Sinorhizobium meliloti DnaJ Is Required for Surface Motility, Stress Tolerance, and for Efficient Nodulation and Symbiotic Nitrogen Fixation

Citation: Brito-Santana, P.; Duque-Pedraza, J.J.; Bernabéu-Roda, L.M.; Carvia-Hermoso, C.; Cuéllar, V.; Fuentes-Romero, F.; Acosta-Jurado, S.; Vinardell, J.-M.; Soto, M.J. Sinorhizobium meliloti DnaJ Is Required for Surface Motility, Stress Tolerance, and for Efficient Nodulation and Symbiotic Nitrogen Fixation. Int. J. Mol. Sci. 2023,24, 5848. https://doi.org/10.3390/ ijms24065848 Academic Editors: Jose J. Pueyo and Elena E. Fedorova Received: 21 February 2023 Revised: 14 March 2023 Accepted: 17 March 2023 Published: 19 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Sinorhizobium meliloti DnaJ Is Required for Surface Motility, Stress Tolerance, and for Efficient Nodulation and Symbiotic Nitrogen Fixation Paula Brito-Santana 1, Julián J. Duque-Pedraza 1, Lydia M. Bernabéu-Roda 1, Cristina Carvia-Hermoso 1, Virginia Cuéllar 1, Francisco Fuentes-Romero 2, Sebastián Acosta-Jurado 3, José-María Vinardell 2 and María J. Soto 1,* 1Estación Experimental del Zaidín, CSIC, Department of Biotechnology and Environmental Protection, 18008 Granada, Spain 2Facultad de Biología, Departamento de Microbiología, Universidad de Sevilla, 41012 Sevilla, Spain 3Centro Andaluz de Biología del Desarrollo, CSIC, Junta de Andalucía, Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, 41013 Seville, Spain *Correspondence: [email protected]; Tel.: +34-958-526-477 Abstract: Bacterial surface motility is a complex microbial trait that contributes to host colonization. However, the knowledge about regulatory mechanisms that control surface translocation in rhizobia and their role in the establishment of symbiosis with legumes is still limited. Recently, 2-tridecanone (2-TDC) was identified as an infochemical in bacteria that hampers microbial colonization of plants. In the alfalfa symbiont Sinorhizobium meliloti, 2-TDC promotes a mode of surface motility that is mostly independent of flagella. To understand the mechanism of action of 2-TDC in S. meliloti and unveil genes putatively involved in plant colonization, Tn5transposants derived from a flagellaless strain that were impaired in 2-TDC-induced surface spreading were isolated and genetically characterized. In one of the mutants, the gene coding for the chaperone DnaJ was inactivated. Characterization of this transposant and newly obtained flagella-minus and flagella-plus dnaJ deletion mutants revealed that DnaJ is essential for surface translocation, while it plays a minor role in swimming motility. DnaJ loss-of-function reduces salt and oxidative stress tolerance in S. meliloti and hinders the establishment of efficient symbiosis by affecting nodule formation efficiency, cellular infection, and nitrogen fixation. Intriguingly, the lack of DnaJ causes more severe defects in a flagellaless background. This work highlights the role of DnaJ in the free-living and symbiotic lifestyles of S. meliloti. Keywords: Rhizobium; plant colonization; surface motility; flagella; chaperone; stress tolerance; nodulation; nitrogen fixation 1. Introduction Rhizobia are soil-dwelling alphaand betaproteobacteria, which are able to establish nitrogen-fixing symbiosis with legumes [ 1 ]. In nitrogen-poor soils, these bacteria can elicit the formation of new organs, known as nodules, on the roots of their host plants. Root nodules are invaded by rhizobia where they differentiate into bacteroids capable of reducing atmospheric dinitrogen into ammonia that is provided to the plant, relieving its dependency on nitrogen fertilizers. Bacteria also benefit from this symbiosis by receiving a carbon source and essential nutrients from the plant, together with a protected environmental niche [ 2 , 3 ]. The development of nitrogen-fixing nodules in legume roots is the outcome of a complex process that involves a highly specific and continuous molecular dialogue between the two symbiotic partners, and of which much knowledge has been gained in the last years [ 1 , 4 – 6 ]. An early event crucial for the establishment of symbiosis is bacterial root colonization. This process involves several steps beginning with the directed movement of bacteria toward the roots, followed by the attachment of bacteria to the root surface Int. J. Mol. Sci. 2023,24, 5848. https://doi.org/10.3390/ijms24065848 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023,24, 5848 2 of 21 (i.e., rhizoplane) and subsequent formation of a biofilm in which cells are embedded in an extracellular matrix that confers protection against adverse conditions [ 7 , 8 ]. In contrast to the well-known processes of nodule formation and nitrogen fixation, the knowledge about mechanisms used by rhizobia for rhizoplane colonization is scarce. Recently, a few genomewide approaches have successfully been adopted to gain insights into this field and motility has been found to play a fundamental role [ 9 – 11 ]. Chemotaxis and motility are not essential for nodulation or nitrogen fixation but are crucial for competitive root colonization, which in turn can influence nodule formation efficiency and competitiveness [ 12 – 14 ]. Flagelladriven swimming motility permits individual bacteria to move in aqueous environments and allows rhizobia to approach its host plant in response to chemoattractants released by legume roots, as well as to find proper sites for infection [13,14]. In nature, microbes are usually associated with biotic and abiotic surfaces. To be able to colonize these niches, bacteria have evolved different motility strategies [ 15 – 18 ]. Swarming is probably the most extensively studied type of surface motility because it is a trait closely connected with biofilm formation and virulence in pathogenic bacteria [ 19 , 20 ]. Swarming is a flagella-driven motility characterized by the rapid coordinated multicellular migration of bacteria across solid surfaces [ 19 ]. Bacteria can also move over surfaces using sliding, a passive appendage-independent spreading in which surfactants and other compounds that diminish friction between cells and surfaces play a fundamental role [ 21 ]. In contrast to swimming, during surface motility, bacteria face important challenges that need to be overcome. Among them, bacteria need to attract water to the surface to allow for flagellar activity, overcome frictional forces, and reduce surface tension [ 17 , 21 , 22 ]. Several physical, chemical, and biological factors are known to influence the bacterial capacity to translocate across surfaces, which convert surface motility into a complex and highly regulated phenotype [ 17 , 18 ]. Rhizobia can move over surfaces by using flagella-dependent and independent mechanisms [ 23 – 29 ]. However, knowledge about the regulatory mechanisms that control surface motility in rhizobia as well as the role of this bacterial trait in the establishment of symbiosis is limited [28–31]. Sinorhizobium meliloti, the alfalfa endosymbiont, can translocate over semisolid surfaces by using flagella-mediated mechanisms, or by sliding promoted by the production of exopolysaccharides and surfactants [ 23 , 28 , 32 – 35 ]. Like in other bacteria, studies on the mechanisms underlying surface motility in S. meliloti revealed a close connection with biofilm formation as these two surface-associated traits were influenced by the same regulatory pathways and chemical cues [ 28 , 29 , 36 ]. The characterization of an S. meliloti mutant that exhibits increased surface motility, defects in biofilm formation, and impaired root colonization, led to the discovery of 2-tridecanone (2-TDC) as an infochemical that affects bacterial surface-associated traits and hampers microbial colonization of plant tissues [ 36 ]. 2-TDC is a volatile methylketone known as a natural insecticide produced in high amounts by wild tomato plants [ 37 ]. Several rhizobacteria, including S. meliloti, can also produce 2-TDC [ 36 , 38 , 39 ]. Our investigations revealed that, without affecting bacterial growth or swimming motility, exogenous application of 2-TDC promoted surface motility and impaired biofilm formation in S. meliloti [ 36 ]. Moreover, the presence of 2-TDC impairs the nodulation of alfalfa by hampering the bacterial ability to efficiently colonize plant roots. Therefore, the mechanistic understanding of the effects caused by 2-TDC on S. meliloti could potentially unveil bacterial genes required for symbiosis. In S. meliloti strain GR4 [ 40 ], 2-TDC promotes a mode of surface motility that is mostly independent of flagella and mediated by an as-yet-unknown mechanism [ 36 ]. To decipher the molecular bases responsible for the volatile-triggered surface spreading, transposants derived from the flagellaless GR4flaAB strain exhibiting limited or no surface motility in the presence of 2-TDC were isolated and genetically characterized. In one of these mutants, the transposon interrupted the dnaJ gene, which potentially codes for a molecular chaperone homologous to the eukaryotic 40 kDa heat shock protein Hsp40. The DnaJ protein belongs to the ATP-dependent chaperone folding system DnaK/DnaJ/GrpE [ 41 ]. DnaJ functions as a chaperone and holdase that transfers unfolded, misfolded, or aggregated proteins Int. J. Mol. Sci. 2023,24, 5848 3 of 21 to DnaK, which is responsible for the folding of the substrate with the participation of the nucleotide exchange factor GrpE. With less efficiency than DnaK, DnaJ can also work autonomously as a foldase of denatured proteins [ 41 – 43 ]. The lack of DnaJ has been associated in some bacteria with the inability to produce flagella or impaired swimming and swarming motilities [ 44 – 46 ]. Moreover, the participation of bacterial DnaJ in biofilm formation, the colonization of eukaryotic hosts, and pathogenicity has been shown [ 47 – 51 ]. The role of DnaJ in legume endosymbionts has scarcely been investigated. Interestingly, the lack of this chaperone affects differently the establishment of symbiosis depending on the rhizobial species [ 52 – 54 ]. In this work, the role of S. meliloti DnaJ in the bacterial response to volatile 2-TDC, as well as in the adaptation to stress conditions and the establishment of symbiosis with alfalfa plants, has been investigated. Our results show that DnaJ is essential in the flagella-independent surface motility triggered by volatile 2-TDC and in the swarming motility exhibited by the wild-type S. meliloti strain GR4, but it plays a minor role in swimming motility. In addition, DnaJ helps S. meliloti cells to adapt to stressful conditions and is required for efficient nodule colonization and symbiotic nitrogen fixation in alfalfa plants. 2. Results 2.1. Isolation and Genetic Characterization of Flagellaless GR4flaAB-Derivative Mutants That Do Not Respond to Volatile 2-TDC To identify S. meliloti genes playing a role in the flagella-independent motility triggered by volatile 2-TDC, strain GR4flaAB was subject to Tn5transposon mutagenesis and the resulting kanamycin-resistant transposants were tested for surface motility on 1% agar minimal medium (MM) in the presence of volatile 2-TDC. After screening 3.885 transposants , five mutants named NS (for nonspreading) were identified as impaired in the response to the volatile, i.e., they exhibited reduced or no translocation across the surface in the presence of airborne 2-TDC in contrast to the parental strain GR4flaAB (Figure S1). To determine the genomic location of each insertion, arbitrary PCR and DNA sequencing were performed (see Section 4). Genetic characterization of the mutants revealed five genes that could potentially be involved in the action mechanism of 2-TDC (Table 1). Table 1. GR4flaAB-derived mutants insensitive to volatile 2-TDC. Transposant Tn5Location 1/Affected Locus GR4/Rm1021 Description of Gene Product NS1 C770_GR4Chr0066/smc02585 (actS) Two-component sensor histidine kinase NS2 C770_GR4Chr0263/smc00334 (cmK) Putative cytidylate kinase NS3 C770_GR4Chr1253/smc01800 (ctaA) Putative heme A synthase NS4 C770_GR4Chr0186/smc02858 (dnaJ) Probable chaperone protein NS5 C770_GR4Chr3081/smc03142 Diguanylate cyclase (GGDEF) domain protein/Hypothetical transmembrane protein 1The name of the affected locus in GR4 is given followed by the orthologous gene in Rm1021. Only one of the five genes identified in our screening, the actS gene, was previously characterized in S. meliloti. The actS gene encodes the sensor histidine kinase of the twocomponent regulatory system ActS/ActR that has been associated with bacterial tolerance to acidic pH, the adaptation to oxidative stress, the regulation of microaerobic respiration, and cell envelope function [ 55 – 58 ]. Transposon insertions in mutants NS2, NS3, and NS4 were located in the cmk,ctaA, and dnaJ genes that code for proteins involved in the phosphorylation of (d)CMP, heme A synthesis, and in chaperoning/protein homeostasis, respectively. Finally, in the NS5 mutant, the transposon was located on a chromosomal gene that in GR4 codes for a protein putatively involved in the metabolism of the second messenger cyclic-dimeric guanosine monophosphate (c-di-GMP). This protein is not produced in the well-known strains Rm1021/Rm2011 due to the truncation of smc03142 by a non-sense mutation [ 59 ]. In this study, we focused on the role of dnaJ, the gene inactivated by Tn5in NS4, in the response of S. meliloti to 2-TDC and its participation in plant colonization. Int. J. Mol. Sci. 2023,24, 5848 4 of 21 2.2. In Silico Analyses of S. meliloti dnaJ The C770_GR4Chr0186 locus affected in the NS4 transposant is orthologous to the smc02858 gene of Rm1021 that is annotated as dnaJ. In the reference Rm1021 strain, dnaJ is flanked upstream by the chaperone Hsp70-encoding dnaK gene and downstream by the smc02859-smc02860 genes that are transcribed in the opposite direction to dnaJ and encode proteins of unknown function. The genomic context of dnaJ in strain GR4 is similar to that of Rm1021 except that a copy of the insertion sequence IS110 is located between dnaJ and the smc02859-smc02860 operon (Figure 1a). In contrast to the genetic organization found in many bacteria [ 42 ], in S. meliloti,grpE (smc01142) is not in the proximity of the dnaK dnaJ genes. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 23 were located in the cmk, ctaA, and dnaJ genes that code for proteins involved in the phosphorylation of (d)CMP, heme A synthesis, and in chaperoning/protein homeostasis, respectively. Finally, in the NS5 mutant, the transposon was located on a chromosomal gene that in GR4 codes for a protein putatively involved in the metabolism of the second messenger cyclic-dimeric guanosine monophosphate (c-di-GMP). This protein is not produced in the well-known strains Rm1021/Rm2011 due to the truncation of smc03142 by a non-sense mutation [59]. In this study, we focused on the role of dnaJ, the gene inactivated by Tn5 in NS4, in the response of S. meliloti to 2-TDC and its participation in plant colonization. 2.2. In Silico Analyses of S. meliloti dnaJ The C770_GR4Chr0186 locus affected in the NS4 transposant is orthologous to the smc02858 gene of Rm1021 that is annotated as dnaJ. In the reference Rm1021 strain, dnaJ is flanked upstream by the chaperone Hsp70-encoding dnaK gene and downstream by the smc02859-smc02860 genes that are transcribed in the opposite direction to dnaJ and encode proteins of unknown function. The genomic context of dnaJ in strain GR4 is similar to that of Rm1021 except that a copy of the insertion sequence IS110 is located between dnaJ and the smc02859-smc02860 operon (Figure 1a). In contrast to the genetic organization found in many bacteria [42], in S. meliloti, grpE (smc01142) is not in the proximity of the dnaK dnaJ genes. Figure 1. In silico analyses of S. meliloti dnaJ. (a) Genomic context of dnaJ in S. meliloti strains Rm1021 and GR4. The arrows represent open reading frames. The transposase encoded by the insertion sequence IS110 in GR4 is shown in red. smc02856 encodes a putative penicillin-binding protein; dnaK and dnaJ code for the Hsp70 and Hsp40 chaperones, respectively; smc02859-smc02860 encodes hypothetical proteins of unknown function. (b) Domain architecture of the S. meliloti DnaJ protein. The conserved HPD motif in the J-domain is shown in bold. The eight cysteines putatively conforming two zinc-binding domains are indicated in red. The location of the Tn5 insertion in the NS4 transposant is shown with a red triangle. In GR4, the dnaJ gene encodes a 379 amino acid protein, which is 100% identical to the protein of Rm1021 and shows a domain architecture typical of class A JDP members (Figure 1b) [42]. The amino terminal J-domain, which is essential for functional interaction with DnaK, contains the invariant tripeptide of histidine, proline, and aspartic acid (HPD motif) [42,43]. Next to the N-terminal J-domain, a glycine/phenylalanine (G/F)-rich region connects the J-domain to a zinc-binding domain (ZBD), which is followed by a C-terminal domain (CTD). The ZBD is a cysteine-rich region with four CXXCXGXG (C, cysteine; G, glycine; X, any amino acid) repeats that coordinate two zinc ions. This domain participates in substrate binding and activation of the DnaK chaperone and also harbors oxidoreductase activity [42,49,60]. The CTD is the main substrate binding domain of DnaJ, although the (G/F)-rich region and the ZBD also contribute to this role [42,43]. In the NS4 mutant, Figure 1. In silico analyses of S. meliloti dnaJ. ( a ) Genomic context of dnaJ in S. meliloti strains Rm1021 and GR4. The arrows represent open reading frames. The transposase encoded by the insertion sequence IS110 in GR4 is shown in red. smc02856 encodes a putative penicillin-binding protein; dnaK and dnaJ code for the Hsp70 and Hsp40 chaperones, respectively; smc02859-smc02860 encodes hypothetical proteins of unknown function. ( b ) Domain architecture of the S. meliloti DnaJ protein. The conserved HPD motif in the J-domain is shown in bold. The eight cysteines putatively conforming two zinc-binding domains are indicated in red. The location of the Tn5insertion in the NS4 transposant is shown with a red triangle. In GR4, the dnaJ gene encodes a 379 amino acid protein, which is 100% identical to the protein of Rm1021 and shows a domain architecture typical of class A JDP members (Figure 1b) [ 42 ]. The amino terminal J-domain, which is essential for functional interaction with DnaK, contains the invariant tripeptide of histidine, proline, and aspartic acid (HPD motif) [ 42 , 43 ]. Next to the N-terminal J-domain, a glycine/phenylalanine (G/F)-rich region connects the J-domain to a zinc-binding domain (ZBD), which is followed by a C-terminal domain (CTD). The ZBD is a cysteine-rich region with four CXXCXGXG (C, cysteine; G, glycine; X, any amino acid) repeats that coordinate two zinc ions. This domain participates in substrate binding and activation of the DnaK chaperone and also harbors oxidoreductase activity [ 42 , 49 , 60 ]. The CTD is the main substrate binding domain of DnaJ, although the (G/F)-rich region and the ZBD also contribute to this role [ 42 , 43 ]. In the NS4 mutant, the Tn5insertion is located in the ZBD of DnaJ affecting residue D 201 , which could lead to a truncated DnaJ protein. The DnaJ protein of S. meliloti shows high identities with DnaJ proteins from different rhizobia such as Sinorhizobium medicae WSM419 (96.6%), Rhizobium etli CFN42 (87.2%), R. tropici CIAT899 (87%), Rhizobium leguminosarum biovar viciae 3841 (86.6%), and B. diazoefficiens USDA 110 (66.1%). It also shows high identities with DnaJ proteins of different pathogenic and beneficial bacteria, such as Agrobacterium tumefaciens (85.3%), Brucella ovis (78.9%), Salmonella enterica subsp. enterica (75.7%), Escherichia coli K12 (55.2%), or Pseudomonas putida (54.7%). Multiple sequence alignments of these proteins reveal that the Int. J. Mol. Sci. 2023,24, 5848 5 of 21 different domains and motifs characteristic of DnaJ are conserved, although the (G/F)-rich region exhibits some variability (Figure S2). 2.3. DnaJ Is Required for Flagella-Dependent and Independent Types of Surface Motility but It Plays a Minor Role in Swimming To confirm that the inactivation of the dnaJ gene caused by the Tn5insertion in the NS4 transposant was responsible for the 2-TDC insensitivity of the mutant, complementation experiments were performed using a plasmid construct that expresses the wild-type dnaJ allele. As shown in Figure 2, the ectopic expression of dnaJ in NS4 (NS4 C) recovered the ability of the mutant to spread over the surface of the medium in the presence of volatile 2-TDC at levels similar to those exhibited by the parental strain GR4flaAB carrying the empty vector (flaAB ev). Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 23 the Tn5 insertion is located in the ZBD of DnaJ affecting residue D 201 , which could lead to a truncated DnaJ protein. The DnaJ protein of S. meliloti shows high identities with DnaJ proteins from different rhizobia such as Sinorhizobium medicae WSM419 (96.6%), Rhizobium etli CFN42 (87.2%), R. tropici CIAT899 (87%), Rhizobium leguminosarum biovar viciae 3841 (86.6%), and B. diazoefficiens USDA 110 (66.1%). It also shows high identities with DnaJ proteins of different pathogenic and beneficial bacteria, such as Agrobacterium tumefaciens (85.3%), Brucella ovis (78.9%), Salmonella enterica subsp. enterica (75.7%), Escherichia coli K12 (55.2%), or Pseudomonas putida (54.7%). Multiple sequence alignments of these proteins reveal that the different domains and motifs characteristic of DnaJ are conserved, although the (G/F)-rich region exhibits some variability (Figure S2). 2.3. DnaJ Is Required for Flagella-Dependent and Independent Types of Surface Motility but It Plays a Minor Role in Swimming To confirm that the inactivation of the dnaJ gene caused by the Tn5 insertion in the NS4 transposant was responsible for the 2-TDC insensitivity of the mutant, complementation experiments were performed using a plasmid construct that expresses the wildtype dnaJ allele. As shown in Figure 2, the ectopic expression of dnaJ in NS4 (NS4 C) recovered the ability of the mutant to spread over the surface of the medium in the presence of volatile 2-TDC at levels similar to those exhibited by the parental strain GR4flaAB carrying the empty vector (flaAB ev). Figure 2. The inactivation of dnaJ in transposant NS4 is responsible for its insensitivity to 2-TDC. Surface motility assays on MM (1% agar) in the presence or absence of volatile 2-TDC. About 20 µL of either a solution containing 1 µmol 2-TDC or ethanol (Control) was applied to the lid of the plates just before incubation. flaAB, strain GR4flaAB; ev, empty vector pJB3; and C, complementing plasmid pJ-dnaJ. Representative pictures of the motilities exhibited after 48 h of incubation are shown. We also constructed in-frame markerless dnaJ deletion mutants derived from the flagellaless strain GR4flaAB and the wild-type strain GR4 (flaABΔdnaJ and GΔdnaJ strains, respectively). No major differences in growth were detected between the dnaJ mutants and their parental strains when serial dilutions of cultures grown to the mid-exponential phase were spotted onto MM plates (Figure S3a). On TY plates, a difference in colony size was observed between the mutants and the parental strains, with the dnaJ mutants developing smaller colonies (Figure S3b). Growth curves performed in liquid MM and TY revealed slower growth of the dnaJ mutants compared to their parental strains (Figure S3c,d). The retarded growth shown by the mutants was mainly due to their longer lag phases, which suggests that the inactivation of dnaJ impairs the adjustment of cells to new environmental conditions. The most affected strain during growth in liquid media was flaABΔdnaJ, whereas GΔdnaJ and NS4 exhibited similar behavior to each other. The differences in growth observed between flaABΔdnaJ and GΔdnaJ indicate that the role of DnaJ is more relevant for the flagellaless strain than for the wild-type strain. Moreover, Figure 2. The inactivation of dnaJ in transposant NS4 is responsible for its insensitivity to 2-TDC. Surface motility assays on MM (1% agar) in the presence or absence of volatile 2-TDC. About 20 µ L of either a solution containing 1 µ mol 2-TDC or ethanol (Control) was applied to the lid of the plates just before incubation. flaAB, strain GR4flaAB; ev, empty vector pJB3; and C, complementing plasmid pJ-dnaJ. Representative pictures of the motilities exhibited after 48 h of incubation are shown. We also constructed in-frame markerless dnaJ deletion mutants derived from the flagellaless strain GR4flaAB and the wild-type strain GR4 (flaAB ∆ dnaJ and G ∆ dnaJ strains, respectively). No major differences in growth were detected between the dnaJ mutants and their parental strains when serial dilutions of cultures grown to the mid-exponential phase were spotted onto MM plates (Figure S3a). On TY plates, a difference in colony size was observed between the mutants and the parental strains, with the dnaJ mutants developing smaller colonies ( Figure S3b ). Growth curves performed in liquid MM and TY revealed slower growth of the dnaJ mutants compared to their parental strains ( Figure S3c,d ). The retarded growth shown by the mutants was mainly due to their longer lag phases, which suggests that the inactivation of dnaJ impairs the adjustment of cells to new environmental conditions. The most affected strain during growth in liquid media was flaAB ∆ dnaJ, whereas G ∆ dnaJ and NS4 exhibited similar behavior to each other. The differences in growth observed between flaAB ∆ dnaJ and G ∆ dnaJ indicate that the role of DnaJ is more relevant for the flagellaless strain than for the wild-type strain. Moreover, the faster growth exhibited by NS4 compared to the deletion mutant flaAB ∆ dnaJ suggests the existence in NS4 of some DnaJ-related activity. The deletion mutants flaAB ∆ dnaJ and G ∆ dnaJ were used in surface motility assays in the presence of volatile 2-TDC. Like NS4, neither of the two dnaJ deletion mutants was able to spread over the surface of the medium in the presence of the volatile (Figure 3a,b). The fact that G ∆ dnaJ had the same phenotype as flaAB ∆ dnaJ in the presence of airborne 2-TDC was not unexpected since 2-TDC stimulates a mode of surface motility that is mostly independent of flagella. Surface translocation in response to 2-TDC was restored in the two deletion mutants carrying the dnaJ-complementing plasmid (Figure 3a,b), clearly Int. J. Mol. Sci. 2023,24, 5848 6 of 21 demonstrating that DnaJ plays an essential role in the flagella-independent surface motility promoted by 2-TDC in S. meliloti. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 23 the faster growth exhibited by NS4 compared to the deletion mutant flaABΔdnaJ suggests the existence in NS4 of some DnaJ-related activity. The deletion mutants flaABΔdnaJ and GΔdnaJ were used in surface motility assays in the presence of volatile 2-TDC. Like NS4, neither of the two dnaJ deletion mutants was able to spread over the surface of the medium in the presence of the volatile (Figure 3a,b). The fact that GΔdnaJ had the same phenotype as flaABΔdnaJ in the presence of airborne 2-TDC was not unexpected since 2-TDC stimulates a mode of surface motility that is mostly independent of flagella. Surface translocation in response to 2-TDC was restored in the two deletion mutants carrying the dnaJ-complementing plasmid (Figure 3a,b), clearly demonstrating that DnaJ plays an essential role in the flagella-independent surface motility promoted by 2-TDC in S. meliloti. Figure 3. Surface motility exhibited by S. meliloti dnaJ deletion mutants and complemented strains. (a) GR4flaAB-derivative strains and (b) GR4-derivative strains were assayed on MM (1% agar) in the presence or absence of volatile 2-TDC. About 20 µL of either a solution containing 1 µmol 2-TDC or ethanol (Control) was applied to the lid of the plates just before incubation. flaAB, strain GR4flaAB; wt, strain GR4; ΔdnaJ, strain GΔdnaJ; ev, empty vector pJB3; and C, complementing plasmid pJ-dnaJ. Representative pictures of the motilities exhibited after 48 h of incubation are shown. Swimming and swarming motilities exhibited by GR4 are abolished in GR4flaAB because of its inability to produce flagella [30]. Surprisingly, the lack of DnaJ also abolished the flagella-dependent swarming motility exhibited by the wild-type strain GR4 under permissive conditions (MM 0.6% agar) (Figure 4a), indicating that DnaJ is not only required for the bacterial response to 2-TDC but seems to have a broader role in surface translocation in S. meliloti. Interestingly, the deletion of dnaJ in GR4 only had a minor impact on swimming motility with a slight reduction in the swimming halo compared with that of the wild-type strain (10 ± 0.02 mm vs. 14 ± 0.02 mm; p < 0.001) (Figure 4b). Compatible with the results obtained in swimming, transmission electron microscopy observations revealed that the GR4-derived dnaJ mutant produced flagella, which were Figure 3. Surface motility exhibited by S. meliloti dnaJ deletion mutants and complemented strains. ( a ) GR4flaAB-derivative strains and ( b ) GR4-derivative strains were assayed on MM (1% agar) in the presence or absence of volatile 2-TDC. About 20 µ L of either a solution containing 1 µ mol 2-TDC or ethanol (Control) was applied to the lid of the plates just before incubation. flaAB, strain GR4flaAB; wt, strain GR4; ∆ dnaJ, strain G ∆ dnaJ; ev, empty vector pJB3; and C, complementing plasmid pJ-dnaJ. Representative pictures of the motilities exhibited after 48 h of incubation are shown. Swimming and swarming motilities exhibited by GR4 are abolished in GR4flaAB because of its inability to produce flagella [ 30 ]. Surprisingly, the lack of DnaJ also abolished the flagella-dependent swarming motility exhibited by the wild-type strain GR4 under permissive conditions (MM 0.6% agar) (Figure 4a), indicating that DnaJ is not only required for the bacterial response to 2-TDC but seems to have a broader role in surface translocation in S. meliloti. Interestingly, the deletion of dnaJ in GR4 only had a minor impact on swimming motility with a slight reduction in the swimming halo compared with that of the wild-type strain (10 ± 0.02 mm vs. 14 ± 0.02 mm; p< 0.001) (Figure 4b). Compatible with the results obtained in swimming, transmission electron microscopy observations revealed that the GR4-derived dnaJ mutant produced flagella, which were indistinguishable from those produced by GR4 (Figure 4c). These results indicate that the lack of DnaJ in S. meliloti does not abolish flagella production or function. The requirement of DnaJ for surface motility, which contrasts with its minor role for swimming, suggests that the chaperone participates in specific mechanisms involved in bacterial translocation across surfaces. Int. J. Mol. Sci. 2023,24, 5848 7 of 21 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 23 indistinguishable from those produced by GR4 (Figure 4c). These results indicate that the lack of DnaJ in S. meliloti does not abolish flagella production or function. The requirement of DnaJ for surface motility, which contrasts with its minor role for swimming, suggests that the chaperone participates in specific mechanisms involved in bacterial translocation across surfaces. Figure 4. Swarming, swimming, and flagella production in the wild-type strain GR4 (wt) and its dnaJ deletion derivative mutant (ΔdnaJ). (a) Surface motility assays on semisolid MM (0.6 %). Representative pictures of the motility exhibited by each strain after 48 h of incubation at 28 °C are shown. (b) Swimming motility assay in BM (0.3%). Pictures were taken 72 h after inoculation. (c) Transmission electron microscopy images showing flagella production of wild-type and dnaJ mutant cells. Scale bars are indicated. 2.4. DnaJ Participates in Salt Stress Tolerance in S. meliloti In Rhizobium tropici, dnaJ has been involved in salt tolerance [54]. To determine if dnaJ in S. meliloti plays a similar role, the growth of the dnaJ deletion mutant strains and the NS4 transposant was tested on MM supplemented with NaCl (300 mM) and compared to that of their parental strains (Figure 5a). In the absence of salt stress (Control), no differences in growth were observed among the different strains. In the NaCl-supplemented medium, the parental strains GR4 and GR4flaAB exhibited a slightly slower growth compared with the control medium, an effect that was noticeable after 5 days of incubation. When the plates were incubated for longer (17 days), no decrease in GR4 and GR4flaAB cell survival was observed in response to salt stress since a similar number of colonyforming units were detected in media with or without the stressor. In contrast, salt stress negatively impacted the growth of the dnaJ mutants, with differences depending on the strain (Figure 5a). Growth was especially impaired in the flaABΔdnaJ mutant, in which almost no growth could be detected after 5 days of incubation. Interestingly, the delay in growth of the NS4 transposant was less severe than for flaABΔdnaJ, indicating that the deletion of dnaJ in the GR4flaAB strain has a stronger impact on salt tolerance than the insertional inactivation of the gene. The GR4-derived dnaJ deletion mutant also showed slower growth than the parental strain in the presence of NaCl, but it was clearly less affected than the GR4flaAB-derived deletion mutant (flaABΔdnaJ). The difference between GΔdnaJ and flaABΔdnaJ was even more obvious when the NaCl-supplemented plates were incubated for longer. Whereas GΔdnaJ hardly showed any decrease in cell survival, in flaABΔdnaJ, the number of cells was reduced by approx. 2–3 orders of magnitude compared to GΔdnaJ or NS4 (Figure 5a). Figure 4. Swarming, swimming, and flagella production in the wild-type strain GR4 (wt) and its dnaJ deletion derivative mutant ( ∆ dnaJ). ( a ) Surface motility assays on semisolid MM (0.6 %). Representative pictures of the motility exhibited by each strain after 48 h of incubation at 28 ◦ C are shown. ( b ) Swimming motility assay in BM (0.3%). Pictures were taken 72 h after inoculation. (c) Transmission electron microscopy images showing flagella production of wild-type and dnaJ mutant cells. Scale bars are indicated. 2.4. DnaJ Participates in Salt Stress Tolerance in S. meliloti In Rhizobium tropici,dnaJ has been involved in salt tolerance [ 54 ]. To determine if dnaJ in S. meliloti plays a similar role, the growth of the dnaJ deletion mutant strains and the NS4 transposant was tested on MM supplemented with NaCl (300 mM) and compared to that of their parental strains (Figure 5a). In the absence of salt stress (Control), no differences in growth were observed among the different strains. In the NaCl-supplemented medium, the parental strains GR4 and GR4flaAB exhibited a slightly slower growth compared with the control medium, an effect that was noticeable after 5 days of incubation. When the plates were incubated for longer (17 days), no decrease in GR4 and GR4flaAB cell survival was observed in response to salt stress since a similar number of colony-forming units were detected in media with or without the stressor. In contrast, salt stress negatively impacted the growth of the dnaJ mutants, with differences depending on the strain (Figure 5a). Growth was especially impaired in the flaAB ∆ dnaJ mutant, in which almost no growth could be detected after 5 days of incubation. Interestingly, the delay in growth of the NS4 transposant was less severe than for flaAB ∆ dnaJ, indicating that the deletion of dnaJ in the GR4flaAB strain has a stronger impact on salt tolerance than the insertional inactivation of the gene. The GR4-derived dnaJ deletion mutant also showed slower growth than the parental strain in the presence of NaCl, but it was clearly less affected than the GR4flaABderived deletion mutant (flaAB ∆ dnaJ). The difference between G ∆ dnaJ and flaAB ∆ dnaJ was even more obvious when the NaCl-supplemented plates were incubated for longer. Whereas G ∆ dnaJ hardly showed any decrease in cell survival, in flaAB ∆ dnaJ, the number of cells was reduced by approx. 2–3 orders of magnitude compared to G ∆ dnaJ or NS4 (Figure 5a). To test whether the salt-sensitive phenotype of the mutants was due to the inactivation of dnaJ, complementation experiments were performed (Figure 5b). Under control conditions and after 5 days of incubation, no significant differences in growth were detected among the mutants, regardless of whether they were carrying the empty vector pJB3 (ev) or the dnaJ-expressing plasmid construct (C). With the same incubation times, salt stress notably reduced the growth of all strains but with a stronger effect in those harboring the empty vector (Figure 5b). A longer incubation revealed that salt stress not only reduced growth rate but also cell survival in the dnaJ mutants carrying the empty vector. Interestingly, the number of dnaJ mutant cells that survived during growth under salt stress conditions was lower for strains carrying the empty vector pJB3 and grown on media supplemented with antibiotics than for the same strains without pJB3 (Figure 5a,b). Most likely, the tetracycline added to the plates to ensure plasmid maintenance and/or the burden of carrying additional DNA represent added stresses for which cells might also require the DnaJ function. These conditions also revealed differences in cell survival between G ∆ dnaJ and NS4 that were not detected when the same strains without plasmid pJB3 were grown in media without antibiotics. The least sensitive strain was G ∆ dnaJ Int. J. Mol. Sci. 2023,24, 5848 8 of 21 followed by NS4. Strain flaAB ∆ dnaJ was the most sensitive with hardly any growth after 17 days of incubation. The salt-sensitive phenotype exhibited by the three mutants was recovered with the ectopic expression of dnaJ from the plasmid construct pJ-dnaJ (C), a complementation effect that also abolished differences between the mutants (Figure 5b). Altogether, these results demonstrate that DnaJ contributes to S. meliloti adaptation to salt stress. Our data also indicate that the role of DnaJ in managing salt stress is more important for the flagellaless strain GR4flaAB than for GR4. Moreover, the increased salt tolerance shown by NS4 compared to the deletion mutant flaAB ∆ dnaJ suggests that the putative DnaJ truncated protein likely produced by the transposant might retain some activity that helps to cope with salt stress. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 23 Figure 5. DnaJ loss-of-function in S. meliloti leads to increased sensitivity against salt stress. (a) Salt stress tolerance of GR4 (wt) and its dnaJ deletion mutant (ΔdnaJ), the flagellaless GR4flaAB (flaAB) and its dnaJ deletion (flaABΔdnaJ), and Tn5 insertion (NS4) mutants. (b) Salt stress tolerance of dnaJ mutants carrying the empty vector (ev) or the dnaJ-expressing plasmid construct (C). Cell suspensions in MM were prepared from mid-log cultures at the same OD 600 , and 10 µL of 10-fold serial dilutions were spotted on MM agar plates supplemented or not with 300 mM NaCl. Plates were incubated at 28 °C for 5 (5 d) or 17 days (17 d). To test whether the salt-sensitive phenotype of the mutants was due to the inactivation of dnaJ, complementation experiments were performed (Figure 5b). Under control conditions and after 5 days of incubation, no significant differences in growth were detected among the mutants, regardless of whether they were carrying the empty vector pJB3 (ev) or the dnaJ-expressing plasmid construct (C). With the same incubation times, salt stress notably reduced the growth of all strains but with a stronger effect in those harboring the empty vector (Figure 5b). A longer incubation revealed that salt stress not only reduced growth rate but also cell survival in the dnaJ mutants carrying the empty vector. Interestingly, the number of dnaJ mutant cells that survived during growth under salt stress conditions was lower for strains carrying the empty vector pJB3 and grown on media supplemented with antibiotics than for the same strains without pJB3 (Figure 5a,b). Most likely, the tetracycline added to the plates to ensure plasmid maintenance and/or the burden of carrying additional DNA represent added stresses for which cells might also require the DnaJ function. These conditions also revealed differences in cell survival between GΔdnaJ and NS4 that were not detected when the same strains without plasmid pJB3 were grown in media without antibiotics. The least sensitive strain was GΔdnaJ followed by NS4. Strain flaABΔdnaJ was the most sensitive with hardly any growth after 17 days of incubation. The salt-sensitive phenotype exhibited by the three mutants was recovered with the ectopic expression of dnaJ from the plasmid construct pJ-dnaJ (C), a complementation effect that also abolished differences between the mutants (Figure 5b). Figure 5. DnaJ loss-of-function in S. meliloti leads to increased sensitivity against salt stress. ( a ) Salt stress tolerance of GR4 (wt) and its dnaJ deletion mutant ( ∆ dnaJ), the flagellaless GR4flaAB (flaAB) and its dnaJ deletion (flaAB ∆ dnaJ), and Tn5insertion (NS4) mutants. ( b ) Salt stress tolerance of dnaJ mutants carrying the empty vector (ev) or the dnaJ-expressing plasmid construct (C). Cell suspensions in MM were prepared from mid-log cultures at the same OD 600 , and 10 µ L of 10-fold serial dilutions were spotted on MM agar plates supplemented or not with 300 mM NaCl. Plates were incubated at 28 ◦C for 5 (5 d) or 17 days (17 d). 2.5. DnaJ Protects S. meliloti against Oxidative Stress The DnaK/DnaJ chaperone protects Salmonella against oxidative stress created by reactive oxygen species that are produced by phagocytes in the innate host response [ 61 ]. In this study, we investigated whether DnaJ plays a role in the protection of S. meliloti against oxidative stress. For that, the growth of the mutants and their parental strains was tested on MM supplemented with different concentrations of hydrogen peroxide (H 2 O 2 ). When 100 µM H 2 O 2 was added to the medium, a slight delay in growth was observed for the three dnaJ mutants compared with the parental strains. Under these conditions, flaAB ∆ dnaJ was the most affected mutant, whereas NS4 and G ∆ dnaJ exhibited similar growth (Figure 6a). No effects on cell survival were observed for any of the strains under these conditions and, Int. J. Mol. Sci. 2023,24, 5848 9 of 21 with longer incubation time, the growth of the mutants was indistinguishable from that of the parental strains. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 23 Altogether, these results demonstrate that DnaJ contributes to S. meliloti adaptation to salt stress. Our data also indicate that the role of DnaJ in managing salt stress is more important for the flagellaless strain GR4flaAB than for GR4. Moreover, the increased salt tolerance shown by NS4 compared to the deletion mutant flaABΔdnaJ suggests that the putative DnaJ truncated protein likely produced by the transposant might retain some activity that helps to cope with salt stress. 2.5. DnaJ Protects S. meliloti against Oxidative Stress The DnaK/DnaJ chaperone protects Salmonella against oxidative stress created by reactive oxygen species that are produced by phagocytes in the innate host response [61]. In this study, we investigated whether DnaJ plays a role in the protection of S. meliloti against oxidative stress. For that, the growth of the mutants and their parental strains was tested on MM supplemented with different concentrations of hydrogen peroxide (H 2 O 2 ). When 100 µM H 2 O 2 was added to the medium, a slight delay in growth was observed for the three dnaJ mutants compared with the parental strains. Under these conditions, flaABΔdnaJ was the most affected mutant, whereas NS4 and GΔdnaJ exhibited similar growth (Figure 6a). No effects on cell survival were observed for any of the strains under these conditions and, with longer incubation time, the growth of the mutants was indistinguishable from that of the parental strains. Figure 6. DnaJ loss-of-function in S. meliloti leads to increased sensitivity against oxidative stress. (a) Oxidative stress tolerance of GR4 (wt) and its dnaJ deletion mutant (ΔdnaJ), the flagellaless GR4flaAB (flaAB) and its dnaJ deletion (flaABΔdnaJ), and Tn5 insertion (NS4) mutants. (b) Oxidative stress tolerance of dnaJ mutants carrying the empty vector (ev) or the dnaJ-expressing plasmid construct (C). Cell suspensions in MM were prepared from mid-log cultures at the same OD 600 , and 10 µL of 10-fold serial dilutions were spotted on MM agar plates supplemented or not with different concentrations of H 2 O 2 . Plates were incubated at 28 °C for 6 (6 d) or 17 days (17 d). When the concentration of H 2 O 2 was increased up to 150 µM, cell survival was significantly reduced in all the strains by three to five orders of magnitude. The decrease in Figure 6. DnaJ loss-of-function in S. meliloti leads to increased sensitivity against oxidative stress. (a) Oxidative stress tolerance of GR4 (wt) and its dnaJ deletion mutant ( ∆ dnaJ), the flagellaless GR4flaAB (flaAB) and its dnaJ deletion (flaAB ∆ dnaJ), and Tn5insertion (NS4) mutants. ( b ) Oxidative stress tolerance of dnaJ mutants carrying the empty vector (ev) or the dnaJ-expressing plasmid construct (C). Cell suspensions in MM were prepared from mid-log cultures at the same OD 600 , and 10 µ L of 10-fold serial dilutions were spotted on MM agar plates supplemented or not with different concentrations of H2O2. Plates were incubated at 28 ◦C for 6 (6 d) or 17 days (17 d). When the concentration of H 2 O 2 was increased up to 150 µ M, cell survival was significantly reduced in all the strains by three to five orders of magnitude. The decrease in cell survival was stronger for the dnaJ mutants than for their parental strains, indicating that DnaJ protects S. meliloti against oxidative stress. As observed for osmotic stress, the dnaJ mutants derived from GR4flaAB were more sensitive than the GR4-derived deletion mutant, highlighting the important role of DnaJ in the flagellaless strain. Interestingly, we found that GR4flaAB was slightly more tolerant to oxidative stress than wild-type GR4 (Figure 6a). This result was confirmed by determining the number of GR4 and GR4flaAB cells that survived after exposure to 1 mM H 2 O 2 for 90 min (Figure S4). In two independent experiments, the percent of GR4flaAB cells that survived the stress was 2.3and 3-fold higher than in GR4 (p< 0.0003), demonstrating the modest but significantly higher tolerance to oxidative stress of the flagellaless strain. Complementation experiments were also performed under these stress conditions (Figure 6b). Similarly, as was observed in response to salt stress (Figure 5b), with the same concentration of stressor (100 µ M H 2 O 2 ), the growth of strains carrying the empty vector on media with an antibiotic was much more severely affected than for the same strains without plasmids, indicating greater bacterial stress. The ectopic expression of dnaJ improved the tolerance of flaAB ∆ dnaJ to H 2 O 2 slightly, but not in NS4 or G ∆ dnaJ. This could indicate Int. J. Mol. Sci. 2023,24, 5848 16 of 21 4.2. Isolation of GR4flaAB Mutants Insensitive to 2-TDC Transposon Tn5mutagenesis of S. meliloti GR4flaAB was carried out as previously described [ 82 ] using strain S17-1 carrying pSUP2021 as the donor. After selection on MM plates supplemented with kanamycin, individual transposants were assayed for surface motility in the presence of volatile 2-TDC, as described below (Section 4.4). Those clones showing impaired surface motility in response to the volatile in three independent experiments were considered insensitive to 2-TDC and were selected for further characterization. The point of insertion for Tn5mutants was determined by arbitrary PCR, as described previously [83], using the primers specified in Table S2. 4.3. Construction of Plasmids and S. meliloti Strains Primers used to obtain the different plasmid constructs and S. meliloti mutant strains are listed in Table S2. To obtain plasmid pJ-dnaJ used in genetic complementation experiments, a 1241-bp fragment containing the S. meliloti GR4 dnaJ gene was amplified from genomic DNA using primers dnaJ-F and dnaJ-R. The resulting PCR product was first cloned into pCR2.1-TOPO and sequenced, and then subcloned as a HindIII fragment into pJB3Tc19 [ 84 ] keeping dnaJ transcription in the same orientation as lacZ. Mutant strains G ∆ dnaJ and flaAB ∆ dnaJ were obtained by allelic replacement of their wild-type dnaJ gene with a markerless in-frame deletion version of the locus. The deleted version of dnaJ was generated by overlap extension PCR using primers dnaJ-1 to dnaJ-3 and dnaJ-R. The resulting 1126-bp fusion product, in which a deletion of 768-bp was created in the coding sequence of dnaJ, was cloned into pCR2.1-TOPO and sequenced. Then, the insert was subcloned into the suicide vector pK18mobsacB as an EcoRI-HindIII fragment. The resulting plasmid pK18- ∆ dnaJ was mobilized to GR4 or GR4flaAB by biparental matings using S17-1, and double cross-over events were selected, as previously described [ 85 ]. PCR amplifications were performed with the proofreading Phusion high-fidelity DNA polymerase (Thermo Scientific, Waltham, Massachusetts, USA). Deletion mutant strains were checked by Southern hybridization using a specific probe. 4.4. Motility Assays Swimming motility was examined on Bromfield medium (BM) (0.04% tryptone, 0.01% yeast extract, and 0.01%CaCl 2· 2H 2 O) containing 0.3% Bacto agar after inoculation of 3µ L droplets of rhizobial cultures grown in TY broth (O.D. 600nm = 1). Surface motility was assayed, as previously described [ 30 , 36 ]. Briefly, S. meliloti cells grown in TY broth to late logarithmic phase (O.D. 600nm = 1–1.2) were pelleted, washed twice in MM, and resuspended in 0.1 volume of the latter medium. Two µ L aliquots of this bacterial suspension (ca. 2×107cells ) were dispensed and allowed to dry for 10 min onto the surface of plates containing 20 mL of semisolid MM containing 0.6% or 1% Noble Agar Difco (BD, Le Pont de Claix, France), which were previously air-dried at room temperature for 15 min. MM (0.6%) was used to test the surface motility of bacterial strains in the absence of 2-TDC, whereas MM (1%) was used to evaluate the response of S. meliloti strains to volatile 2TDC. For assays in the presence of volatile 2-TDC, 20 µ L of a 50 mM 2-TDC solution prepared in ethanol were applied onto the lid of the plate just before sealing with parafilm and incubation face-down. For control treatments, the same volume of pure ethanol was applied to the plates. 4.5. Stress Tolerance Assays S. meliloti cells grown in TY broth to an OD 600nm of 0.5–0.7 were pelleted, washed twice in MM, and resuspended in the same volume of the latter medium. These bacterial suspensions were serially diluted and 10 µ L of each dilution were spotted on solid MM plates supplemented with 300 mM NaCl (osmotic stress) or with different concentrations of H 2 O 2 (oxidative stress), as indicated. To determine cell survival after exposure to H 2 O 2 , S. meliloti cultures were grown in MM broth up to an OD 600nm of 0.5–0.7 and then diluted 1:100 in MM. These bacterial suspensions were split in half and H 2 O 2 was applied to one- Int. J. Mol. Sci. 2023,24, 5848 17 of 21 half of the cultures to a final concentration of 1 mM. The reaction mixtures were incubated for 90 min at 30 ◦ C. The survival rate was calculated by determining the colony-forming units (CFU) obtained on TY after plating serial dilutions of nontreated cultures and cultures exposed to H2O2. These assays were performed with three replicates per experiment. 4.6. Plant Assays Alfalfa seeds (Medicago sativa L. cv. Victoria) were surface-sterilized and germinated, as previously described [ 86 ]. For nodulation kinetics/infectivity tests, alfalfa seedlings were grown in hydroponic cultures under axenic conditions in glass tubes containing nitrogen-free nutrient solution (one plant per tube) [ 86 ]. Ten-day-old plants (a total of 20–24 replicates ) were inoculated with 1 mL of a rhizobial suspension containing 5×106cells . Prior to this inoculation, bacteria were grown to the exponential phase ( OD600 = 0.5–0.6 ) in TY broth and diluted 100-fold in sterile water. After inoculation, the number of nodules per plant was recorded daily. To obtain plant material for nitrogenase activity (see Section 4.7) and nodules for microscopy (Section 4.8), alfalfa plants were grown in Leonard jars, as previously described [ 30 ]. In this case, ten seedlings per jar were placed equidistantly from the center and immediately inoculated by applying 5 mL of a rhizobial suspension containing 1×103cells/mL to the center of the jar. The appearance and dry weight of the aerial part of plants, morphology, and the number of nodules, as well as nodule fresh weight and ARA were determined one month after inoculation. 4.7. Acetylene Reduction Activity (ARA) Nitrogenase activity was determined by acetylene reduction, as described previously [ 87 ]. Four nodulated alfalfa roots were incubated at room temperature in vials containing C 2 H 2 (10%, vol/vol) in air and sealed with serum caps. Aliquots of 0.1 mL were taken after 1 h of incubation and analyzed for ethylene in a Hewlett Packard 5890 gas chromatograph (Spring, TX, USA) equipped with a Poropak R column. 4.8. Microscopy Studies Bacterial cells were observed using transmission electron microscopy (TEM). Cells were obtained from colonies grown on 1% agar MM plates. Carbon-coated Formvar grids were placed for 5 min on top of a drop of water previously applied to the colony. The grids were then washed twice in water for 1 min and stained with 2% (wt/vol) uranyl acetate for 3 min. The grids were allowed to air-dry for at least 1 h and visualized using a JEOL JEM-1011 transmission electron microscope with a 100-kV beam at the Microscopy Service of the Estación Experimental del Zaidín, Granada, Spain. Images were captured using an Orius Gatan charge-coupled-device camera. Optical microscopy studies of alfalfa nodules were performed, as described previously [ 88 ]. Briefly, entire and half sections of nodules (30 days postinoculation) were immediately fixed in 4% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for 1 h under vacuum conditions overnight at 4 ◦ C. Samples were washed several times in 0.1 M cacodylate buffer, pH 7.2, dehydrated in acetone at progressively higher concentrations and embedded in Spurr resin (low viscosity embedding kit, by Dr. Spurr). The semithin sections (0.6–1 µ m) were obtained on a Leica EM UC7 ultramicrotome, stained with Toluidine blue, and viewed in an Olympus BX61 light microscope. The number of infected versus noninfected cells was determined using transverse sections taken from the middle region of at least three different nodules induced by each strain. 4.9. Bioinformatic Analysis Domain architecture of S. meliloti DnaJ was obtained using InterPro [ 89 ]. Multiple sequence alignment of DnaJ proteins was performed using MUSCLE hosted by EMBLEBI [90]. Int. J. Mol. Sci. 2023,24, 5848 18 of 21 Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24065848/s1. Author Contributions: Conceptualization, M.J.S.; formal analysis, P.B.-S., L.M.B.-R. and F.F.-R.; investigation, P.B.-S., J.J.D.-P., L.M.B.-R., C.C.-H., V.C., F.F.-R., S.A.-J., J.-M.V. and M.J.S.; writing—original draft preparation, M.J.S.; writing—review and editing, P.B.-S., J.J.D.-P., L.M.B.-R., C.C.-H., V.C., F.F.-R., S.A.-J., J.-M.V. and M.J.S.; visualization, P.B.-S., J.J.D.-P., L.M.B.-R., F.F.-R., S.A.-J. and M.J.S.; supervision, J.-M.V. and M.J.S.; funding acquisition, J.-M.V. and M.J.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”, grant numbers PGC2018-096477-B-I00, PID2019-107634R, and PID2021-123540NB-I00. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank Alicia Rodríguez for technical assistance at the Microscopy Service of the Estación Experimental del Zaidín (CSIC), Granada, Spain, and the Microscopy Research Service of the “Centro de Investigación, Tecnología e Innovación” (CITIUS) of the University of Seville. We also thank Pieter van Dillewijn for his critical reading of the manuscript and helpful discussions. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. Poole, P.; Ramachandran, V.; Terpolilli, J. Rhizobia: From saprophytes to endosymbionts. Nat. Rev. Microbiol. 2018 ,16, 291–303. [CrossRef] [PubMed] 2. Udvardi, M.; Poole, P.S. Transport and metabolism in legume-rhizobia symbioses. Annu. Rev. Plant. 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