids express the nitrogenase enzyme converting N 2 to ammonia that is secreted to the plant for amino acid synthesis and, in return, the plant provides carbon sources and energy in the form of dicarboxylic acids ( 2 ). For the establishment of symbiosis, plant- and bacteria-dependent determinants are required to adjust the behavior of both partners ( 3 , 4 ). Inside the nodules, bacteroids address a change in their physiology that leads to an adaptation to nitrogen fixation ( 2 – 4 ). Plants provide an adequate ultra-microaerobic environment in the nodule that is required for the synthesis and functioning of the oxygen-sensitive nitrogenase enzyme. Rhizobia sense the low oxygen signal by an adapted regulatory circuit to adjust the expression of nif and fix genes to this condition ( 5 , 6 ). Legumes of the Inverted Repeat-Lacking Clade (IRLC) ( 7 ), including relevant crops such as alfalfa, vetch, lentil, or pea, produce a family of antimicrobial peptides, called nodule-specific cysteine-rich (NCR) peptides, that induce a terminal bacteroid differentiation ( 8 ). The NCR peptides provoke an alteration of membrane permeability, inhibition of cell division, and DNA amplification coupled to cell elongation in vegetative cells, and similar modifications have been observed in bacteroids ( 9 , 10 ). The cationic NCR peptides have been shown to exhibit antimicrobial activity in vegetative cells leading to a disruption of the integrity of the cell membranes causing a loss of membrane potential ( 11 – 14 ). Bacterial intracellular components have been identified as targets of the NCR peptides in vegetative cells and bacteroids ( 12 ), indicating that these peptides have the potential to modulate the bacteroid physiology. The number and the composition of the NCR peptides family vary among IRLC legumes ( 15 ), and several bacterial adaptations have been proposed to face exposure to NCR peptides. It has been suggested that these peptides play a role in the microsymbiont discrimination and specificity of the symbiosis ( 3 , 8 ).

Rhizobium leguminosarum bv. viciae ( Rlv ) UPM791 strain is a member of the genospecies E included in the recently described Rhizobium leguminosarum species complex ( 16 ). Rlv effectively nodulates IRLC legume genera belonging to the Viciae tribe ( Pisum, Vicia, Lathyrus, and Lens ) ( 17 ). However, it has been shown that the symbiotic performance is not equally effective in all the combinations, and differences associated with the plant genotype have been reported ( 18 ). Likewise, previous studies have shown that the expression of Rlv UPM791 hydrogenase, a metalloenzyme that catalyzes the oxidation of H 2 evolved during the N 2 fixation, is dependent on the legume host, and permissive ( Pisum , Vicia , and Lathyrus ) and non-permissive ( Lens ) hosts have been defined ( 19 ). A proteomics-based approach identified a relevant number of proteins differentially expressed in bacteroids induced by Rlv UPM791 in pea and lentil nodules, among which stress-related proteins, including small heat shock proteins (sHSPs), were identified ( 20 ). Rlv UPM791 encodes seven sHSPs, one of which was observed to be expressed at higher levels in pea bacteroids (RLV_1399) and three of them were overexpressed in lentil bacteroids (RLV_0502, RLV_0817, and RLV_0818) ( 20 ). All the host-dependent sHSPs identified in Rlv UPM791 are encoded in plasmids ( 21 ) suggesting that these proteins might be involved in the adaptation and survival of rhizobia to the symbiotic environment offered by the different legume hosts. Stress-response proteins upregulated in bacteroids were previously identified by transcriptomic and proteomic analyses in nodule bacteria, indicating that bacteroids are subjected to specific stress symbiotic conditions ( 22 – 24 ). The bacterial tolerance responses to sources of stress in the symbiotic environment have been postulated to be critical for survival within the host and symbiotic performance ( 4 , 25 – 27 ). A key trait inside the nodule is the extremely low oxygen tensions, maintained in the low nanomolar range by high-affinity leghemoglobins ( 8 ). Rhizobia adapt to such quasi-anoxic conditions by modifying their electron transport chains through the incorporation of high-affinity terminal oxidases. In the case of Rlv UPM791, the response to microaerobic conditions is carried out through the action of FnrN. Rhizobial FnrN proteins are members of the CRP/FNR superfamily transcriptional regulators. These regulators control gene expression in response to low oxygen concentrations by binding to anaerobox operators in response to the redox status of a haem group bound to the protein ( 6 ). Rlv UPM791 contains two functional copies of FnrN (FnrN1 and FnrN2 [ 28 ]) and an autoregulation mechanism for their binding to promoter anaerobox sequences has been previously demonstrated ( 29 ).

sHSPs are molecular chaperones that maintain protein homeostasis preventing irreversible aggregation of denatured proteins, thus facilitating the substrate protein to be refolded by ATP-dependent chaperone systems ( 30 , 31 ). Alternatively, irreversibly damaged proteins are degraded by ATP-dependent proteolytic systems ( 32 – 34 ). Biochemically, sHSPs are characterized by their low molecular mass (12–43 kDa) and by a conserved α -crystallin domain (ACD) flanked by highly variable and non-conserved N-terminal (NTE) and C-terminal (CTE) extensions ( 35 , 36 ). sHSPs are present in the three domains of life ( 35 ), with a special relevance in multicellular eukaryotes, accounting for more than 30 sHSPs in plants ( 37 , 38 ). By contrast, most bacterial genomes encode only 1–2 sHSPs but, interestingly, legume endosymbiotic bacteria harbor multiple versions of these proteins ( 35 , 37 , 39 ), likely reflecting the different stresses associated with changes in their lifestyle. Previous studies showed that Bradyrhizobium diazoefficiens sHSPs are divided into two classes according to their primary sequence ( 39 ). Class A includes bacterial members closely related to Escherichia coli IbpA and IbpB and highly similar along their sequences. Class B comprises proteins more divergent in sequence and phylogenetic origin, and prokaryotic and eukaryotic members fall into this class.

Functional studies of sHSPs in plant-associated bacteria are scarce. A role of an sHSP in cyst desiccation resistance in Azotobacter vinelandii has been demonstrated ( 40 ), and further works have revealed that these proteins promote Agrobacterium tumefaciens virulence through the stabilization of a component of the virulence system ( 41 ). In addition, several heat-regulated sHSPs whose expression is under the control of ROSE (repression of heat shock gene expression) elements have been identified in rhizobia ( 32 , 39 ), but the connection of the regulation of these proteins with the symbiosis had not been identified. Previous studies have identified a significant number of cytosolic proteins involved in diverse cellular functions interacting with sHSPs under heat-stress conditions ( 32 , 39 ), but the analysis of their interactors in legume bacteria has not been addressed so far. In this work, the role of sHSP RLV_1399 in the optimization of the symbiosis of Rlv UPM791 with pea plants has been demonstrated. In addition, the rlv_1399 gene is transcribed from an FnrN-dependent promoter activated under microaerobic free-living conditions. Overexpression of RLV_1399 improves microaerobic culture tolerance to the presence of hydrogen peroxide or antimicrobial NCR peptides suggesting that this protein might protect rhizobia against adverse nodule conditions. The identification of a wide range of bacteroid proteins as co-eluting with RLV_1399 indicates that sHSPs might be a general binding protein stabilizing denatured proteins under symbiotic conditions. The identification of nitrogenase structural subunits as the main proteins associated with this sHSP suggests a relevant role of RLV_1399 for obtaining optimal levels of nitrogen fixation by the Rhizobium /legume symbiosis.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions

Strains and plasmids used in this study are listed in Table 1 . R. leguminosarum strains were routinely grown at 28°C in yeast mannitol broth (YMB) ( 42 ), tryptone-yeast extract (TY) ( 43 ), Rhizobium minimal (Rmin) medium ( 44 ), or universal minimal salt (UMS) medium ( 45 ). E. coli strains were grown at 37°C in LB medium ( 46 ). E. coli DH5 α was used for standard cloning procedures ( 47 ) and E. coli S17.1 ( 48 ) for conjugative plasmid transfer between E. coli and R. leguminosarum . Antibiotic concentrations used were as follows (µg mL −1 ): ampicillin, 100; kanamycin, 50; gentamicin, 5; tetracycline, 5 (for R. leguminosarum ) or 10 (for E. coli ).

Determination of bacterial tolerance to oxidative stress and NCR peptides

To study tolerance to oxidative stress, bacterial cultures were grown aerobically in a UMS medium up to OD 600 ≈ 0.6 and diluted in a fresh medium to an initial OD 600 = 0.1. Cell cultures were incubated in 100-well Honeycomb plates (Growth Curves Ltd., Piscataway, New Jersey, United States) with constant, double orbital shaking in a Bioscreen C Pro device (Growth Curves Ltd., Piscataway, New Jersey, United States) at 28°C and 1% O 2 , with optical density measurement intervals of 30 min for 144 h.

To carry out NCR peptides tolerance assays, four NCR peptides in processed form were chemically synthesized (Proteogenix): two peptides identified in pea nodules (G35 and G39) and two identified in lentil nodules (L36 and L40) and showing the highest levels of expression in these legumes ( 20 ). Sequences of the peptides are included in Table S1 . The lyophilized peptides were resuspended in DMSO at 28 mg mL −1 as stock concentrations and diluted with ultrapure water at the required working concentrations. Bacterial cultures were grown microaerobically (1% O 2 ) in UMS medium as described ( 53 ) up to OD 600 = 0.1. Then, microaerobic cultures were diluted in fresh medium to OD 600 = 0.01, and NCR peptides were added at the final concentrations assayed. The mix was incubated for 2 h at 28°C, fivefold serially diluted in 0.9% NaCl and, for each dilution, 5 µL were spotted on TY plates. Growth was evaluated after 72 h of incubation at 28°C.

Mutant and plasmid construction

DNA manipulations, including purification, restriction, ligation, agarose gel electrophoresis, PCR amplification, and transformation into E. coli cells were carried out by standard methods ( 54 ). Primers used for mutant and plasmid constructions are included in Table S1 .

A mutant derivative bearing a deletion in the rlv_1399 gene was generated in Rlv UPM791 using the pK18 mob suicide vector ( 50 ). For this purpose, a DNA region internal to rlv_1399 was PCR-amplified using primers rlv_1399_F/rlv_1399_R and genomic DNA from UPM791 as a template. The resulting DNA fragment was cloned in PCR 2.1.-TOPO vector, sequenced, and moved into pK18 mob vector as a BamHI-XbaI restriction fragment. The resulting plasmid, pK18mob.1399, was transferred into Rlv UPM791 by conjugation, and single-crossover-positive colonies were selected by plating on the Rmin medium supplemented with kanamycin. Insertion was verified by PCR using appropriate primers, and the transconjugant strain carrying the mutation in rlv_1399 was named UPM1421.

For the construction of pLMB1399 ST and pBBR1399 ST plasmids, the rlv_1399 gene, including its promoter region, was PCR amplified from Rlv UPM791 genomic DNA using primers PromC_rlv_1399_51_BamHI_F/rlv_1399_51_XbaI_Strep_R and Prom_rlv_1399_KpnI_F/ rlv_1399_51_XbaI_Strep_R, respectively. The reverse primers included the sequence coding for Strep Tag II peptide (WSHPQFEK) for in-frame fusion of the tag sequence to the 3′-end of the gene. PCR products were cloned separately into pBluescript-II KS+ plasmid using BamHI-XbaI or KpnI-XbaI sites and sequenced. Then, the BamHI-XbaI fragment was cloned upstream of the promoterless gusA reporter gene in plasmid pLMB51 ( 51 ) rendering pLM1399 ST plasmid. The KpnI-XbaI restriction fragment was ligated into the broad host range vector pBBR1MCS-5 ( 52 ) resulting in plasmid pBBR1399 ST .

Plant assays

Pea ( Pisum sativum L. cv. Frisson) and lentil ( Lens culinaris cv. Magda) seeds were surface-sterilized, pregerminated, and seedlings were planted in sterilized Leonard jar-type assemblies with vermiculite as substrate and inoculated with 1 mL of early stationary phase bacterial cultures as previously described ( 55 ). Plants were grown in a greenhouse under bacteriologically controlled conditions with a nitrogen-free plant nutrient solution ( 56 ) at 18/25°C (night/day) temperatures and 16/8 h of light/dark photoperiod. 21 and 28 days post-inoculation of pea and lentil plants, respectively, shoots were collected and dried (60°C for 48 h), and shoot dry weight was determined. Shoot total nitrogen content was measured using a TruMac C/N analyzer (Leco Corporation). Nitrogenase activity in nodulated roots was determined by the acetylene reduction assay ( 57 ).

RNA extraction and quantitative real-time RT-PCR

RNA extraction from nodule samples and cDNA synthesis were carried out as previously described ( 58 ). RNA concentration was quantified with a Nanodrop spectrophotometer and tested for possible DNA contamination by PCR using the RNA samples as templates and primers specific for rpoD gen ( Table S1 ). RNA integrity was confirmed by electrophoresis in a 1% agarose gel. Quantitative real-time reverse transcription polymerase chain reactions (qRT-PCR) were performed with the Power SYBR Green master mix (Applied Biosystems) and primers rlv_1399_qPCR_F/rlv_1399_qPCR_R for rlv_1399, rlv_0502_qPCR_F/rlv_0502_qPCR_R for rlv_0502, rlv_0817_qPCR_F/rlv_0817_qPCR_R for rlv_0817, rlv_0818_qPCR_F/rlv_0818_qPCR_R for rlv_0818, hupL_qPCR_F/hupL_qPCR_R for hupL, and rpoD_qPCR_F/rpoD_qPCR_R for rpoD genes ( Table S1 ).

β -Glucuronidase activity assays

β -Glucuronidase activity assays were performed as described ( 59 ) using X-GlcA (5-bromo 4- chloro-3-indolyl- β -D-glucuronide) as substrate, and values of activity were expressed as OD 420 min −1 (mg protein) −1 . Protein contents of vegetative cell cultures were determined following the bicinchoninic acid method ( 60 ) after alkaline digestion of cells at 90°C in 2 N NaOH for 10 min using bovine serum albumin as the standard.

Purification of RLV_1399-StrepTag II fusion protein

Protein purification was carried out from 5 g of pea nodules. Bacteroids were suspended in 4 mL of buffer W (100 mM Tris–HCl, pH 8, 150 mM NaCl) containing a protease inhibitor mixture (Complete-mini; Roche Diagnostics GmbH), and membrane-free soluble extracts were obtained as previously described ( 53 ). Bacteroids were disrupted by three passages through a French pressure cell (SLM Aminco, Silver Spring, MD) at 100 MPa, and soluble fractions were cleared of cell debris and membranes by ultracentrifugation at 135,000 × g at 4°C for 1 h. The soluble extract was added to a 0.2 mL Strep Tactin Superflow column (IBA, Göttingen, Germany) operated by gravity flow. The column was washed 30 times with 0.2 mL of buffer W to remove unbound proteins, and the tagged protein was eluted by the addition of 600 µL (6 × 100 µL) of buffer W supplemented with 2.5 mM D-desthiobiotin. Relevant eluted fractions were collected, pooled, and concentrated using a centrifugal filter device (Amicon Ultra 0.5 mL, 3K).

Protein gel electrophoresis and western immunoblot

Proteins were resolved by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized in gels by Coomassie brilliant blue G-250 staining or by immunological detection by western blot assays as previously described ( 56 ). NifDK and NifH proteins were detected immunologically using antiserum raised against A. vinelandii NifDK (1:8,000 dilution) and NifH (1:4,000 dilution), both a gift from Dr. L. Rubio. Blots were developed using a secondary goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate (1:3,000 dilution) and a chromogenic substrate (bromochloroindolyl phosphate-nitro blue tetrazolium) as recommended by the manufacturer (Bio-Rad Laboratories, Inc. Hercules, CA, USA). For identification of Strep-tag fusion RLV_1399 protein, Strep Tactin conjugated to alkaline phosphatase (1:2,500; IBA, Göttingen, Germany) was used. The images of the Coomassie-stained gels and western blots were analyzed using the ImageJ software ( 61 ).

Protein extract in-gel digestion and proteomic analysis

Protein extracts were subjected to in-gel tryptic digestion and desalted as previously described ( 62 ). The desalted protein digests were dried, resuspended in 10 mL of 0.1% formic acid, and analyzed by RP-LC-MS/MS in an Easy-nLC 1200 system coupled to an ion trap LTQ-Orbitrap-Velos-Pro hybrid mass spectrometer (Thermo Scientific) ( 63 ). Peptide identification from raw data was carried out using the PEAKS Studio Xpro search engine (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada). Database search was performed against Uniprot- Rhizobium leguminosarum bv. viciae UPM791 (7244 entries) + Pisum sativum NCRs (52 entries) (decoy-fusion database). The following constraints were used for the searches: tryptic cleavage after Arg and Lys (semi-specific), up to two missed cleavage sites, and tolerances of 20 ppm for precursor ions and 0.6 Da for MS/MS fragment ions, and the searches were performed allowing optional Met oxidation and Cys carbamidomethylation. False discovery rates (FDR) for peptide spectrum matches (PSM) and protein were limited to 0.01. Only those proteins with at least two unique peptides being discovered from LC/MS/MS analyses were considered reliably identified. A similar procedure was used for the identification of specific proteins in excised bands obtained from gels.

Estimation of protein abundance was carried out through a comparison of RPSM values, calculated as RPSM = (PSM/number of predicted tryptic peptides) × 100 ( 20 ). The number of predicted tryptic peptides for each protein was calculated using the Expasy PeptideCutter tool ( 64 ).

Phylogenetic analysis

Protein sequences obtained from the GenBank database were aligned using Clustal Omega v.1.2.4 ( 65 ). The phylogenetic tree of the protein alignment was inferred using IQ-Tree v.1.6.12 ( 66 ) with model testing parameter (-m TEST).

Statistical analysis

Statistical analysis was performed using an analysis of variance (ANOVA) linear model test, following a completely random design. Multiple comparisons of means were analyzed by Fisher’s protected least significant difference method. The analysis was performed using Statgraphics 19 software.

RESULTS

Phylogenetic analysis of rhizobial sHSPs

Rlv UPM791 genome encodes 7 sHSPs, five of which are encoded in plasmids (RLV_0361, RLV_0502, RLV_0817, RLV_0818, and RLV_1399) and two in the chromosome (RLV_2751 and RLV_6296) ( 21 ). To study the potential diversification in their functional role that might account for the high number of sHSPs, a sequence alignment of sHSPs from Rlv UPM791 and other rhizobia was performed, and a phylogenetic tree was generated. In this analysis, the four sHSPs encoded in the genome of Rhizobium johnstonii 3841 strain, together with the six sHSPs from R. leguminosarum bv. trifolii WSM1325, and the five sHSPs encoded in Rhizobium etli CFN42 and Ensifer meliloti Rm1021, respectively, were included. In addition, 12 sHSPs from B. diazoefficiens USDA110 strain were also considered in the analysis as these proteins have been previously studied to a large extent ( 32 , 39 , 67 ).

The phylogenetic tree has shown two clearly differentiated branches. One branch includes Class A proteins previously described in B. diazoefficiens HspA, HspB, HspD, HspE, and HspH ( 39 ) together with Rlv UPM791 RLV_2751, RLV_6296, RLV_0361, RLV_0502, and RLV_0818 proteins ( Fig. 1 ). On the other hand, RLV_1399 and RLV_0817 were clustered together with B. diazoefficiens HspC and HspF, which were classified as Class B proteins ( 39 ). Most of the rhizobial sHSPs analyzed grouped with B. diazoefficiens Class A proteins which are more conserved, with an average percentage of amino acid identity of 76.9 ± 3.6, bearing shorter N-terminal extensions (31 ± 3 amino acids) ( Fig. S1 ). Class B includes proteins that are more divergent in length and sequence (sequence identity 33.2% ± 5.3%) with longer N-terminal extensions (53 ± 3 amino acids). This branch comprises RLV_1399 and RL1883 (its homolog in R. johnstonii 3841), one R. etli sHSP encoded in the chromosome, and two E. meliloti plasmid-encoded sHSPs ( Fig. 1 ). B. diazoefficiens also contains three additional sHSPs that, together with HspC and HspF, belong to this class. The phylogenetic tree also shows that some sHSPs from different rhizobial species are clustered together on specific branches and correspond to those encoded in the chromosome. In particular, RLV_2751 and RLV_6296 form two distinctive groups that include the corresponding homologs of the Rhizobium species analyzed suggesting that these proteins are highly conserved. Together, these data indicate that Rlv UPM791 sHSPs are spread in different branches that might reflect their potential differential function. In addition, sequence comparison suggests sHSPs duplication and diversification events from an ancestor gene.

RLV_1399 contributes to symbiotic performance in pea plants

The host-dependent sHSP RLV_1399 was shown to be an abundant protein in pea bacteroids, with protein expression levels twofold higher in peas than in lentil bacteroids ( 20 ). In addition, the expression of the gene encoding RL1883, the homolog to RLV_1399 in R. johnstonii 3841, was highly induced in pea bacteroids (>400-fold) in comparison with the levels of expression in free-living cultures ( 24 ). These observations led us to study the potential role of the host-specific RLV_1399 in the symbiosis of Rlv UPM791 with pea and lentil plants. Inoculation of pea plants with UPM791 derivative rlv_1399 -deleted mutant (UPM1421 strain) resulted in a significant decrease on shoot dry weight and nitrogen accumulation values in comparison with those observed with the wild-type strain in this legume ( Table 2 ). On the contrary, the symbiotic performance of the wild-type and mutant strains was statistically similar in lentil plants. The impaired phenotype of Rlv UPM1421 in pea was complemented by the expression of wild-type copies of RLV_1399 in pBBR1399 ST plasmid. It has to be noted that the introduction of extra copies of the rlv_1399 gene into the wild-type strain did not result in an improvement of the symbiotic performance in either legume host, indicating that the levels of the bacterial sHSP in pea and lentil bacteroids are not limiting for optimal symbiotic role. Furthermore, the expression of RLV_1399 from pBBR1399 ST plasmid in wild-type strain resulted in a reduction of its symbiotic performance in pea plants, suggesting that there may be a dosage effect of Rlv1399 expression, with excess expression being detrimental. Altogether, the data indicate a role of the pea-specific sHSP in the symbiotic performance of Rlv UPM791 with pea plants.

To elucidate whether the defective phenotype observed in pea plants inoculated with the Rlv UPM1421 strain had an effect on the nitrogen fixation process, the levels of nitrogenase activity were determined in pea and lentil nodules induced by wild-type and mutant strains. In this experiment, the levels of nitrogenase activity in pea nodules induced by the rlv_1399 mutant were significantly lower than those observed in the wild-type strain (1559.4 ± 70.4 and 1012.2 ± 86.9 nmoles ethylene. h −1 (g nodule fresh weight) −1 for wild-type and mutant strains, respectively). On the other hand, the levels of nitrogenase activity in lentil nodules were similar for both strains (1570.95 ± 80.1 and 1497.45 ± 12.8 nmoles ethylene. h −1 (g nodule fresh weight) −1 for wild-type and mutant strains, respectively). These data indicate that the functional role of sHSP RLV_1399 affects the level of nodule nitrogenase activity in the Rlv UPM791 symbiosis with pea plants.

Regulation analysis of Rlv UPM791 sHSP expression

We first investigated whether the control of the observed host-dependent expression of sHSPs in the Rlv UPM791 strain was exerted at the level of transcription. Quantification of the levels of transcripts corresponding to sHSPs previously identified through proteomic analysis (RLV_1399, RLV_0502, RLV_0817, and RLV_0818) revealed that the expression of rlv_1399 gene was upregulated in pea vs lentil bacteroids, whereas high upregulation in lentil was observed for the three remaining sHSPs ( Fig. 2 ). In these experiments, transcripts of the gene encoding hydrogenase large subunit HupL, known to exhibit a strong host-dependent regulation ( 19 ), showed of 80-fold increase in pea vs lentil expression. These results confirm that the expression of sHSPs is dependent on the legume host and is exerted at the transcriptional level.

To get insight into the regulatory mechanisms governing sHSP expression, we focused our study on the rlv_1399 gene. Analysis of the sequence of the non-coding region 5′ upstream of the rlv_1399 gene revealed the existence of a highly conserved anaerobox palindromic sequence (TTGA-n 6 -TCAA) centered at position −87.5 regarding the translation start site of the gene ( Fig. 3 ). A second imperfect anaerobox was also found further upstream in the promoter region at position −143.5 regarding translation start site.

The identification of a potential FnrN-type promoter upstream of the rlv_1399 gene prompted us to investigate whether rlv_1399 was microaerobically induced in free-living cells in a FnrN-dependent manner. To carry out this study, a transcriptional fusion of the whole gene and its coding region to a promoterless gusA reporter gene was constructed using pLMB51 plasmid. The resulting plasmid, pLMB1399 ST , also contained a Strep Tag sequence at the 3′-end of the cloned gene for monitoring purposes. The plasmid was transferred into UPM791 and DG2 ( fnrN1fnrN2 ) strains by conjugation, and the levels of β -glucuronidase activity in aerobically and microaerobically grown cells were analyzed. The results showed that levels of reporter activity observed in UPM791(pLMB1399 ST ) grown under microaerobic conditions were ca . 50-fold higher than those detected under aerobiosis ( Fig. 4A ). By contrast, the same fusion induced only basal levels of reporter activities in FnrN-deficient DG2 strain grown under microaerobic conditions. Analysis of crude cell extracts through immunoblot using AP-conjugated Strep -Tactin led to the identification of a band with the expected size of sHSP RLV_1399 ST in the wild-type microaerobic cells ( ca . 25 kDa, Fig. 4B ). This band was absent in both the DG2 strain grown under the same conditions and in microaerobic cells of UPM791 carrying empty vector included as negative control. In addition, the specific immunoreactive band was not detected when bacterial cultures of the wild-type and DG2 strains were grown under aerobic conditions. Together, these data indicate that the expression of the rlv_1399 gene is controlled by FnrN in response to low oxygen concentrations under free-living conditions. It has to be noted that an additional, unspecific band of ca . 17 kDa was identified in the immunoblots of the crude cell extracts, likely corresponding to a constitutively expressed biotinylated protein. This band was detected in all the strains including UPM791 (pLMB51), negative control, and FnrN-deficient DG2.

RLV_1399 improves cell tolerance against oxidative stress and NCR antimicrobial peptides

The microsymbiont is exposed to several stress conditions in the nodule, including the presence of reactive oxygen species produced by both plant and bacteria ( 68 , 69 ), and NCR antimicrobial peptides in IRLC-legumes ( 8 ). To study the effect of RLV_1399 protein on the viability of cells exposed to oxidative stress, the Rlv UPM1421 derivative strain harboring pLMB1399 ST plasmid, expressing the protein under the control of its promoter, was grown microaerobically in the presence of H 2 O 2 (2 mM) mimicking the oxidative conditions existing in the nodule. A control strain harboring the empty vector pLMB51 was also included in the analysis. The results indicated that both bacterial strains exhibited similar growth in the absence of the oxidative agent ( Fig. 5 ). The addition of H 2 O 2 (2 mM) resulted in a significant inhibition of growth in both strains. Interestingly, a higher degree of growth inhibition was observed in the UPM1421(pLMB51) strain exposed to H 2 O 2 , whereas the presence of wild-type copies of rlv_1399 gene in pLMB1399 ST plasmid resulted in partial reversion of the mutant phenotype. A similar growth pattern was observed in the wild-type strain in the absence of the oxidative agent, whereas in the presence of H 2 O 2 , a faster recovery was observed associated with the over-expression of RLV_1399 ( Fig. S2 ). These data indicate that sHSP RLV_1399 improves the growth of microaerobic cells exposed to H 2 O 2 , suggesting that this protein might protect the bacteroids against oxidative stress conditions in the nodule.

The potential role of the RLV_1399 protein in the protection of bacterial cultures against NCR peptides was also studied. In these analyses, the most abundant anionic and cationic NCR peptides previously identified to exhibit the highest levels of expression in nodules from pea (G35, pI: 9.03; G39, pI: 5.56) and lentil (L36, pI: 8.11; L40, pI: 6.19) plants ( 20 ) were tested for their antimicrobial activity. For this purpose, microaerobic (1% O 2 ) bacterial cultures were exposed to different concentrations of NCR peptides for 2 h, and cell viability was determined by the drop plate method. The results indicated that the presence of cationic peptides G35 and L36 diminished cell viability in a concentration-dependent manner ( Fig. 6 ). G35 and L36 abolished colony formation in rlv_1399 -deficient UPM1421(pLMB51) strain at 40 µM and 30 µM, respectively. The expression of wild-type copies of the rlv_1399 gene in pLMB1399 ST plasmid resulted in an improvement of cell viability at 20 µM of both cationic peptides. These results indicate that the cationic NCR peptides exhibit antimicrobial activity against Rlv UPM791 and, also, that sHSP RLV_1399 improves the tolerance of bacterial cultures exposed to antimicrobial peptides under these conditions. On the other hand, the anionic peptides G39 and L40 did not show antimicrobial activity against bacterial cultures, even at high peptide concentrations (up to 300 µM, Fig. 6 )

Identification of sHSP RLV_1399-interacting proteins under symbiotic conditions

To further understand the functional role of RLV_1399 in symbiosis, the potential repertoire of proteins protected by this sHSP in pea bacteroids has been studied. To carry out these analyses, pull-down experiments with soluble extracts from bacteroids induced by UPM1421(pBBR1399 ST ) in pea nodules were performed. Soluble fractions were loaded onto Strep Tactin columns, and the pooled desthiobiotin-eluted fractions were concentrated and resolved by SDS-PAGE gels followed by Coomassie blue staining ( Fig. 7A ). Analysis of the Strep -Tactin eluates revealed the presence of multiple bands in the gel. The most prominent band corresponded to the expected size of sHSP RLV_1399 ST ( ca . 25 kDa), which was absent in the eluate from UPM1421(pBBR1MCS-5) included as a negative control. This band was subjected to LC-MS/MS analysis after trypsin digestion. In this analysis, only peptides from RLV_1399 were detected (data not shown).

SDS-PAGE analysis revealed that RLV_1399 ST co-purified with multiple potential candidate targets. The eluate was digested with trypsin and subsequently analyzed by LC-MS/MS. The list of 218 proteins consistently identified in the sHSP complexes from two independent replicates is shown in Table S2, where the number of peptide spectrum matches (PSMs) with the in silico digested proteome deduced from Rlv UPM791 Uniprot database is also included. The 50 most abundant potential sHSP interactors, based on the highest normalized RPSM parameter (see Material and Methods), fell into five main functional categories: nitrogenase-related proteins, stress-related proteins, carbon and nitrogen metabolism, and a variety of proteins involved in essential cellular processes ( Table 3 ).

The shortlist of the highly abundant proteins coeluting with RLV_1399 includes the structural nitrogenase subunits, which were identified as the most abundant potential interactors of this sHSP. In addition, proteins required for nitrogenase function and biosynthesis (Nif and Fix proteins) were also detected in the analysis but at lower levels. Regarding stress-related proteins, other sHSPs (RLV_6296, RLV_0818, RLV_0502, RLV_2751, and RLV_0817) were identified, suggesting that RLV_1399 might form heterocomplexes with these proteins. RLV_0502 and RLV_0817 were not detected in our previous direct proteomic analysis carried out in pea bacteroids ( Table 3 ; [ 20 ]), suggesting that these sHSPs copurified with RLV_1399 are enriched in the eluate fraction. The current analysis has also revealed that RLV_1399 potentially participates in complexes with subunits of ATP-dependent chaperones (GroEL/GroES and DnaK/DnaJ/GrpE) suggesting an involvement of the sHSP in the protein quality control system in bacteroids ( 33 , 36 ). Also, two universal stress proteins (USPs) have been observed to be highly abundant in the proteomic analysis showing that RLV_1399 might participate in a complex stress-response network. Furthermore, a redoxin family protein, which includes proteins involved in maintaining redox homeostasis and protecting cells against oxidative stress ( 70 ), was also identified.

The highly abundant substrate proteins co-eluting with RLV_1399 also included proteins related to carbon and nitrogen metabolism, as a component of the pyruvate dehydrogenase enzyme, and other enzymes as those participating in the tricarboxylic acid cycle (TCA) pathway (succinate-CoA ligase), glyoxylate shunt (succinate semialdehyde dehydrogenase), and extracellular polysaccharides synthesis (UDP-glucose 6-dehydrogenase) ( 71 , 72 ). In addition, enzymes participating in amino acid synthesis and metabolism have been also observed, such as glutamate synthase or D-3-phosphoglycerate dehydrogenase ( 73 ). Furthermore, DNA replication-, transcription-, and translation-related proteins were present in the top list of sHSP-coeluting proteins, including DNA-directed RNA polymerase subunit β , DNA gyrase subunit β , together with ribosomal proteins and elongation factors Tu and G.

Other prominent potential RLV_1399 substrates included the enzyme glutamyl-tRNA reductase involved in the biosynthesis of delta-aminolevulinic acid, the precursor of tetrapyrrole required for haem groups ( 74 ). The potential complex identified might be relevant to keep in a competent state haem proteins as cytochromes, essential for bacteroid respiration, contributing to the survival of the bacteroid as previously suggested ( 20 ). Furthermore, we also detected highly prevalent proteins participating in oxidative respiration, such as components of the ATP synthase, and electron transfer flavoproteins likely required for bacteroid respiration under microaerobic conditions. Our analysis also identified one component of the Suf machinery that mediates the iron-sulfur cluster assembly in proteins involved in a number of biological processes, such as electron transfer, redox sensing, and catalysis, playing an important role under oxidative stress and iron starvation limitation ( 75 ). Furthermore, HupL, the large structural subunit of the NiFe-hydrogenase, was also detected. Finally, it is interesting to note that, although at position 63, a relevant sHSP-binding partner is the cell division protein FstZ, which is required for the formation of contractile Z-ring prior to the cell division ( 76 ).

The data obtained suggest that sHSP RLV_1399 protects a wide diversity of bacteroid proteins against symbiotic stress conditions, being the nitrogenase enzyme one of the most abundant of these potential sHSPs-targets. Given the known role of sHSPs in stabilizing unfolded proteins, we can hypothesize that the “common theme” for this wide range of proteins is their denatured status, likely as a consequence of the different intracellular stresses.

To obtain additional confirmation of the existence of complexes involving the sHSP and NifDKH proteins, desthiobiotin-eluate fractions were further studied by immunoblot analysis using the corresponding antisera. Immunoblot membranes developed with a Strep- Tactin-AP conjugated showed the presence of a strong band of the expected size of RLV_1399 ST protein that was absent in the eluted fraction from UPM1421(pBBR1MCS-5) included as negative control ( Fig. 7B ). Similar immunoblots were performed with NifDK antiserum revealing specific bands coeluted with RLV_1399 ST of the size corresponding to NifD and NifK proteins that were absent in the eluate from the UPM1421 carrying the empty vector. Immunoblot analysis was also carried out with NifH antiserum. This analysis identified the presence of an immunoreactive band of the size of NifH in the soluble and eluate fractions of bacteroids of the strain expressing RLV_1399 ST . This band was also present in the soluble fraction of the strain included as negative control but was not observed in the eluate fraction. These data confirm that RLV_1399 participates in complexes with NifDK and NifH proteins.

We were also interested in determining the level of enrichment of these proteins in the eluate fractions. To carry out these studies, we estimated the percentage of the band intensity from the eluate fraction corresponding to RLV_1399 ST protein in relation to the total proteins from soluble fraction stained by Coomassie blue ( Fig. 7A ), obtaining a value of around 50% ( Fig. 7C ). Further analyses were carried out on the plots generated from the images of the immunoblots. Measurements of plot peak areas relative to the amount of protein subjected to immunoblots analysis resulted in increments of 2.5- and 3.8-fold for NifDK and NifH proteins, respectively, in the eluate fraction in relation to the soluble fraction ( Fig. 7D ). For RLV_1399 ST , this value was also estimated, obtaining increments of 10.7-fold in the eluate fractions.

Most of the proteins detected in the RLV-1399-associated protein extract, including nitrogenase components, had been detected previously in the analysis of a bacteroid full extract ( 20 ). It is interesting to note that the rank of the identified proteins is quite different in both extracts in a number of cases ( Table 3 ; Table S2 ). When different, proteins are usually ranked higher in the co-eluted fraction, which is consistent with an RLV_1399-dependent enrichment.

DISCUSSION

In this work, we study symbiotic sHSPs from Rlv UPM791 as molecular chaperones that might prevent protein aggregation and maintain protein homeostasis in response to specific symbiotic stress conditions within the nodule. The phylogenetic analysis of rhizobial sHSPs reveals a probable evolution by gene duplication followed by sequence divergence from an ancient common ancestor as was stated in sHSP evolution studies from different phyla and plants ( 35 , 38 ).

Our data reveal that the host-dependent sHSP expression is exerted at the transcriptional level. Similar results were obtained with R. johnstonii 3841 homologs for RLV_1399 (RL1883) and RLV_6296 (RL4089), both shown to be differentially upregulated in pea and vetch ( 24 ) and abundant sHSPs in pea and lentil bacteroids in our system ( 20 ). The variable composition of the NCR peptides in IRLC legume nodules ( 15 , 20 , 77 ) suggests that rhizobia might be exposed to different NCR peptides in different hosts as previously reported ( 8 ), and this fact could be connected to the host-dependent stress responses in bacteroids. This is also in line with an earlier report in which upregulated genes encoding E. meliloti homologs to lentil-specific sHSPs RLV_0817 and RLV_0818 were differentially induced in bacterial cultures exposed to NCR247 and NCR355 peptides ( 11 ). In addition, these authors also showed that proteins involved in oxidative stress response (glutathione S-transferase and thioredoxine) were also induced at different levels in an NCR peptide-dependent manner.

Reporter gene analysis has revealed that the rlv_1399 gene is induced under microaerobic conditions in a FnrN-dependent manner. Previous studies showed that the simultaneous disruption of the two fnrN gene copies present in Rlv UPM791 suppressed microaerobic induction of hypBFCDE , encoding hydrogenase accessory genes, and fixNOQP , encoding for cbb3 -type cytochrome oxidase required for bacteroid respiration ( 28 ). The situation is different in the case of R. johnstonii 3841, for which FixK controls microaerobic expression in free-living cells and FnrN acts as the main regulator in symbiosis ( 78 ). Analysis of the relative mRNA levels of Rlv UPM791 fnrN1 and fnrN2 genes revealed that these genes are expressed at different levels in the bacteroid, probably due to their location at different replicons as previously stated ( 21 , 28 , 29 ). We have not observed a host effect on gene transcription of fnrN genes (data not shown). The presence of two anaeroboxes in the promoter region of rlv_1399 might be related with the maintenance of precise sHSP levels in the cells, similar to the autoregulation mechanism described for fnrN1 and fnrN2 genes and shown to provide appropriate levels of these transcriptional regulators in the cell ( 29 ).

To investigate whether anaeroboxes are present in the promoter regions of genes encoding sHSPs in other rhizobial species, a BLAST analysis of the DNA sequence upstream of the rlv_1399 gene and the 100 nucleotides of their coding sequence was performed. The results identified two Fnr-binding sites in the promoter region of genes encoding to RLV_1399 homologous in other Rlv strains (BIHB 1217, RCAM0610, 3841, RCAM2802, and RCAM0626) and R. johnstonii 3841 strain. The presence of two anaeroboxes has been also identified in R. leguminosarum bv. trifolii (4B, 3B, 23B, 22B, RCAM1365, CC275e, 9B, TA1, and 31B strains), R. leguminosarum (Ta6k, Ta9K, Ta6, Ta1k, ATCC14479, and Tp73_4 strains), Rhizobium sp. (C104 and TAL182 strains), and Rhizobium ruizarguesonis TA1 strain. In Rhizobium binae BLR195 strain only, the anaerobox proximal to the translational start site is conserved. The identified genes show a sequence identity of over 90% with RLV_1399 protein. These data suggest that similar regulatory mechanisms of sHSP expression might be conserved in different strains.

It has been previously suggested that FnrN might provide a flexible adaptation to environments in which oxygen concentration changes rapidly ( 21 , 28 , 29 , 79 ). This might allow an effective and rapid expression of RLV_1399 sHSP to stabilize the bacteroid proteome under the ultra-low oxygen tensions prevalent in symbiotic conditions. Heat-regulated expression of several sHSP has been described in bradyrhizobia ( 32 , 39 ), and ROSE elements have been identified in UPM791 lentil-sHSPs, but a similar control of sHSP expression by an Fnr-like protein had not been described so far.

The symbiotically deficient phenotype associated with the rlv_1399 mutant in pea plants suggests that RLV_1399 might be involved in the adaptation and survival of rhizobia to the symbiotic environment in pea nodules. Previous work revealed that a R. johnstonii 3841 mutant in RL1883, the homolog to RLV_1399, was not affected by nitrogen fixation in pea plants ( 24 ). The different phenotypes observed might be related to redundancy of functions and a change in the balance of the sHSP in the bacteroids induced by different strains.

Our data are consistent with previous works that showed that the overexpression of Deinococcus radiodurans sHSP20 and E. coli IbpA/IbpB increases bacterial tolerance to oxidative stress in E. coli ( 80 , 81 ). In addition, a role for sHSP in maintaining redox homeostasis and preventing oxidative enzyme inactivation under these conditions has been also reported ( 81 – 83 ). This is also consistent with the enzymes involved in the response to oxidative stress as potential interactors of RLV_1399 under symbiotic conditions. Regarding the NCR peptides, our results are in line with previous works in which it was observed that the growth of bacterial cultures of E. meliloti exposed to Medicago truncatula cationic NCR peptides was affected in a concentration-dependent manner ( 10 – 12 ). The antimicrobial efficacy of two M. truncatula cationic NCR peptides was previously demonstrated to vary among a wide range of bacteria suggesting either a different mode of action or different sensitivity of proteases to these peptides ( 11 , 13 ). This might explain the different inhibitory concentrations observed for the cationic NCR peptides in Rlv UPM791. The protective role of RLV_1399 might be related to keeping proteins in a soluble state, as antimicrobial activities of NCR peptides have been associated with bacterial targets, including components of the bacterial membranes and a wide range of proteins involved in different cellular functions ( 3 , 8 , 10 , 12 , 13 ) previously shown to be substrates of bacterial sHSPs ( 84 – 88 ). By contrast, anionic NCR peptides did not show detectable antimicrobial activity ( 8 ).

Other sHSPs as potential interactors of RLV_1399 suggest the existence of heterooligomers. Although it has been shown that heterooligomer formation occurs among members of the same class in bacteria ( 67 , 89 ), some human sHSPs have been reported for their ability to interact with members of both classes ( 30 ). Furthermore, the proteomic analysis suggests a potential cooperation involving RLV_1399 in stabilizing denatured proteins and its participation in the cell’s protein quality system, as described for E. coli IbpB ( 33 , 83 , 88 , 90 ). The observation of universal stress proteins as potential interactors of sHSP indicates that their stabilization might be important in the nodule. These proteins have been reported to facilitate colonization and pathogenicity in bacterial pathogens ( 91 – 93 ) and the role of these proteins in an adaptation to the symbiotic environment has been previously suggested ( 20 ). Similar stress responses have been observed under symbiotic conditions and in NCR peptides-treated bacterial cultures. In those cases, upregulated stress-related functions, including those involved in the detoxification of reactive oxygen species, have been identified ( 11 , 23 , 24 ). These responses might operate in our system to face stress conditions required for rhizobia survival in the bacteroid.

Structural nitrogenase subunits (NifKDH) were included among the 50 most abundant in pea-bacteroids ( 20 ). A non-specific interaction with RLV_1399 based only on abundance is unlikely, as 17 of such 50 proteins were not detected as potential substrates of RLV_1399. Although the specific targets of rhizobia sHSP have not been previously studied, the NCR247 complex isolated from M. truncatula bacteroids induced by E. meliloti contained both nitrogenase subunits and GroEL suggesting the existence of an NCR-nitrogenase interaction via GroEL ( 12 ). In addition, GroEL is required for the nitrogenase structural subunit assembly in other bacteria, and a role in the formation of complexes with other Nif proteins involved in FeMoCo synthesis has also been suggested ( 94 – 96 ).

Multiple proteins involved in a diversity of cellular functions were also previously shown to be targeted and protected from thermal inactivation by sHSPs in other bacteria and yeast ( 83 , 86 , 88 , 97 – 99 ). In addition, cell division protein FtsZ was previously shown to interact with IbpA in other bacteria, and an additional role in assisting FtsZ polymerization contributing to cell division under unfavorable conditions was suggested ( 100 ). The observation of this complex in our system is also consistent with the role of NCR247 in cell division inhibition via interaction with FtsZ ( 12 ).

The diversity of targets that potentially bind to RLV_1399 suggests that this protein might be a general binding protein for denatured substrates of proteins originating under microaerobic and symbiotic conditions. It has to be noted, however, that some of the proteins identified might be indirectly bound to RLV_1399 via other proteins that interact directly with the sHSP. Previous reports have stated that the abundance of proteins is not the factor that determines substrate binding to sHSP but it is their tendency to aggregation under stress conditions ( 83 ). In addition, hydrophobic sites from sHSPs have been shown to contribute to substrate interaction, in which non-conserved NTE and CTE play the major role, associated with the promiscuity in the substrate recognition by these proteins ( 30 , 34 , 36 , 101 ). The independent evolution of NTE and CTE ( 35 ) has also been suggested to be responsible for the variations in the substrates interacting with sHSPs from different species ( 30 ).

ACKNOWLEDGMENTS

This work was supported by funds from Spain's MICINN (PID2021-124344OB-I00) project to J.M.P., Spain. L.D.-S. is a recipient of an FPI scholarship (PRE2019-091327) from the MICINN, Spain.

Contributor Information

José Manuel Palacios, Email: jose.palacios@upm.es.

Marta Albareda, Email: marta.albareda@upm.es.

Gladys Alexandre, University of Tennessee at Knoxville, Knoxville, Tennessee, USA.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.01385-24 .

aem.01385-24-s0001.pdf

Sequence alignment of rhizobial sHSPs.

aem.01385-24-s0002.pdf

Effect of RLV_1399 expression on the tolerance of Rlv UPM791 to oxidative stress.

aem.01385-24-s0003.pdf

Peptides and primers used in this work.

aem.01385-24-s0004.xlsx

Proteins identified in sHSP complexes.

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REFERENCES

1. Oldroyd GED. 2013. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 11:252–263. doi: 10.1038/nrmicro2990

2. Poole PS, Ramachandran V, Terpolilli J. 2018. Rhizobia: from saprophytes to endosymbionts. Nat Rev Microbiol 16:291–303. doi: 10.1038/nrmicro.2017.171

3. Mergaert P. 2018. Role of antimicrobial peptides in controlling symbiotic bacterial populations. Nat Prod Rep 35:336–356. doi: 10.1039/c7np00056a

4. Ledermann R, Schulte CCM, Poole PS. 2021. How rhizobia adapt to the nodule environment. J Bacteriol 203:e0053920. doi: 10.1128/JB.00539-20

5. Zamorano-Sánchez D, Girard L. 2015. FNR-like proteins in rhizobia: past and future, p 155–166. In Brujin FJ (ed), Biological nitrogen fixation. John Wiley and Sons, United kingdom.

6. Rutten PJ, Poole PS. 2019. Oxygen regulatory mechanisms of nitrogen fixation in rhizobia. Adv Micro Physiol 75:325–389. doi: 10.1016/bs.ampbs.2019.08.001

7. Wojciechowski MF, Lavin M, Sanderson MJ. 2004. A phylogeny of legumes (Leguminosae) based on analysis of the plastid matK gene resolves many well-supported subclades within the family. Am J Bot 91:1846–1862. doi: 10.3732/ajb.91.11.1846

8. Downie JA, Kondorosi E. 2021. Why should nodule cysteine-rich (NCR) peptides be absent from nodules of some groups of legumes but essential for symbiotic N-fixation in others? Front Agron 3:654576. doi: 10.3389/fagro.2021.654576

9. Mergaert P, Uchiumi T, Alunni B, Evanno G, Cheron A, Catrice O, Mausset AE, Barloy-Hubler F, Galibert F, Kondorosi A, Kondorosi E. 2006. Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis. Proc Natl Acad Sci U S A 103:5230–5235. doi: 10.1073/pnas.0600912103

10. Van de Velde W, Zehirov G, Szatmari A, Debreczeny M, Ishihara H, Kevei Z, Farkas A, Mikulass K, Nagy A, Tiricz H, Satiat-Jeunemaître B, Alunni B, Bourge M, Kucho K, Abe M, Kereszt A, Maroti G, Uchiumi T, Kondorosi E, Mergaert P. 2010. Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 327:1122–1126. doi: 10.1126/science.1184057

11. Tiricz H, Szucs A, Farkas A, Pap B, Lima RM, Maróti G, Kondorosi É, Kereszt A. 2013. Antimicrobial nodule-specific cysteine-rich peptides induce membrane depolarization-associated changes in the transcriptome of Sinorhizobium meliloti. Appl Environ Microbiol 79:6737–6746. doi: 10.1128/AEM.01791-13

12. Farkas A, Maróti G, Durgő H, Györgypál Z, Lima RM, Medzihradszky KF, Kereszt A, Mergaert P, Kondorosi É. 2014. Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms. Proc Natl Acad Sci U S A 111:5183–5188. doi: 10.1073/pnas.1404169111

13. Mikuláss KR, Nagy K, Bogos B, Szegletes Z, Kovács E, Farkas A, Váró G, Kondorosi É, Kereszt A. 2016. Antimicrobial nodule-specific cysteine-rich peptides disturb the integrity of bacterial outer and inner membranes and cause loss of membrane potential. Ann Clin Microbiol Antimicrob 15:43. doi: 10.1186/s12941-016-0159-8

14. Farkas A, Pap B, Kondorosi É, Maróti G. 2018. Antimicrobial activity of NCR plant peptides strongly depends on the test assays. Front Microbiol 9:2600. doi: 10.3389/fmicb.2018.02600

15. Montiel J, Downie JA, Farkas A, Bihari P, Herczeg R, Bálint B, Mergaert P, Kereszt A, Kondorosi É. 2017. Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides. Proc Natl Acad Sci U S A 114:5041–5046. doi: 10.1073/pnas.1704217114

16. Young JPW, Moeskjær S, Afonin A, Rahi P, Maluk M, James EK, Cavassim MIA, Rashid MH, Aserse AA, Perry BJ, et al. 2021. Defining the Rhizobium leguminosarum species complex. Genes (Basel) 12:111. doi: 10.3390/genes12010111

17. Laguerre G, Louvrier P, Allard M-R, Amarger N. 2003. Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes. Appl Environ Microbiol 69:2276–2283. doi: 10.1128/AEM.69.4.2276-2283.2003

18. Den Herder G, Parniske M. 2009. The unbearable naivety of legumes in symbiosis. Curr Opin Plant Biol 12:491–499. doi: 10.1016/j.pbi.2009.05.010

19. Brito B, Toffanin A, Prieto RI, Imperial J, Ruiz-Argüeso T, Palacios JM. 2008. Host-dependent expression of Rhizobium leguminosarum bv. viciae hydrogenase is controlled at transcriptional and post-transcriptional levels in legume nodules. Mol Plant Microbe Interact 21:597–604. doi: 10.1094/MPMI-21-5-0597

20. Durán D, Albareda M, García C, Marina A-I, Ruiz-Argüeso T, Palacios J-M. 2021. Proteome analysis reveals a significant host-specific response in Rhizobium leguminosarum bv. viciae endosymbiotic cells. Mol Cell Proteomics 20:100009. doi: 10.1074/mcp.RA120.002276

21. Sánchez-Cañizares C, Jorrín B, Durán D, Nadendla S, Albareda M, Rubio-Sanz L, Lanza M, González-Guerrero M, Prieto RI, Brito B, Giglio MG, Rey L, Ruiz-Argüeso T, Palacios JM, Imperial J. 2018. Genomic diversity in the endosymbiotic bacterium Rhizobium leguminosarum. Genes (Basel) 9:60. doi: 10.3390/genes9020060

22. Djordjevic MA, Chen HC, Natera S, Van Noorden G, Menzel C, Taylor S, Renard C, Geiger O, Weiller GF, Sinorhizobium DNA Sequencing Consortium . 2003. A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation. Mol Plant Microbe Interact 16:508–524. doi: 10.1094/MPMI.2003.16.6.508

23. Djordjevic MA. 2004. Sinorhizobium meliloti metabolism in the root nodule: a proteomic perspective. Proteomics 4:1859–1872. doi: 10.1002/pmic.200300802

24. Karunakaran R, Ramachandran VK, Seaman JC, East AK, Mouhsine B, Mauchline TH, Prell J, Skeffington A, Poole PS. 2009. Transcriptomic analysis of Rhizobium leguminosarum biovar viciae in symbiosis with host plants Pisum sativum and Vicia cracca. J Bacteriol 191:4002–4014. doi: 10.1128/JB.00165-09

25. Gourion B, Berrabah F, Ratet P, Stacey G. 2015. Rhizobium-legume symbioses: the crucial role of plant immunity. Trends Plant Sci 20:186–194. doi: 10.1016/j.tplants.2014.11.008

26. Schwartzman JA, Ruby EG. 2016. Stress as a normal cue in the symbiotic environment. Trends Microbiol 24:414–424. doi: 10.1016/j.tim.2016.02.012

27. Hawkins JP, Oresnik IJ. 2022. The Rhizobium-legume symbiosis: co-opting successful stress management. Front Plant Sci 12:796045. doi: 10.3389/fpls.2021.796045

28. Gutiérrez D, Hernando Y, Palacios JM, Imperial J, Ruiz-Argüeso T. 1997. FnrN controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium leguminosarum biovar viciae UPM791. J Bacteriol 179:5264–5270. doi: 10.1128/jb.179.17.5264-5270.1997

29. Colombo MV, Gutiérrez D, Palacios JM, Imperial J, Ruiz-Argüeso T. 2000. A novel autoregulation mechanism of fnrN expression in Rhizobium leguminosarum bv viciae. Mol Microbiol 36:477–486. doi: 10.1046/j.1365-2958.2000.01867.x

30. Haslbeck M, Weinkauf S, Buchner J. 2019. Small heat shock proteins: simplicity meets complexity. J Biol Chem 294:2121–2132. doi: 10.1074/jbc.REV118.002809

31. Obuchowski I, Karaś P, Liberek K. 2021. The small ones matter-sHsps in the chaperone network. Front Mol Biosci 8:666893. doi: 10.3389/fmolb.2021.666893

32. Nocker A, Krstulovic NP, Perret X, Narberhaus F. 2001. ROSE elements occur in disparate rhizobia and are functionally interchangeable between species. Arch Microbiol 176:44–51. doi: 10.1007/s002030100294

33. Mogk A, Deuerling E, Vorderwülbecke S, Vierling E, Bukau B. 2003. Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation. Mol Microbiol 50:585–595. doi: 10.1046/j.1365-2958.2003.03710.x

34. Basha E, O’Neill H, Vierling E. 2012. Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions. Trends Biochem Sci 37:106–117. doi: 10.1016/j.tibs.2011.11.005

35. Kriehuber T, Rattei T, Weinmaier T, Bepperling A, Haslbeck M, Buchner J. 2010. Independent evolution of the core domain and its flanking sequences in small heat shock proteins. FASEB J 24:3633–3642. doi: 10.1096/fj.10-156992

36. Mogk A, Ruger-Herreros C, Bukau B. 2019. Cellular functions and mechanisms of action of small heat shock proteins. Annu Rev Microbiol 73:89–110. doi: 10.1146/annurev-micro-020518-115515

37. Han M-J, Yun H, Lee SY. 2008. Microbial small heat shock proteins and their use in biotechnology. Biotechnol Adv 26:591–609. doi: 10.1016/j.biotechadv.2008.08.004

38. Waters ER. 2013. The evolution, function, structure, and expression of the plant sHSPs. J Exp Bot 64:391–403. doi: 10.1093/jxb/ers355

39. Münchbach M, Nocker A, Narberhaus F. 1999. Multiple small heat shock proteins in rhizobia. J Bacteriol 181:83–90. doi: 10.1128/JB.181.1.83-90.1999

40. Cocotl-Yañez M, Moreno S, Encarnación S, López-Pliego L, Castañeda M, Espín G. 2014. A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii. Microbiology (Reading) 160:479–487. doi: 10.1099/mic.0.073353-0

41. Hwang H-H, Liu Y-T, Huang S-C, Tung C-Y, Huang F-C, Tsai Y-L, Cheng T-F, Lai E-M. 2015. Overexpression of the HspL promotes Agrobacterium tumefaciens virulence in Arabidopsis under heat shock conditions. Phytopathology 105:160–168. doi: 10.1094/PHYTO-05-14-0133-R

42. Vincent JM. 1970. A manual for the practical study of root-nodule bacteria. International Biological Programme. Blackwell Scientific Publications, Oxford.

43. Beringer JE. 1974. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198. doi: 10.1099/00221287-84-1-188

44. O’Gara F, Shanmugam KT. 1976. Regulation of nitrogen fixation by Rhizobia. Export of fixed N 2 as NH 4 + . Biochim Biophys Acta 437:313–321. doi: 10.1016/0304-4165(76)90001-5

45. Wheatley RM, Ramachandran VK, Geddes BA, Perry BJ, Yost CK, Poole PS. 2017. Role of O 2 in the growth of Rhizobium leguminosarum bv. viciae 3841 on glucose and succinate. J Bacteriol 199:e00572-16. doi: 10.1128/JB.00572-16

46. Bertani G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300. doi: 10.1128/jb.62.3.293-300.1951

47. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580. doi: 10.1016/S0022-2836(83)80284-8

48. Simon R, Priefer UB, Pühler A. 1983. Vector plasmids for in-vivo and in-vitro manipulations of gram-negative bacteria, p 98–106. In Pühler A (ed), Molecular genetica of the bacteria-plant interactions. Springer-Verlagg, Berlin.

49. Leyva A, Palacios JM, Ruiz-Argüeso T. 1987. Conserved plasmid hydrogen-uptake (hup)-specific sequences within Hup + Rhizobium leguminosarum strains. Appl Environ Microbiol 53:2539–2543. doi: 10.1128/aem.53.10.2539-2543.1987

50. Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73. doi: 10.1016/0378-1119(94)90324-7

51. Tett AJ, Rudder SJ, Bourdès A, Karunakaran R, Poole PS. 2012. Regulatable vectors for environmental gene expression in Alphaproteobacteria. Appl Environ Microbiol 78:7137–7140. doi: 10.1128/AEM.01188-12

52. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176. doi: 10.1016/0378-1119(95)00584-1

53. Albareda M, Manyani H, Imperial J, Brito B, Ruiz-Argüeso T, Böck A, Palacios J-M. 2012. Dual role of HupF in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum. BMC Microbiol 12:256–6. doi: 10.1186/1471-2180-12-256

54. Sambrook J, Russle DW.. 2001. Molecular cloning: a laboratory manuals. Cold Spring Harbor Laboratory Press, New York.

55. Sánchez-Cañizares C, Rey L, Durán D, Temprano F, Sánchez-Jiménez P, Navarro A, Polajnar M, Imperial J, Ruiz-Argüeso T. 2011. Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus. Syst Appl Microbiol 34:207–215. doi: 10.1016/j.syapm.2010.11.020

56. Brito B, Palacios JM, Hidalgo E, Imperial J, Ruiz-Argüeso T. 1994. Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits. J Bacteriol 176:5297–5303. doi: 10.1128/jb.176.17.5297-5303.1994

57. Ruiz-Argüeso T, Hanus J, Evans HJ. 1978. Hydrogen production and uptake by pea nodules as affected by strains of Rhizobium leguminosarum. Arch Microbiol 116:113–118. doi: 10.1007/BF00406025

58. Ballesteros-Gutiérrez M, Albareda M, Barbas C, López-Gonzálvez Á, Dunn MF, Palacios JM. 2023. A host-specific diaminobutyrate aminotransferase contributes to symbiotic performance, homoserine metabolism, and competitiveness in the Rhizobium leguminosarum/Pisum sativum system. Front Microbiol 14:1182563. doi: 10.3389/fmicb.2023.1182563

59. Lodwig E, Kumar S, Allaway D, Bourdes A, Prell J, Priefer U, Poole PS. 2004. Regulation of L-alanine dehydrogenase in Rhizobium leguminosarum bv. viciae and its role in pea nodules. J Bacteriol 186:842–849. doi: 10.1128/JB.186.3.842-849.2004

60. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85. doi: 10.1016/0003-2697(85)90442-7

61. Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. doi: 10.1038/nmeth.2089

62. Moreno M-L, Escobar J, Izquierdo-Álvarez A, Gil A, Pérez S, Pereda J, Zapico I, Vento M, Sabater L, Marina A, Martínez-Ruiz A, Sastre J. 2014. Disulfide stress: a novel type of oxidative stress in acute pancreatitis. Free Rad Biol Med 70:265–277. doi: 10.1016/j.freeradbiomed.2014.01.009

63. Alonso R, Pisa D, Marina AI, Morato E, Rábano A, Rodal I, Carrasco L. 2015. Evidence for fungal infection in cerebrospinal fluid and brain tissue from patients with amyotrophic lateral sclerosis. Int J Biol Sci 11:546–558. doi: 10.7150/ijbs.11084

64. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF. 1999. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552. doi: 10.1385/1-59259-584-7:531

65. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. doi: 10.1038/msb.2011.75

66. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268–274. doi: 10.1093/molbev/msu300

67. Studer S, Narberhaus F. 2000. Chaperone activity and homo- and hetero-oligomer formation of bacterial small heat shock proteins. J Biol Chem 275:37212–37218. doi: 10.1074/jbc.M004701200

68. Santos R, Hérouart D, Sigaud S, Touati D, Puppo A. 2001. Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction. Mol Plant Microbe Interact 14:86–89. doi: 10.1094/MPMI.2001.14.1.86

69. Matamoros MA, Becana M. 2020. Redox control of the legume-Rhizobium symbiosis. Adv Bot Res 94:67–96. doi: 10.1016/bs.abr.2019.09.011

70. Voth W, Jakob U. 2017. Stress-activated chaperones: a first Line of defense. Trends Biochem Sci 42:899–913. doi: 10.1016/j.tibs.2017.08.006

71. Poole PS, Allaway D. 2000. Carbon and nitrogen metabolism in Rhizobium. Adv Micro Physiol 43:117–163. doi: 10.1016/S0065-2911(00)43004-3

72. Lodwig E, Poole PS. 2003. Metabolism of Rhizobium bacteroids. Crit Rev Plant Sci 22:37–78. doi: 10.1080/713610850

73. Dunn MF. 2015. Key roles of microsymbiont amino acid metabolism in rhizobia-legume interactions. Crit Rev Microbiol 41:411–451. doi: 10.3109/1040841X.2013.856854

74. O’Brian MR. 1996. Heme synthesis in the Rhizobium-legume symbiosis: a palette for bacterial and eukaryotic pigments. J Bacteriol 178:2471–2478. doi: 10.1128/jb.178.9.2471-2478.1996

75. Garcia PS, Gribaldo S, Py B, Barras F. 2019. The SUF system: an ABC ATPase-dependent protein complex with a role in Fe-S cluster biogenesis. Res Microbiol 170:426–434. doi: 10.1016/j.resmic.2019.08.001

76. Du S, Lutkenhaus J. 2019. At the heart of bacterial cytokinesis: the Z ring. Trends Microbiol 27:781–791. doi: 10.1016/j.tim.2019.04.011

77. Montiel J, Szűcs A, Boboescu IZ, Gherman VD, Kondorosi É, Kereszt A. 2016. Terminal bacteroid differentiation is associated with variable morphological changes in legume species belonging to the inverted repeat-lacking clade. Mol Plant Microbe Interact 29:210–219. doi: 10.1094/MPMI-09-15-0213-R

78. Rutten PJ, Steel H, Hood GA, Ramachandran VK, McMurtry L, Geddes B, Papachristodoulou A, Poole PS. 2021. Multiple sensors provide spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis. PLoS Genet 17:e1009099. doi: 10.1371/journal.pgen.1009099

79. Schlüter A, Patschkowski T, Unden G, Priefer UB. 1992. The Rhizobium leguminosarum FnrN protein is functionally similar to Escherichia coli Fnr and promotes heterologous oxygen-dependent activation of transcription. Mol Microbiol 6:3395–3404. doi: 10.1111/j.1365-2958.1992.tb02207.x

80. Kitagawa M, Matsumura Y, Tsuchido T. 2000. Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli. FEMS Microbiol Lett 184:165–171. doi: 10.1111/j.1574-6968.2000.tb09009.x

81. Singh H, Appukuttan D, Lim S. 2014. Hsp20, a small heat shock protein of Deinococcus radiodurans, confers tolerance to hydrogen peroxide in Escherichia coli. J Microbiol Biotechnol 24:1118–1122. doi: 10.4014/jmb.1403.03006

82. Kitagawa M, Miyakawa M, Matsumura Y, Tsuchido T. 2002. Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants. Eur J Biochem 269:2907–2917. doi: 10.1046/j.1432-1033.2002.02958.x

83. Fu X, Shi X, Yan L, Zhang H, Chang Z. 2013. In vivo substrate diversity and preference of small heat shock protein IbpB as revealed by using a genetically incorporated photo-cross-linker. J Biol Chem 288:31646–31654. doi: 10.1074/jbc.M113.501817

84. Török Z, Goloubinoff P, Horváth I, Tsvetkova NM, Glatz A, Balogh G, Varvasovszki V, Los DA, Vierling E, Crowe JH, Vigh L. 2001. Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding. Proc Natl Acad Sci U S A 98:3098–3103. doi: 10.1073/pnas.051619498

85. Basha E, Lee GJ, Breci LA, Hausrath AC, Buan NR, Giese KC, Vierling E. 2004. The identity of proteins associated with a small heat shock protein during heat stress in vivo indicates that these chaperones protect a wide range of cellular functions. J Biol Chem 279:7566–7575. doi: 10.1074/jbc.M310684200

86. Haslbeck M, Braun N, Stromer T, Richter B, Model N, Weinkauf S, Buchner J. 2004. Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae. EMBO J 23:638–649. doi: 10.1038/sj.emboj.7600080

87. Maitre M, Weidmann S, Dubois-Brissonnet F, David V, Covès J, Guzzo J. 2014. Adaptation of the wine bacterium Oenococcus oeni to ethanol stress: role of the small heat shock protein Lo18 in membrane integrity. Appl Environ Microbiol 80:2973–2980. doi: 10.1128/AEM.04178-13

88. Marklund EG, Zhang Y, Basha E, Benesch JLP, Vierling E. 2018. Structural and functional aspects of the interaction partners of the small heat-shock protein in Synechocystis. Cell Stress Chaperones 23:723–732. doi: 10.1007/s12192-018-0884-3

89. Ratajczak E, Zietkiewicz S, Liberek K. 2009. Distinct activities of Escherichia coli small heat shock proteins IbpA and IbpB promote efficient protein disaggregation. J Mol Biol 386:178–189. doi: 10.1016/j.jmb.2008.12.009

90. Veinger L, Diamant S, Buchner J, Goloubinoff P. 1998. The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network. J Biol Chem 273:11032–11037. doi: 10.1074/jbc.273.18.11032

91. Liu W-T, Karavolos MH, Bulmer DM, Allaoui A, Hormaeche R, Lee JJ, Khan CMA. 2007. Role of the universal stress protein UspA of Salmonella in growth arrest, stress and virulence. Microb Pathog 42:2–10. doi: 10.1016/j.micpath.2006.09.002

92. Seifart Gomes C, Izar B, Pazan F, Mohamed W, Mraheil MA, Mukherjee K, Billion A, Aharonowitz Y, Chakraborty T, Hain T. 2011. Universal stress proteins are important for oxidative and acid stress resistance and growth of Listeria monocytogenes EGD-e in vitro and in vivo. PLoS One 6:e24965. doi: 10.1371/journal.pone.0024965

93. O’Connor A, Jurado-Martín I, Mysior MM, Manzira AL, Drabinska J, Simpson JC, Lucey M, Schaffer K, Berisio R, McClean S. 2023. A universal stress protein upregulated by hypoxia has a role in Burkholderia cenocepacia intramacrophage survival: implications for chronic infection in cystic fibrosis. Microbiologyopen 12:e1311. doi: 10.1002/mbo3.1311

94. Govezensky D, Greener T, Segal G, Zamir A. 1991. Involvement of GroEL in nif gene regulation and nitrogenase assembly. J Bacteriol 173:6339–6346. doi: 10.1128/jb.173.20.6339-6346.1991

95. Ribbe MW, Burgess BK. 2001. The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase. Proc Natl Acad Sci U S A 98:5521–5525. doi: 10.1073/pnas.101119498

96. Rubio LM, Ludden PW. 2005. Maturation of nitrogenase: a biochemical puzzle. J Bacteriol 187:405–414. doi: 10.1128/JB.187.2.405-414.2005

97. Kuczynska-Wisnik D, Kçdzierska S, Matuszewska E, Lund P, Taylor A, Lipinska B, Laskowska E. 2002. The Escherichia coli small heat-shock proteins IbpA and IbpB prevent the aggregation of endogenous proteins denatured in vivo during extreme heat shock. Microbiol (Reading) 148:1757–1765. doi: 10.1099/00221287-148-6-1757

98. Bepperling A, Alte F, Kriehuber T, Braun N, Weinkauf S, Groll M, Haslbeck M, Buchner J. 2012. Alternative bacterial two-component small heat shock protein systems. Proc Natl Acad Sci U S A 109:20407–20412. doi: 10.1073/pnas.1209565109

99. Ungelenk S, Moayed F, Ho C-T, Grousl T, Scharf A, Mashaghi A, Tans S, Mayer MP, Mogk A, Bukau B. 2016. Small heat shock proteins sequester misfolding proteins in near-native conformation for cellular protection and efficient refolding. Nat Commun 7:13673. doi: 10.1038/ncomms13673

100. Chernova LS, Vedyaykin AD, Bogachev MI, Fedorova MS, Ivanov VA, Vishnyakov IE, Kayumov AR. 2022. The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii. Biochim Biophys Acta Gen Subj 1866:130220. doi: 10.1016/j.bbagen.2022.130220

101. Haslbeck M, Vierling E. 2015. A first line of stress defense: small heat shock proteins and their function in protein homeostasis. J Mol Biol 427:1537–1548. doi: 10.1016/j.jmb.2015.02.002

Associated Data

Supplementary Materials

aem.01385-24-s0001.pdf

Sequence alignment of rhizobial sHSPs.

aem.01385-24-s0002.pdf

Effect of RLV_1399 expression on the tolerance of Rlv UPM791 to oxidative stress.

aem.01385-24-s0003.pdf

Peptides and primers used in this work.

aem.01385-24-s0004.xlsx

Proteins identified in sHSP complexes.