The Type VI Secretion System of Sinorhizobium fredii USDA257 Is Required for Successful Nodulation With Glycine max cv Pekin

1 of 17 Microbial Biotechnology, 2025; 18:e70112 https://doi.org/10.1111/1751-7915.70112 Microbial Biotechnology RESEARCH ARTICLE OPEN ACCESS The Type VI Secretion System of Sinorhizobium fredii USDA257 Is Required for Successful Nodulation With Glycine max cv Pekin PedroJoséReyes-Pérez1 | IreneJiménez-Guerrero1 | AnaSánchez-Reina1 | CristinaCivantos1 | NataliaMoreno-deCastro1 | FranciscoJavierOllero1 | JacintoGandullo2 | PatriciaBernal1 | FranciscoPérez-Montaño1 1Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain | 2Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Sevilla,Spain Correspondence: Irene JiménezGuerrero ([email protected]) | Patricia Bernal ([email protected]) | Francisco PérezMontaño ([email protected]) Received: 4 December 2024 | Revised: 29 January 2025 | Accepted: 3 February 2025 Funding: This work was funded by grants from the State Subprogram for Knowledge Generation from the Spanish Minister of Science, Innovation and Universities (MICIU), the Spanish State Research Agency (AEI) and the European Union (UE) (MICIU/AEI/10.13039/501100011033). Keywords: competition| legume| nodulation| rhizobium| symbiosis| T6SS effectors| type VI secretion system ABSTRACT The symbiotic relationship between rhizobia and legumes is critical for sustainable agriculture and has important economic and environmental implications. In this intricate process, rhizobial bacteria colonise plant roots and induce the formation of specialised plant organs, the nodules. Within these structures, rhizobia fix environmental nitrogen into ammonia, significantly reducing the demand for synthetic fertilisers. Multiple bacterial secretion systems (TXSS, Type X Secretion System) are involved in establishing this symbiosis, with T3SS being the most studied. While the Type 6 Secretion System (T6SS) is known as a “nanoweapon” commonly used by diderm (formerly gramnegative) bacteria for interbacterial competition and potentially manipulating eukaryotic cells, its precise role in legume symbiosis remains unclear. Sinorhizobium fredii USDA257, a fastgrowing rhizobial strain capable of nodulating diverse legume plants, possesses a single T6SS cluster containing genes encoding structural components and potential effectors that could target plant cells and/or act as effectorimmunity pairs. Our research reveals that this T6SS can be induced in nutrientlimited conditions and, more importantly, is essential for successful nodulation and competitive colonisation of Glycine max cv Pekin. Although the system did not demonstrate effectiveness in eliminating competing bacteria invitro, its active presence within root nodules suggests a sophisticated role in symbiotic interactions that extends beyond traditional interbacterial competition. 1 | Introduction Rhizobia are αand ßProteobacteria soilborne microorganisms frequently found on leguminous plant roots and rhizosphere. This environment is highly appropriate for rhizobia development and, eventually, they form a symbiotic relationship with the leguminous plant. The rhizosphere protects rhizobia from desiccation, extreme temperatures and light stress. At the same time, legumes supply bacteria with nutrients exuded from the roots, including amino acids, organic acids, sugars, aromatic compounds and secondary metabolites (Walker et al. 2003). Flavonoids are secondary metabolites exudate by legume plants that initiate the molecular dialogue between them and rhizobia. This dialogue culminates with the formation of new root organs called nodules, where the bacterial reduction of atmospheric nitrogen to the most needed form (ammonia) takes place (Oldroyd et al. 2011). Therefore, when recognised by the appropriate rhizobia, This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2025 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd. 2 of 17 Microbial Biotechnology, 2025 flavonoids induce the production of rhizobial signal molecules, called nodulation factors (Nod factors). Nod factors can be specifically recognised by legume plants, initiating the nodulation process that culminates with nodule development, its occupation and the transformation of the rhizobium into a nitrogenfixing cell, the bacteroid (Oldroyd2013; Zipfel and Oldroyd2017). Besides the molecular recognition mediated by flavonoids and Nod factors, the success of the nodulation process also depends on secretion system effectors. Thus, in the last 15 years, effector proteins secreted through the rhizobial type III secretion system (T3SS) have been proven important, and in some cases essential, for the symbiotic performance of several rhizobial genera, such as Sinorhizhobium, Rhizobium, Bradyrhizobium and Mesorhizobium (JiménezGuerrero et al. 2022, 2021; LópezBaena et al. 2016). Interestingly, a novel secretion system, the type VI secretion system (T6SS), has been described over the past two decades, and may play a complementary role to the effects observed by T3SS effectors. This machinery was first identified in Rhizobium leguminosarum (Bladergroen etal.2003), although the term T6SS was not established until 2006 when the T6SS of Vibrio cholerae and Pseudomonas aeruginosa were simultaneously characterised (Mougous etal.2006; Pukatzki etal.2006). The T6SS is present in ca. 25% of diderm (formerly gramnegative) bacteria, mainly in the Pseudomonadota (formerly Proteobacteria) phylum, where the α- , βand γproteobacteria classes are included (Boyer etal.2009). Generally, this system secretes effectors/ toxins into prokaryotic cells, playing a critical role in interbacterial competition (Ho etal.2014). However, some T6SS effectors target eukaryotic cells and can manipulate the host during an infective process (Hachani etal.2016). The specific role of T6SS effectors in rhizobial symbiosis is understudied, although recent work suggests that these proteins could exert neutral, positive, or negative effects, depending on the symbiotic pair. Thus, it has been reported that the T6SS of Paraburkholderia phymatum and Azorhizobium caulinodans do not appear to be directly implicated in the symbiotic effectiveness between these rhizobia and the legumes Vigna unguiculata and Sesbania rostrata, respectively. However, both T6SSs are involved in symbiotic competitiveness against other rhizobial species for nodulation (De Campos etal.2017; Lin etal.2018). In contrast, in both Rhizobium etli Mim1 and Bradyrhizobium sp. LmicA16, the T6SS is required for efficient nodulation with Phaseolus vulgaris and Lupinus spp., respectively (SalineroLanzarote etal.2019; Tighilt etal.2022). In the case of R. etli Mim1, this system is expressed at high cell densities, in the presence of root exudates and within hostplant nodules (SalineroLanzarote etal.2019). Interestingly, one of the T6SS effectors secreted by this system, Re78, is an antimicrobial toxin involved in interbacterial competition and nodule occupancy (De Sousa etal.2023). On the contrary, Rhizobium leguminosarum RBL5787 is unable to form nitrogenfixing nodules on peas (Pisum sativum) due to the presence of a functional T6SS (Bladergroen etal.2003). Structurally, the T6SS is a multiprotein complex composed of 13 main constituents. The genes encoding these proteins are grouped into genetic clusters and named tss (type six secretion) (Ho etal.2014). In some cases, an additional set of genes, named tag genes (type six accessory genes), encodes accessory proteins with regulation and finetuning functions (Aschtgen etal.2010; Bernal etal.2021; Hsu etal.2009; Lin etal.2018; Santin etal.2018). The T6SS is structured into three main compartments: the membrane complex formed by TssJ, TssL and TssM, the baseplate and the tail, that is formed by an inner tube (Hcp), surrounded by a contractile sheath and ended in a needleshaped tip (VrgG and PAAR). The T6SS effectors can be transported inside the tube, or connected to the tip, being released into the intracellular environment of the target cell upon sheath contraction. (Allsopp and Bernal 2023). Genes encoding effector/toxin proteins can be found within the T6SS cluster or are scattered throughout the genome. These genes are usually located downstream of those encoding VgrG, Hcp, and/or PAAR proteins. In some cases, T6SS effectors are encoded by the same gene that encodes Hcp, VrgG or PAAR proteins at the 3' end. Structural proteins with a Cterminal cytotoxic domain are commonly termed as “specialised” Hcp, VrgG or PAAR (Allsopp and Bernal2023). Antibacterial T6SS effectors can target the cell envelope (peptidoglycan hydrolases, phospholipases and poreforming effectors), or bacterial cytoplasm (nuclease and cofactor degrader effectors) (Allsopp and Bernal2023; GonzálezMagaña etal.2022). Bacteria with a functional T6SS produce immunity proteins to protect from sister cell attacks and selfintoxication. T6SS effectors delivered into eukaryotic host cells are less widespread than antimicrobial effectors, but the few identified to date are involved in different steps of host manipulation to promote bacterial infection (Hachani etal.2016). Sinorhizobium fredii USDA257, hereafter USDA257, is a fastgrowing rhizobium that was isolated from wild soybean (Glycine soja), but is also able to form nitrogenfixing nodules in a wide variety of legume species, such as G. max, S. rostrata, V. radiata, P. vulgaris, Cajanus cajan, Lotus japonicus and L. burtii (Pueppke and Broughton1999). USDA257 is one of the most versatile rhizobia, along with other strains of the same species, S. fredii NGR234 and S. fredii HH103, and is, therefore, a model organism in many laboratories. Interestingly, among these strains, only USDA257 contains both the T3SS and T6SS machinery. The T3SS of USDA257 has been extensively studied, playing a prominent role in symbiosis as well as in determining its nodulation host range (Staehelin and Krishnan 2015). In USDA257, legume recognition of T3SS effectors can exert positive (induction of nodulation) or negative (inhibition of nodulation) extreme effects, depending on the plant cultivar (JiménezGuerrero etal.2022). However, the ecological and physiological functions of the T6SS are completely unknown in USDA257 in particular and in the Sinorhizobium genera in general. In this study, we identified and characterised the USDA257 T6SS, which exhibits structural proteins with novel characteristics, implying an apparatus with a distinctive assembly and a definite set of T6SS effectors. We showed that USDA257 T6SS is induced in nutrientlimited media during the stationary phase of growth and, more importantly, when this strain colonises legume root nodules. Plant assays demonstrated that USDA257 utilises this protein secretion system to improve symbiotic effectiveness and competitiveness in G. max cv Pekin. 17517915, 2025, 3, Downloaded from https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70112 by Readcube (Labtiva Inc.), Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3 of 17 2 | Materials and Methods 2.1 | Bacterial Strains and Growth Conditions Bacterial strains used in this work are listed in Table S1. Rhizobial strains used in this study were grown at 28°C on tryptone yeast (TY) medium (Beringer 1974), yeast extract mannitol (YM) medium (Vincent 1970) or minimal (MM) medium (Robertsen etal.1981) with two different mannitol concentrations (3 or 10 g mL−1, YM3/MM3 or YM10/MM10, respectively). Agrobacterium tumefaciens and Pectobacterium carotovorum strains were cultured in lysogeny broth (LB) (Lennox LB 5 g L−1 NaCl) and agar (1.5% w/v) (Sambrook etal.1989) at 28°C. Antibiotics were used at (μg mL−1): carbenicillin (Cb), 100 for Escherichia coli, rifampicin (Rif), 50 for S. fredii; tetracycline (Tc), 10 for S. fredii and E. coli; kanamycin (Km), 50 for S. fredii and 25 for E. coli; ampicillin (Ap) 100 for S. fredii and E. coli; gentamycin (Gm), 10 for A. tumefaciens, P. carotovorum, S. fredii and E. coli; spectomycin (Spc), 50 for S. fredii and E. coli; piperacillin (Pip), 15 for S. fredii and E. coli. Genistein, a Sinorhizobium fredii nod geneinducing flavonoid, was dissolved in ethanol at a concentration of 1 μg mL−1 to obtain a final concentration of 3.7 μM. 2.2 | Construction of Plasmids and Bacterial Strains Plasmids and primers used in this study are listed in TablesS2 and S3. To construct USDA257 tssA mutant, an internal fragment of this gene was amplified using primers P1 and P2, digested with EcoRI and BamHI and cloned into the pK18mob suicide vector, which was previously digested with the same enzymes, obtaining plasmid pMUS1480. This construct was employed to generate the tssA mutant strain in USDA257 by inserting the pMUS1480 vector into the USDA257 chromosome via single recombination at the tssA locus. The mutant was confirmed by Southern blot, PCR and sequencing. For Southern blot hybridization, DNA was blotted onto HybondN nylon membranes (Amersham, UK), and the DIGDNA labeling method from Roche (Switzerland) was used following the manufacturer's instructions. The Southern blot results confirmed that the band hybridising with the probe was approximately 3.7 kb larger in the mutant strain than in the wildtype strain, as expected after the insertion of the 3.7 kb pMUS1480 vector (FigureS1). To analyse the expression profile of the T6SS cluster, the promoter region of USDA257 ppkA gene was amplified with primers P3 and P4 (TableS3) resulting in a 575 bps fragment that was digested with EcoRI and XbaI and cloned into the mcs of plasmid pMP220, upstream the lacZ gene, using the same enzymes and obtaining plasmid pMP220::PppkA. The USDA257 wildtype strain was conjugated with plasmids pMP220::PppkA and pMP240 (de Maagd etal.1988), which contain transcriptional fusions of the ppkA promoter from USDA257 and the nodA promoter of R. leguminosarum, respectively, to the lacZ gene. The strain carrying the pMP240 plasmid served as a positive control, whereas USDA257, containing the empty plasmid pMP220 (Spaink etal.1987), was used as a negative control. To study the expression of USDA257 T6SS during the symbiotic process, we constructed a strain with a dual reporter system that expressed both constitutive GFPand T6SSresponsive mRFP (pBBR4::Pkan::GFPPppkA::mRFP). Construction of the dualreporter vector was performed using a previously developed system (Samal and Chatterjee2021). The Samal and Chatterjee system is based on the pBBR1MCS4 plasmid, in which a constitutively expressed GFP gene (Pkan::GFP) was divergently cloned into an mRFP gene preceded by the promoter region of the eng gene from Xanthomonas (Peng). To construct the T6SS responsive mRFP reporter, we first removed Peng from the original vector using EcoRI to produce a promoterless (Pw/o) mRFP gene in the control vector pBBR4::Pkan::GFPPw/o::mRFP. Then, we cloned the promoter region of the USDA257 ppkA gene (PppkA), amplified using primers P5P6 (Table S3) at the EcoRI site to engineer the vector pBBR4::Pkan::GFPPppkA::mRFP. The insertion was confirmed by PCR using primers P7P8 and sequenced by Macrogen Inc. These vectors were transferred into the USDA257 strain to study the expression of T6SS in the nodules. All plasmids described in this section were transferred from E. coli to Sinorhizobium strains by triparental conjugation (Simon 1984) using E. coli DH5α harbouring the plasmid pRK2013 as the helper strain (Figurski and Helinski 1979). Recombinant DNA techniques were performed according to the general protocols of (Sambrook etal.1989). For the production of the antibody against USDA257 Hcp protein, the hcp gene was cloned into an expression vector that allowed the production of a Cterminal 6xHis tagged protein. First, the hcp gene without the stop codon was amplified by PCR with primers P9 and P10 (TableS3) and cloned into the entry Gateway pDONR207 vector, which was replicated in E. coli DB3.1. The insertion was confirmed by PCR and sequenced using the primers P11 and P12. The fragment was then subcloned into the destination pETDEST42 Gateway vector following the manufacturer's instructions (Invitrogen, USA). Then, the generated plasmid pET42hcp was transferred to E. coli BL21 (DE3) by transformation (Sambrook etal.1989) for protein expression. 2.3 | Bioinformatical Analysis Sequences of 160 TssB proteins from 153 strains, belonging to 12 genera were obtained and compiled using the BLASTp tool from the NCBI website (TableS4; Boratyn etal. 2013). Sequences were aligned using ClustalW software (Sievers et al. 2011), and the phylogenetic tree was constructed using MEGA7 (Kumar et al. 2016), applying the Maximum Likelihood algorithm and a JTT matrix model (bootstrap value = 500). The phylogenetic tree was customised using the iTOL website (Letunic and Bork2016). Amino acid sequence searches were performed using SMART (Letunic etal.2015) and Pfam (Finn etal.2016). The Protein Homology/Analogy Recognition Engine (Phyre2) server was used to perform a templatebased approach to predict protein structural homology (Kelley etal.2015). An additional analysis to identify structural homologues was performed using the Foldseek algorithm (Van Kempen et al. 2024), which is a structural 17517915, 2025, 3, Downloaded from https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70112 by Readcube (Labtiva Inc.), Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 17 Microbial Biotechnology, 2025 alignment tool based on a structural protein alphabet of tertiary interactions that operates using predicted models from the Alphafold database (Abramson etal.2024). The PSORTb server was used to predict the subcellular locations of the proteins (Yu etal.2010). Conserved domains to identify orphan effector genes were identified using the Batch CDSearch Tool from the NCBI website using the VgrG, Hcp, PAAR, MIX, FIX, RIX, and PIX queries. Linear comparisons of multiple genomic T6SS loci (vgrG regions) were performed using clinker and clustermap.js (Gilchrist and Chooi2021). 2.4 | βGalactosidase Assays Overnight cultures of S. fredii strains carrying the pMP220 plasmid and derived pMP220::PppkA and pMP240 were diluted to a final turbidity (A600) of 0.01 in fresh medium containing tetracycline, and cultures were grown at 28°C and 180 rpm. At different time points, aliquots were taken to measure βgalactosidase activity in permeabilized whole cells, as described by (Zaat etal.1987). Units of βgalactosidase activity were calculated according to the method described by Miller(1972). At least three independent assays were performed for each case, and the standard errors of the mean were calculated. 2.5 | RNA Extraction and qRTPCR Experiments Total RNA was isolated using a High Pure RNA Isolation Kit (Roche, Switzerland), according to the manufacturer's instructions. Verification of the amount and quality of total RNA samples was carried out using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA) and a Qubit 2.0 Fluorometer (Invitrogen, USA). Four independent total RNA extractions were performed for each condition. The (DNAfree) RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative PCR was performed using a LightCycler 480 (Roche, Switzerland) with the following conditions: 95°C for 10 min; 95°C for 30 s; 50°C for 30 s; 72°C for 20 s; 40 cycles, followed by a melting curve profile from 60°C to 95°C to verify the specificity of the reaction. The USDA257 16S rRNA gene was used as an internal control to normalise gene expression. The foldchanges in four biological samples with three technical replicates for each condition were obtained using the ΔΔCt method (Pfaffl2001). The selected genes and primers (P13– P16) are listed in TableS3. 2.6 | S. fredii Hcp Antibody Production The E. coli BL21(DE3) strain carrying the plasmid pET42hcp was inoculated in 5 mL of LB supplemented with ampicillin and incubated overnight at 37°C and 200 rpm. The culture was then transferred to 200 mL of fresh medium and incubated under the same conditions. When OD600nm reached 0.6, protein expression was induced with 1 mM isopropyl βD1thiogalactopyranoside (IPTG). The cultures were then grown for 4 h at 37°C and 200 rpm. Cells were harvested by centrifugation (5.000 g, 20 min, 4°C) and the pellet was resuspended in a buffer containing 50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 10 mM imidazole, 1 mg/mL lysozyme and a Protease Inhibitor Cocktail used following the manufacturer's instructions (SigmaAldrich, USA). The suspension was incubated for 30 min at RT and sonicated on ice five times for 30 s, with 30 s cooling intervals between sonication treatments. Cell debris was eliminated by centrifugation (10.000 g, 30 min, 4°C). The clarified lysate was filtered with a 0.45 nm filter and incubated in a column containing 2.5 mL of NiSepharose resin (Protino NiNTA, MachereyNagel, Duren, Germany). This column was previously equilibrated with 10 volumes of NPI buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole), allowing the binding of Histagged Hcp to Protino NiNTA agarose. The column was washed with 10 volumes of buffer NPI containing 100 mM imidazole, and Histagged Hcp protein was eluted from the resin with 1 mL of buffer NPI containing 500 mM imidazole. Following the manufacturer's instructions, Histagged Hcp protein was washed with PBS and concentrated using an Amicon Ultra Centrifugal Filter Unit (Millipore Sigma, Burlington, MA, USA). The expression and purification of Histagged Hcp were verified by SDSPAGE and confirmed by western blotting using a Histag monoclonal antibody (Cell Signalling Technology, USA). Polyclonal antibody production was carried out by the “Centro de Experimentación Animal Óscar Pintado” from the University of Seville (Spain) following the procedure described by (Vidal etal.1980) (1 rabbit; 4 protein injections –500, 125, 125 and 125 μg– and 2 bleedings). 2.7 | Purification and Analysis of Extracellular Proteins Extracellular and intracellular proteins were recovered following the protocol described by Hachani etal.(2011), with some modifications. Briefly, 20 mL of the different rhizobial cultures grown on an orbital shaker (200 rpm) at 28°C for 48 h with an adjusted A600 of 1 were centrifuged for 20 min at 10.000 g at 4°C. Bacterial pellets were normalised and resuspended in 200 μL of sample buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS [w/v], 10% glycerol [v/v], 5% βmercaptoethanol [w/v], and 0.001% bromophenol blue [w/v]). To eliminate any remaining cells in the supernatant, three additional sequential centrifugations (20 min, 10.000 g, 4°C) were performed. One volume of 1.8 mL from each culture supernatant was collected and precipitated with trichloroacetic (TCA) acid overnight at 4°C. The mixtures were centrifuged for 30 min at 16.000 g and 4°C. Dried pellets, previously washed with 90% acetone, were resuspended in the sample buffer. For immunostaining, proteins were separated on SDS 20%–4% (w/v) polyacrylamide gels (BioRad, USA) and electroblotted onto ImmunBlot polyvinylidene difluoride membranes (BioRad) using a Mini TransBlot electrophoretic transfer cell (BioRad). Membranes were blocked with TBS containing 5% (w/v) milk powder and then incubated with the previously described antibody raised against USDA257 Hcp protein diluted 1:1000 in the same solution. An antirabbit HRPlinked antibody (Cell Signalling Technology, USA) was used as a secondary antibody, developed using HRP Immobilon Forte (Merck, Germany) according to the manufacturer's instructions, and visualised using an ImageQuant LAS 500 imaging workstation instrument (GE Healthcare, USA). 17517915, 2025, 3, Downloaded from https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70112 by Readcube (Labtiva Inc.), Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 5 of 17 2.8 | Interbacterial Competition Assays In vitro competition assays between USDA257 and different rhizobia and plant pathogens were performed as previously described (Civantos etal.2024), with some modifications. Briefly, overnight bacterial cultures were washed and adjusted to an OD600 of 1.0 in sterile PBS, and mixed at a 1:1 ratio between USDA257 as the attacker and A. tumefaciens, P. carotovorum and S. fredii HH103 as preys. 100 μL of the mixtures were plated on YM3 (for A. tumefaciens and P. carotovorum) or MM3 media (for S. fredii HH103) and incubated at 30°C for 24 or 48 h, respectively. Subsequently, the competitions were collected using an inoculating loop and resuspended in 1 mL of sterile PBS. The outcome of the competition was quantified by counting the colonyforming units (CFUs) using antibiotic selection of the input (time = 0 h) and output (time = 24–48 h). A. tumefaciens and P. carotovorum prey strains harboured the plasmid pRL662, which confers resistance to gentamicin and was used for antibiotic selection, whereas USDA257 was naturally resistant to streptomycin. Three independent biological experiments were performed. 2.9 | Plant Tests To evaluate symbiotic effectiveness in nodulation assays, wildtype and mutant strains were grown in YM3 medium. Surfacesterilised seeds of G. max cv Pekin were pregerminated and placed in sterilised Leonard jars, containing Fårhaeus Nfree solution (Vincent 1970). Germinated seeds were inoculated with 1 mL of bacterial culture at an OD600 of 0.6. Growth conditions were 16 h at 26°C in the light and 8 h and 18°C in the dark, with 70% humidity. Nodulation parameters were evaluated after 6 weeks. The shoots were dried at 70°C for 48 h and then weighed. Nodulation experiments were performed three times, with five technical replicates for each treatment. Competition experiments for nodulation (competitiveness) on G. max cv Pekin were performed using the parental and the T6SS mutant strains of USDA257. These bacteria were grown to 109 cells mL−1, and four to five Leonard jar assemblies containing two plant seedlings were inoculated with 1 mL of a mixture of bacterial competitors at ratios of 1:1, 1:10 and 10:1. Plants were grown for 6 weeks in a plant growth chamber under the growth conditions described above. To identify bacteria occupying the nodules, 100 G. max cv Pekin nodules from each treatment were surface sterilised by immersing them in 5% [w/v] sodium hypochlorite for 5 min, followed by five washing steps in sterilised distilled water. The effectiveness of the surfacesterilising treatment was checked by inoculating TY plates with 20μl aliquots from the last washing step. Individual surface sterilised nodules were crushed in 30 μL of sterilised distilled water, and 20 μL aliquots were used to inoculate TY plates. Nodule occupancy was determined by assessing the ratio of differential antibiotic resistance of isolates (only rifampicin for the wildtype strain and rifampicin and kanamycin for the T6SS mutant). At least 10 colonies from each isolate were analysed to check the possibility of nodules containing both inoculants. For nodule occupancy visualisation by fluorescence microscopy, 30dayold L. burttii nodules formed in plants inoculated with USDA257 harbouring the dual fluorescent reporter, were embedded in 6% agarose in water and sliced in thick layer sections (50 μm) using a Leica VT 1000S vibratome (Wetzlar, Germany). Sections of nodules were stained with 0.04% calcofluor and observed by using a Leica Stellaris 8 SPE Confocal Microscope (Leica Microsystems) (Jena, Germany) fluorescence microscope as previously described (Kawaharada etal.2017). The image's contrast and intensity were adjusted using ImageJ software (Schindelin etal.2015). 2.10 | Statistical Analysis The statistical tests performed in this work are indicated in the figure legends and were done using Prism 8 (GraphPad, La Jolla, CA, USA). 3 | Results 3.1 | GenomeWide Screening for T6SSs in N2Fixing Bacteria A phylogenetic analysis determines the presence of a T6SS cluster in at least 160 N2fixing bacterial species from 13 different genera all belonging to the Phylum Pseudomonadota, including the main genera containing rootnoduleforming bacteria (TableS4). The selected species belong to the order Hyphomicrobiales (better known as Rhizobiales) from the αProteobacteria class and the order Bulkholderiales from the βProteobacteria class. We have included welldescribed Agrobacterium and Pseudomonas T6SSs to identify and locate the previously described T6SS phylogenetic groups (Bernal etal.2018). The tree displayed in Figure1 contains 160 TssB proteins, showing the phylogenetic distribution of selected rhizobia T6SSs. Our analysis shows that rhizobial T6SSs are distributed among the five main clades previously described (Boyer etal.2009). Most of them, 140 (87.5%) belong to groups 3 (54, 33.75%) and 5 (86, 53.75%) (Figure1). Group 3 contains a great variety of species from the Ensifer (=Sinorhizobium), Rhizobium, Bradyrhizobium and Mesorhizobium genera among others, as well as the firstdiscovered P. aeruginosa H1T6SS (Mougous etal.2006). Importantly for this work, The T6SS of USDA257 belongs to phylogenetic Group 3. Group 5 contains mostly Rhizobium and Azorhizobium species and includes the wellstudied A. tumefaciens T6SS (Ma et al. 2014; Wu etal.2008). Although phylogeneticallydistance to the abovementioned genera Cupriavidus and Parabulkholderia species can be found in both groups. Minority Groups 2 and 4 principally contain Paraburkholderia species (2, 1.25% each) and Group 1 (14, 9.75%) contains predominantly species of the Methylobacterium genus (Figure1). The number of T6SS clusters in a strain could range from 1 to 5. In some bacteria groups, namely Pseudomonas, they commonly contain 2 or 3 but in a broader group such as phytobacteria, only an estimated 7% of strains contain more than one cluster (Bernal etal.2018, 2017). This number is even smaller among rhizobial T6SSs where most strains contain only a single T6SS cluster (TableS4; Figure1). 17517915, 2025, 3, Downloaded from https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70112 by Readcube (Labtiva Inc.), Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 17 Microbial Biotechnology, 2025 3.2 | The Reference Rhizobial Strain S. fredii USDA257 Possesses a Complete T6SS Cluster Inspection of the USDA257 genome revealed 26 T6SSrelated ORFs located in a chromosomal cluster (Figure2A; TableS5). Thirteen genes encode the structural proteins required for a functional T6SS, including the membrane complex (TssJLM), the base plate (TssKEFG), the tail (TssBC and Hcp) and the ATPase that recycles the system (ClpV). We further identified genes encoding a previously described regulatory phosphorylation cascade (Mougous etal.2006), including the threonine kinase/phosphatase pair (PpkAPppA), the phosphorylation substrate (Fha) and TagF, a posttranslational repressor that regulates T6SS via Fha interaction (Lin etal.2018). TssJ, TssL and TssM are the core components of the T6SS membrane complex that docks the system to the cell envelope (Allsopp and Bernal2023). According to in silico predictions, the USDA257 TssL (DotU and OmpA domains) and TssM (three IcmF domains) are anchored to the inner membrane through one and three transmembrane helices, respectively, and form a channel through the cell envelope by interaction with each other and the outer membrane lipoprotein TssJ (SciN domain) (Figure2B). In an indepth analysis of USDA257 T6SS components, we identified, in addition to the three core components of the membrane complex, two associated accessory proteins named TagM and TagN (Figure2B). TagM, a 821 amino acids protein, shares a certain degree of homology with both TssL and TssM (Figure2B; TableS5; FigureS2). This protein contains a FIGURE 1 | Phylogenetic tree of T6SSs of nitrogenfixing bacteria. Maximum likelihood tree with 500 bootstrap replicates was built with Mega 7 software for the core component protein TssB. T6SS cluster nomenclature shows the main phylogenetic clusters (Boyer etal.2009). Branches with black circles indicate a confidence level higher than 0.75. Five phylogenetic groups are highlighted: Group 1 (green), Group 2 (red), Group 3 (yellow), Group 4 (blue) and Group 5 (purple). The bacterium of the genus Bacteroides represents the tree root. The position of S. fredii USDA257 T6SSs is marked with a grey arrow. 17517915, 2025, 3, Downloaded from https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70112 by Readcube (Labtiva Inc.), Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 7 of 17 cytosolic DotU domain in the Nterminal part, a single transmembrane domain and two structural IcmF domains. Its transmembrane domain and the absence of a specific signal peptide indicate this protein might be anchored to the inner membrane (Figure2B). TagN, a 248 amino acids protein, contains a signal peptide in the Nterminal end and an OmpA peptidoglycan (PG)- binding domain in the Cterminal part of the protein similarly to TssL (Figure2B; TableS5; FigureS2). Lastly, we have identified a gene encoding an additional accessory protein we have named tagY (Figure 2A). TagY presents homology with FIGURE 2 | The T6SS of S. fredii USDA257. (A) Organisation of the T6SS cluster of USDA257. Genes that encode the components of the membrane complex (tssJLM) and accessory proteins (tagMLY) are represented in beige. Genes coding for components of the base plate (tssEFGK) are shown in purple and the tssA gene in grey. Genes tssB and tssC encoding the contractile sheath are displayed in light green. The hcp gene, that encodes the protein forming the inner tube is represented in dark blue. Genes that encode regulatory proteins (tagF, fha, ppkA and pppA) are shown in light blue. The clpV gene which codes for the ClpV ATPase is displayed in red. Genes coding for possible effectors of the system (tsre1, tsre2 and tsrx) are shown in light brown. Genes that encode possible adapters (tsar1 and tsar2) are represented in dark green. Lately, the vgrG and OBfold genes and the paar domain are displayed in dark brown. (B) Schematic representation of TssJ, TssM, TssL, TagM and TagN protein domains. Colour used to display the different domains are in brackets as follows: DotU cytosolic domain (dark green), transmembrane domain (light blue), IcmF structural domain (dark blue), OmpA peptidoglycanbinding domain (red) and signal peptide (light green). OM: Outer membrane, IM: Inner membrane, PG: Peptidoglycan layer. (C) Sequence similarities and structural alignments as predicted by AlphaFold including RMSD values and TMscores of Tse7Tsre1 and Tsi7TsrX pairs. P. aeruginosa PAO1 proteins are displayed in blue and S. fredii USDA257 proteins in yellow. 17517915, 2025, 3, Downloaded from https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70112 by Readcube (Labtiva Inc.), Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 of 17 Microbial Biotechnology, 2025 an M15 peptidase, a Dalanyl Dalanine carboxypeptidase that is involved in bacterial cell wall biosynthesis (Lessard and Walsh1999). 3.3 | The S. fredii USDA257 T6SS Cluster Harbours Genes Encoding Putative Effectors Frequently, genes encoding T6SS effectors are genetically linked to vgrG and hcp genes, which can be found within a T6SS cluster or, in different numbers, scattered through the genome. For instance, P. aeruginosa PAO1 genome contains 10 vgrG and 5 hcp genes (Hachani etal.2016). The in silico study of the USDA257 genome has revealed single copies of vrgG and hcp genes in the T6SS cluster (Figure2A). The hcp gene is found surrounded by the structural genes tssC and tssE and there is no evidence of genes encoding putative effectors in the hcp proximity. On the other hand, vrgG is located at the end of the cluster and genetically linked to putative T6SS effectors and adaptors, displaying a similar genetic architecture to P. aeruginosa PAO1 and P. fluorescens F113 vrgG1b clusters (Durán etal.2021; Pissaridou etal.2018). These Pseudomonas vgrG clusters encoded a VrgG, an oligonucleotidebinding (OB)- fold, a DUF2169 adaptor, a thiolaselike protein, a PAAR protein with a Cterminal cytotoxic domain, an immunity protein and a heat repeatcontaining protein. The set of genes of PAO1 and F113 only differ in the sequence of the toxic domain and the cognate immunity pair (P. aeruginosa Tse7Tsi7 and P. fluorescens Tfe6Tfi6), a common characteristic of these genetic islands, previously described by Pissaridou etal.(2018). In USDA257, genes downstream vrgG are inverted relative to the P. aeruginosa and P. fluorescens clusters, but aside from this inversion, the genetic organisation of the region is conserved. In this way, USDA257 contains the genes encoding VrgG, the OBfold protein, the DU2169 adaptor that we have named Tsar (tsar, type six adaptor rhizobium), followed by the thiolaselike protein, an evolved PAAR protein named Tsre1 (tsre, type six rhizobial effector) with a Cterminal domain of unknown function and, lastly, a protein of unknown function that we have named Tsrx (Figure2A). According to Phyre2, which predicts structures by comparison with crystalized proteins that serve as structural templates (Kelley etal. 2015), Tsrx shows structural homology with a glycoside hydrolase enzyme GH74 with a xyloglucan binding domain from Caldicellulosiruptor lactoaceticus 6A (69% alignment, 99.3% confidence). In addition, the predicted model of USDA257 Tsrx protein was collected from the Alphafold database and queried using the FoldSeek tool, which predicts the structural homology based on the tertiary interactions of proteins in a sequenceindependent manner (Van Kempen etal.2024). Using this approach, Tsrx presented high structural homology with the immunity protein Tsi7 from P. aeruginosa PAO1 (from amino acid 2 to 340; Evalue of 3.77e14, Figure2C, right panel). Foldseek also identified structural homology between Tsrx and the glycoside hydrolase enzyme previously identified using Phyre2 (from amino acid 11 to 338) but with a much lower Evalue than the homology with Tsi7 (Evalue 1.10e1 vs. 3.77e14). The Cterminal domain of the evolved PAAR protein Tsre1 does not display any structural homology using Phyre2. Still, it is predicted to be structurally homologous to the DNase toxin Tse7 of P. aeruginosa PAO1 using AlphaFold and Foldseek tools (amino acids 3 to 321, EValue 5.25e24, Figure2C, left panel). Moreover, we identified another Tsar adaptor (DUF2169 adaptor) and one potential orphan effector that consists of an evolved PAAR protein (Tsre2) whose genes were located separately from the main T6SS cluster (Figure 2A, left). This orphan PAAR protein lacked sequence similarity to the Tsre1 effector in its Nterminal and Cterminal domains, and Phyre2 analysis revealed no structural homology with any known proteins. This putative effector protein is conserved across some Sinorhizobium species, including S. americanum and S. sojae, but it is absent or significantly divergent in other bacteria. Using FoldSeek, we identified a PAARlike DUF4150 domaincontaining protein from Methylobacterium sp. yr596 with limited structural homology. Specifically, structural similarity was observed only in the Nterminal PAAR region (amino acids 38–168), with a statistically significant Evalue of 1.11e16. Interestingly, no genes encoding a putative immunity protein were found within this genetic orphan T6SS cluster. To obtain a broader perspective of the functioning and distribution among rhizobia of the elements that vary in the vgrG cluster, that is, tsre1 and tsrx genes, we performed a comparison of the USDA257 vgrG cluster across 18 representative rhizobial strains. Synteny mapping of clusters, sorted by their vgrG similarity, is displayed in Figure3A, revealing sequence conservation across the entire genetic cluster. The genes encoding the VgrG and the OBfold proteins exhibited high sequence conservation, whereas those encoding Tsre1 and Tsrx displayed the greatest variability. Curiously, the 3´ end of the DUF2169 adaptor gene and the 5' end of the gene encoding the thiolaselike protein showed reduced conservation, potentially suggesting effector/ toxin specificity (Figure3A). A neighbourjoining phylogeny of the Tsre1like and Tsrxlike proteins from these rhizobial strains grouped USDA257 with Sinorhizobium americanum strains CCGM7 and CFNEI 73 in both cases, whereas most Tsre1 and Tsrx Rhizobium phaseoli versions are distributed in two separated branches, one grouping proteins from strains R620, R650, R611, N771, N671, N261, R723 and Brasil 5, and the other one clustering proteins from strains N841, R630, N831, N931 and R744 (Figure3B,C). Alignment of the different Tsre1 versions revealed a high degree of similarity in the Nterminal (PAARlike) domain, whereas the Cterminal domain showed notable divergence across all versions (FigureS3). As expected, the Tsrx versions exhibited a lower degree of similarity throughout the whole protein sequence (FigureS4). Intriguingly, despite their low sequence identity, structural homology predictions using both Phyre2 and Foldseek tools suggest that Tsre1like and Tsrxlike proteins exhibit significant structural similarity across different protein variants (TableS6). 3.4 | The T6SS of S. fredii USDA257 Is Functional and Induced in Minimal Medium at Stationary Phase of Growth To determine the conditions under which the T6SS of S. fredii USDA257 is expressed, we constructed transcriptional fusion of the promoter region of the T6SS structural operon (ppkA) to the promoterless lacZ. We measured the expression level of USDA257 T6SS structural operon by ßgalactosidase assays (See Material and Methods for more details). We tested USDA257 cultures 30 h postinoculation in selected rich, 17517915, 2025, 3, Downloaded from https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70112 by Readcube (Labtiva Inc.), Wiley Online Library on [12/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 9 of 17 standard and minimal media for growing rhizobia, that is, TY, YM, and MM respectively (Figure4A). YM is a standard medium containing moderate amounts of yeast extract, which provides essential organic nitrogen and micronutrients. In contrast, MM is a minimal medium that exclusively contains glutamate as the nitrogen source. Both YM and MM media can be prepared using different concentrations of mannitol as the carbon source. T6SS gene expression was highest in the minimal medium MM3 (3 g L−1 of mannitol) and lowest in the rich medium TY. Compared to the strain carrying the empty plasmid, gene expression was approximately 3fold and 1.8fold in these media, respectively (Figure4A). To confirm these findings, we followed a complementary approach, measuring the transcriptional activation of the USDA257 ppkA gene by quantitative PCR 48 h postinoculation. qRTPCR experiments revealed a similar transcriptional activation pattern for the USDA257 ppkA gene. The highest transcriptional levels were observed in the MM3 medium, demonstrating approximately 8fold higher expression compared to cultures grown in the TY medium. (Figure4B). To further investigate T6SS regulation, we performed ßgalactosidase assays at different stages of the USDA257 growth curve, inoculating the bacterium in the MM3 medium as the inducing condition. Interestingly, the PppkA::lacZ fusion in the wildtype strain indicated a gradual upregulation of T6SS expression over time, with a plateau in βgalactosidase activity observed at approximately 54 h FIGURE 3 | In silico analysis of rhizobial vrgG clusters and putative effectors. (A) Genome sequence alignment of the vgrG regions demonstrated the divergence of tsre1like and tsrxlike genes in 18 rhizobial strains using clinker & clustermap.js (Gilchrist and Chooi2021). Clusters are sorted by homology degree with respect to the vgrG gene from S. fredii USDA257 (Blastn E value). Numbers indicate the homology degree (identity) among genes. (B and C) Neighbourjoining tree of rhizobia T6SS putative effectors with 500 bootstrap replicates. Branches with black circles indicate a confidence level higher than 0.75. Analyses of the PAAR effector (Tsrelike) (B) and the Tsrxlike proteins (C) by sequence similarity were performed by CLUSTALW in the MEGA7 software. In bold, representative Tsre1like and Tsrxlike proteins of each branch (USDA257group: Orange; N841group: Green; R620group: Blue). 17517915, 2025, 3, Downloaded from https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70112 by Readcube (Labtiva Inc.), Wiley Online Library on [12/03/2025]. 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