Abstract Several genes coding for proteins with metal ion-inducible autocleavage (MIIA) domains were identified in type III secretion system tts gene clusters from draft genomes of recently isolated Bradyrhizobium spp. MIIA domains have been first described in the effectors NopE1 and NopE2 of Bradyrhizobium diazoefficiens USDA 110. All identified genes are preceded by tts box promoter motifs. The identified proteins contain one or two MIIA domains. A phylogenetic analysis of 35 MIIA domain sequences from 16 Bradyrhizobium strains revealed four groups. The protein from Bradyrhizobium sp. LmjC strain contains a single MIIA domain and was designated MdcE (MdcELmjC). It was expressed as a fusion to maltose-binding protein (MalE) in Escherichia coli and subsequently purified by affinity chromatography. Recombinant MalE-MdcELmjC-Strep protein exhibited autocleavage in the presence of Ca2+, Cu2+, Cd2+ and Mn2+, but not in the presence of Mg2+, Ni2+ or Co2+. Site-directed mutagenesis at the predicted cleavage site abolished autocleavage activity of MdcELmjC. An LmjC mdcE− mutant was impaired in the ability to nodulate Lupinus angustifolius and Macroptilium atropurpureum. MIIA domain, metal ion-induced autocleavage, effector, type III secretion, Bradyrhizobium INTRODUCTION Rhizobia are able to establish a symbiosis with a wide variety of legume host plants by inducing the formation of specific plant organs, the nodules. Rhizobia reside inside these nodules and reduce atmospheric nitrogen to ammonia, which is used as a nutrient by the host plant. The rhizobia–legume symbiosis is initiated by a complex and coordinated signal exchange between both partners (Oldroyd 2013). Rhizobial extracellular compounds, e.g. polysaccharides and secreted proteins, are known to affect symbiosis at different stages (Krishnan et al.2003; Jones and Walker 2008; Downie 2010; Margaret et al.2013). For the secretion of proteins, rhizobia use a number of different secretion systems (Fauvart and Michiels 2008; Downie 2010; Nelson and Sadowsky 2015; Green and Mecsas 2016). In some rhizobia, a functional type III secretion system (T3SS) was identified (Deakin and Broughton 2009; Staehelin and Krishnan 2015; Deng et al.2017). The components of the rhizobial T3SS are encoded in a 30–47 kb gene cluster located either in a symbiotic island on the chromosome or on a symbiotic plasmid (Tampakaki 2014). The so-called T3SS gene cluster contains about 10 conserved genes coding for core components of the secretion system, and further genes coding for effector proteins and other T3SS-related proteins. The organization and gene content for the structural core components are conserved, while the number and distribution of genes coding for other proteins secreted via the T3SS designated Nops (nodulation outer proteins) are variable (Deakin and Broughton 2009; Tampakaki 2014). A conserved component of rhizobial T3SS clusters is the regulator gene ttsI, which has a nod box in its promoter. TtsI induces the expression of the T3SS gene cluster in a NodD- and flavonoid-dependent manner. Host plant roots secrete flavonoids, which interact with the rhizobial NodD regulator to activate symbiotic genes with a conserved nod box in promoter regions (Viprey et al.1998; Perret et al.1999; Krause, Doerfel and Göttfert 2002; López-Baena et al.2008). TtsI binds to a conserved promoter motif, the tts box and induces transcription of downstream genes (Krause, Doerfel and Göttfert 2002; Marie et al.2004; Wassem et al.2008; Zehner et al.2008). This promoter motif is frequently used for the identification of TtsI-regulated genes and for prediction of genes coding for putative effectors in rhizobial genomes (Zehner et al.2008; Sánchez et al.2009; Yang et al.2010; Kimbrel et al.2013). Mutational studies revealed that type III secretion plays an important role in defining the host range of rhizobia (Meinhardt et al.1993; Krishnan et al.2003; Skorpil et al.2005). Furthermore, it was shown that individual T3SS-dependent effectors can affect the interaction with different host plants (Marie et al.2003; Skorpil et al.2005; Dai et al.2008; López-Baena et al.2008; Okazaki et al.2010; Wenzel et al.2010, 2013; Jiménez-Guerrero et al.2015; Staehelin and Krishnan 2015, 2017). Bradyrhizobium diazoefficiens USDA 110 secretes at least 14 proteins into the supernatant of bacterial cultures in a type III secretion-dependent manner (Süβ et al.2006; Zehner et al.2008; Hempel et al.2009). The NopE1 and NopE2 proteins of USDA 110 are type III-secreted effector proteins that were shown to be translocated into plant cells (Wenzel et al.2010; Kimbrel et al.2013). The presence and secretion of NopE proteins strongly restricts the nodulation of B. diazoefficiens with Vigna radiata KPS2 (Wenzel et al.2010). NopE1 and NopE2 are homologous proteins sharing about 77% sequence identity. Both proteins contain two conserved domains of ∼170 amino acids with non-enzymatic metal ion-inducible autocleavage (MIIA) activity (Wenzel et al.2010; Schirrmeister et al.2013). In this study, we identify a novel family of proteins bearing a single MIIA domain. The autocleavage activity of one such protein with a single MIIA domain from LmjC strain was studied. A mutation was generated in the corresponding gene of LmjC and its ability to nodulate different legumes was examined. MATERIALS AND METHODS Bacterial strains, plasmids and culture conditions The main characteristics of bacterial strains and plasmids used in this work are shown in Table S1 in the online supplementary material. Bradyrhizobium sp. LmjC is an extra-slow-growing strain isolated from Lupinus mariae-josephae (Sánchez-Cañizares et al.2011). The bacteria were grown at 28°C in yeast mannitol (Vincent 1970). Escherichia coli was used for protein expression [BL21(DE3) cells] and for cloning purposes (DH10B cells). Cells were grown in Luria Bertani broth (LB) (Sambrook, Fritsch and Maniatis 1989). Antibiotic concentrations used were as follows (in mg L−1) ampicillin, 100; kanamycin, 50; and spectinomycin, 100. Purification and autocleavage activity of LmjC MdcE protein expressed in E. coli The mdcE gene sequence from Bradyrhizobium sp. LmjC was amplified by PCR from genomic DNA using Pfu polymerase (Thermo Scientific, Darmstadt, Germany) and primers MalE_mdcE_for and strep_mdcE_rev, which includes an additional strep-tag sequence (Table S3 in the online supplementary material). The fragment was cloned into pMal-c5X (New England Biolabs, Frankfurt am Main, Germany) using NdeI and EcoRI restriction sites (Table S1). Cloning resulted in plasmid pMNES.6 encoding the fusion protein consisting of MalE (maltose-binding protein), MdcE and a C-terminal Strep tag II. Plasmid pMNES.6 was transformed into E. coli BL21 (DE3). Induction of expression was performed as described previously (Wenzel et al.2010). After induction with IPTG (100 μM), cultures were grown at 30°C for 4 h. Cell crude extracts were obtained by sonication using high salt TKE buffer (50 mM Tris, 200 mM KCl, 10 mM EDTA, pH 8.0) (Wenzel et al.2010). The fusion protein was purified by affinity chromatography with MBPTrap HP columns (GE Healthcare, Freiburg, Germany) using TKE buffer for loading and 10 mM maltose in TKE for elution. The autocleavage activity of the fusion protein was analyzed by incubating the purified protein in TKE buffer with solutions of CaCl2, CdCl2, CoCl2, CuCl2, NiCl2, MnCl2 or MgCl2 (final concentration 25 mM) for 30 min at room temperature. A control protein sample was incubated identically with buffer alone. The reaction was stopped by addition of 100 mM EDTA. All samples were analyzed by SDS-PAGE and gels were stained with Roti-Blue Quick (Carl Roth, Karlsruhe, Germany). Site-directed mutagenesis of mdcELmjC For site-directed mutagenesis of mdcELmjC, the QuikChange protocol (Agilent, Waldbronn, Germany) was applied using pMNES.6 as template and the primers LmjC-DA-for and LmjC-DA-rev (Table S3). This resulted in a change of aspartic acid residue D176 to alanine at the cleavage site, generating MdcELmjCD176A. A new ApaI restriction site was introduced at the site of mutation. The resulting plasmid pMNES.DA was confirmed by sequencing. Generation of a LmjC mdcE-deleted mutant DNA fragments from flanking regions of mdcE and a spectinomycin-resistance gene marker were ligated with Gateway technology (ThermoFisher). One fragment, with partial sequence of the 5'-mdcE gene (361 bp) and 351 bp of the upstream sequence, was amplified by PCR with primers MdcE4 (with an EcoRI target at the end) and MdcE3; a second fragment, with the 3' region of mdcE (391 bp) and 247 bp of the downstream sequence, was amplified with primers MdcE1 (also with an EcoRI target at the end) and MdcE2. A third amplicon with a spectinomycin-resistance gene was obtained with primers attB4r-Sp-sF and attB3r-Sp2R from plasmid pHP45Ω (Prentki and Krisch 1984). The construct containing the spectinomycin resistance gene and end sequences of MdcE was checked by sequencing, excised with EcoRI and introduced into the pK18mobsacB vector (Schäfer et al.1994) (Table S1). The latter was conjugated to Bradyrhizobium sp. LmjC and colonies with double recombination events were obtained by SacB-based selection (Schäfer et al.1994). The mutant was confirmed by Southern blot analysis. Genomic DNA of the wild type and mutant strains were digested with EcoRI and hybridized with a DIG-labeled mdcE probe obtained by PCR amplification using primers mdcE1 and mdcE4. The size of the hybridizing band in the mutant showed an increase in size corresponding to the interposon. Sequences of primers used are in Table S3. Plant assays Seeds were surface-disinfected with diluted bleach as previously reported (Sánchez-Cañizares et al.2011) and axenically germinated on 1% agar plates. Seedlings were transferred into sterilized Leonard jars containing vermiculite and Jensen's solution. Each jar contained two plants that were grown under bacteriologically controlled conditions for 4–8 weeks, depending on the legume host, 4 weeks for Lupinus angustifolius, 5 weeks for L. cosentinii, L. micranthus, Macroptilium atropurpureum and Lotus corniculatus, 6 weeks for L. luteus and L. mariae-josephae, and 8 weeks for Retama sphaerocarpa. Bacterial suspensions (2 mL,108–109 cells mL−1) were added to the seedlings. Non-inoculated jars were used as negative nodulation controls. At least three different replicates with four plants per strain and per legume host were performed. Bioinformatics analyses and accession numbers Proteins with MIIA domains were identified from Bradyrhizobium sp. genomic sequence drafts obtained in our group using Illumina HiSeq 2000, 500 bp paired-end libraries, 100 bp reads and 7 million reads and from Bradyrhizobium genomes available at NCBI and JGI Databases. Strains and accession numbers for sequences used in this study are listed in Table S2 in the online supplementary material. The two MIIA domain sequences of NopE1 from B. diazoefficiens USDA 110 were used as queries for BLAST sequence searches. Promoter sequence analysis was performed with the program fuzznuc of the EMBOSS package (Rice, Longdenand and Bleasby 2000). MIIA domains in the deduced proteins were identified by the Motif Scan tool of MyHits (Pagni et al.2007; Johnson et al.2008). Sequences were aligned using CLUSTALW (Thompson, Gibson and Higgins 2002; Larkin et al.2007). Phylogenetic and molecular evolutionary analyses were conducted using MEGA 7 (Kumar, Stecher and Tamura 2016). The Neighbor-Joining method (Saitou and Nei 1987) was employed to infer the phylogenetic distance and the evolutionary distance was computed using the p-distance method (Nei and Kumar 2000). Phylogenetic trees were made after 1000 bootstrap replications. Prediction of extracellular localization of proteins was performed with PsortB (Yu et al.2010). The accession number of the T3SS of LmjC strain is MG266265. RESULTS Identification of MIIA domain coding sequences in the genus Bradyrhizobium A BLAST search for MIIA domains was performed on protein sequences in rhizobial databases and also in Bradyrhizobium draft genomes obtained in our group. Among the rhizobia, MIIA domains were identified only in strains belonging to the Bradyrhizobium genus. Most of the domains were in proteins similar (>60% identity) to Bradyrhizobium diazoefficiens NopE1 or NopE2. Remarkably, seven proteins, with <20% identity to NopE1 or NopE2, containing only one domain, were identified: six from strains isolated from Lupinus mariae-josephae (Lmj strains) and one, B. elkanii WSM1741, isolated from Rynchosia minima (Fig. 1b, and Fig. S1 and Table S2 in the online supplementary material). These proteins are encoded by genes located within T3SS clusters (see below) and they were designated MdcE (MIIA domain-containing proteins as NopE). Figure 1. View largeDownload slide MIIA domain-containing proteins and Bradyrhizobium strains. (a) Schematic representation of NopE and MdcE proteins of different types. NopE1USDA 110 and NopE2USDA 110 refer to the proteins NopE1 and NopE2 from B. diazoefficiens USDA 110 containing two MIIA domains (grey boxes). MdcELmjC refers to the MdcE protein from B. sp. LmjC containing a single MIIA domain. MIIA-N, MIIA-C1, MIIA-C2 and MIIA-S depict the groups of identified MIIA domain sequences according to phylogenetic analysis. Numbers indicate the amino acid positions flanking the proteins and MIIA domains. (b) Venn diagram indicates Bradyrhizobium strains coding for the different types of MIIA domain-containing proteins. Figure 1. View largeDownload slide MIIA domain-containing proteins and Bradyrhizobium strains. (a) Schematic representation of NopE and MdcE proteins of different types. NopE1USDA 110 and NopE2USDA 110 refer to the proteins NopE1 and NopE2 from B. diazoefficiens USDA 110 containing two MIIA domains (grey boxes). MdcELmjC refers to the MdcE protein from B. sp. LmjC containing a single MIIA domain. MIIA-N, MIIA-C1, MIIA-C2 and MIIA-S depict the groups of identified MIIA domain sequences according to phylogenetic analysis. Numbers indicate the amino acid positions flanking the proteins and MIIA domains. (b) Venn diagram indicates Bradyrhizobium strains coding for the different types of MIIA domain-containing proteins. A representative selection of 35 genes coding for MIIA domains was further analyzed. All of them were preceded by a tts box (Table S4 in the online supplementary material). In all analyzed strains possessing a single gene coding for a protein with a MIIA domain, it was localized inside the predicted T3SS gene cluster (Table S2). In contrast, strains isolated from Glycine max have two genes: one, nopE1, is localized inside the T3SS gene cluster, and the second gene, nopE2, is outside of the cluster. In LmjM3, LmjG2 and LmjTa10 strains coding for two proteins containing MIIA domains, both genes are located within the T3SS gene cluster. However, it has to be noted that the localization of the second gene, could not be described in some cases, due to the lack of enough genomic sequence information. The predicted NopE proteins containing two MIIA domains showed an overall sequence identity between 60 and 99%. The second group, formed by proteins containing only one MIIA domain (Fig. 1a), presented a higher intragroup identity of 83–97%. In contrast, the sequence identity between the two groups of MIIA-containing proteins was very low (15–18%) (Fig. S1). The length of MIIA domains was similar in all sequences and spanned between 160 and 175 amino acids. All identified MIIA domains contained the conserved cleavage site motif (GD’PH) previously identified in NopE1 of B. diazoefficiens USDA 110 (Wenzel et al.2010). According to the constructed tree, the MIIA domains identified in Bradyrhizobium strains can be classified into four groups (Fig. 2). The domains originating from proteins with their two MIIA domains grouped consistently with its N-terminal and C-terminal position. The MIIA domains of the designated N-terminal MIIA group (MIIA-N) shared between 76–100% sequence identity. The group of C-terminal MIIA domains could be divided further into two groups. One group (MIIAC1) contained the C-terminal MIIA domains of NopE1 from B. diazoefficiens USDA 110, B. sp. ORS285, B. sp. WSM1743 and B. japonicum strains, sharing over 81% identity. The other group included the C-terminal MIIA domain of NopE2 (MIIA-C2) from B. diazoefficiens USDA 110, and the sequences identified in several Bradyrhizobium species (Fig. 1b and Fig. 2). The protein domains of this MIIA-C2 group show over 84% sequence identity. The amino acid sequences of the groups MIIA-C1 and MIIA-C2 shared between 69 and 78% identities. The fourth group of MIIA domains according to the phylogenetic tree originated from the proteins containing only one MIIA domain grouped with only seven sequences sharing 92% sequence identity (Fig. 1; MIIA-S). The single MIIA domain was significantly different from the other described MIIA domains and shared only 18–24% sequence identity with N- and C-terminal MIIA domains. This low sequence similarity raised the question of whether the metal ion-inducible autocleavage activity was conserved in this group of MdcE proteins. Figure 2. View largeDownload slide Neighbor-joining phylogenetic tree based on the MIIA domain sequences from Bradyrhizobium strains. Bootstrap values greater than 60% are indicated at nodes. The MIIA sequence of VIC_001052 from Vibrio coralliilyticus ATCC-BAA450 was used as outgroup. To show a compact tree avoiding very similar sequences, collapsed groups for N-terminal and C-terminal MIIA domains include protein sequences from: B. japonicum strains USDA 4, USDA 6, USDA 38, USDA 122, USDA 123, USDA 124, USDA 135, WSM1743 and B. diazoefficiens USDA 110. Scale bar shows number of amino acid substitutions per site. Figure 2. View largeDownload slide Neighbor-joining phylogenetic tree based on the MIIA domain sequences from Bradyrhizobium strains. Bootstrap values greater than 60% are indicated at nodes. The MIIA sequence of VIC_001052 from Vibrio coralliilyticus ATCC-BAA450 was used as outgroup. To show a compact tree avoiding very similar sequences, collapsed groups for N-terminal and C-terminal MIIA domains include protein sequences from: B. japonicum strains USDA 4, USDA 6, USDA 38, USDA 122, USDA 123, USDA 124, USDA 135, WSM1743 and B. diazoefficiens USDA 110. Scale bar shows number of amino acid substitutions per site. Characterization of a MdcE protein with a single MIIA domain The MdcE protein of B. sp. LmjC is encoded by a 1281 bp gene (accession number KT274200) and is located within the predicted T3SS gene cluster (Fig. S2 in the online supplementary material). Upstream of the coding sequence a tts box promoter motif was identified, located 172 nt from the potential translational start site (Table S4 in the online supplementary material). The protein showed low sequence similarity to NopE1 and NopE2 of B. diazoefficiens USDA 110, which was restricted to the MIIA domain. Apart from that MdcELmjC showed no significant sequence similarity to any characterized protein in the databases. Web applications, EffectiveDB (Eichinger et al.2015) and BPBAac (Wang et al.2011), designed to predict T3SS effectors, were used to analyze LmjC MdcE proteins and NopEs from USDA 110. None of them were predicted to be secreted, although it has been shown experimentally that NopEs from USDA 110 are secreted T3SS-dependent effectors (Süβ et al.2006). PsortB analysis predicted an extracellular localization for MdcELmjC. The MIIA domain (165 amino acids) is in the central part of the protein and shows 28% sequence similarity with both N-terminal and C-terminal MIIA domains of NopE1. The domain identified in MdcELmjC possesses the conserved cleavage site motif GD’PH (Fig. S1). The protein MdcELmjC was expressed as a recombinant MalE-MdcE-Strep fusion protein (91 kDa) in E. coli. The fusion protein was soluble in the cytoplasm and was purified by affinity chromatography. Autocleavage of the fusion protein was observed when incubated in the presence of Ca2+ ions, resulting in two fragments with compatible sizes (29 and 62 kDa) predicted from the autocleavage site (GD’PH). The conserved aspartate residue at position 176 in MdcELmjC was changed to alanine by site-specific mutagenesis of mdcELmjC. The resulting protein variant MalE-MdcELmjC(D176A)-Strep was purified and subsequently analysed in the autocleavage assay. The protein showed no autocleavage in the presence of calcium ions (Fig. 3). Figure 3. View largeDownload slide Calcium-induced autocleavage of MdcELmjC. (a) Scheme of the fusion protein MalE-MdcELmjC-Strep showing the internal MIIA domain and the predicted cleavage site. Sizes of the resulting cleavage products FN and FC are shown. (b) Analysis of the autocleavage activity of purified MalE-MdcELmjC-Strep and MalE-MdcELmjC(D176A)-Strep variant. For each sample, 8 μg of protein was loaded on a 15% polyacrylamide gel after incubation with (+) and without (−) calcium ions. Asterisk marks the full-length protein, arrowheads indicate: FN, N-terminal fragment (62 kDa); FC, C-terminal fragment (29 kDa). M, Prestained Protein Ladder (Thermo Scientific). The gel was stained with Roti-Blue Quick (Carl Roth) after electrophoresis. Figure 3. View largeDownload slide Calcium-induced autocleavage of MdcELmjC. (a) Scheme of the fusion protein MalE-MdcELmjC-Strep showing the internal MIIA domain and the predicted cleavage site. Sizes of the resulting cleavage products FN and FC are shown. (b) Analysis of the autocleavage activity of purified MalE-MdcELmjC-Strep and MalE-MdcELmjC(D176A)-Strep variant. For each sample, 8 μg of protein was loaded on a 15% polyacrylamide gel after incubation with (+) and without (−) calcium ions. Asterisk marks the full-length protein, arrowheads indicate: FN, N-terminal fragment (62 kDa); FC, C-terminal fragment (29 kDa). M, Prestained Protein Ladder (Thermo Scientific). The gel was stained with Roti-Blue Quick (Carl Roth) after electrophoresis. In order to test whether autocleavage of MdcELmjC could be induced by divalent metal ions other than calcium, the purified MalE-MdcELmjC-Strep protein was incubated with different metal ion solutions (Fig. 4). In the presence of Mn2+, Cu2+ and Cd2+ ions, autocleavage could be induced at similar rates to those observed with Ca2+. Residual full-length protein was still observed in these samples after 30 min. In contrast, the protein was not cleaved in the presence of Mg2+, Ni2+ or Co2+ ions. Figure 4. View largeDownload slide Metal ion-induced autocleavage of MalE-MdcELmjC-Strep protein. The purified protein was incubated without (−) and with different metal ion solutions at a final concentration of 25 mM. Autocleavage was analyzed by SDS-PAGE (NuPAGE, 8% Bis-Tris gel; Life Technologies). Asterisk and arrowheads are as in Fig. 3. Figure 4. View largeDownload slide Metal ion-induced autocleavage of MalE-MdcELmjC-Strep protein. The purified protein was incubated without (−) and with different metal ion solutions at a final concentration of 25 mM. Autocleavage was analyzed by SDS-PAGE (NuPAGE, 8% Bis-Tris gel; Life Technologies). Asterisk and arrowheads are as in Fig. 3. Legume host-range analysis A previous report showed that NopE proteins from B. diazoefficiens USDA 110 are responsible for a host-specific phenotype (Wenzel et al.2010). To test whether MdcELmjC could influence nodulation, a mdcE-deleted mutant strain was created, and the effect of the mutation on nodule formation was examined on different legume hosts (Table 1). Effective red nodules were induced by both the wild type and the MdcE− mutant on various lupins, namely L. mariae-josephae, L. cosentinii, L. micranthus and R. sphaerocarpa. In contrast, a difference between wild type and mutant strains was observed in three independent biological assays with L. angustifolius and with Macroptilium atropurpureum: while the wild type strain produced white nodules that do not fix nitrogen, the mdcE− mutant strain did not induce any nodules (Table 1). The behavior of both strains was also similar with L. luteus (white nodules with some plants) and with Lotus corniculatus (absence of nodules). Table 1. Legume host-range and nodulation analysis of Bradyrhizobium sp. LmjC mdcE mutant. Legume strain Lmj Lan Llu Lco Lmi Lcor Rsp Mat LmjC +/Ra +*/W +*/W +/R +/R − +/R +/W 11.4 ± 1.3b 3.5 ± 1.5 3.0 ± 2.1 15.3 ± 3.6 49.8 ± 4.8 10.5 ± 2.3 3.3 ± 2.2 LmjC mdcE− +/R − +*/W +/R +/R − +/R − 10.2 ± 3.5 3.1 ± 1.7 12.5 ± 1.0 54.5 ± 11.8 11.1 ± 5.9 Legume strain Lmj Lan Llu Lco Lmi Lcor Rsp Mat LmjC +/Ra +*/W +*/W +/R +/R − +/R +/W 11.4 ± 1.3b 3.5 ± 1.5 3.0 ± 2.1 15.3 ± 3.6 49.8 ± 4.8 10.5 ± 2.3 3.3 ± 2.2 LmjC mdcE− +/R − +*/W +/R +/R − +/R − 10.2 ± 3.5 3.1 ± 1.7 12.5 ± 1.0 54.5 ± 11.8 11.1 ± 5.9 a Nodules b average nodules/plant ± standard deviation * only 35–50% plants were nodulated. +, presence of nodules; −, absence of nodules; R, red nodules; W, white nodules; Lmj, Lupinus mariae-josephae; Lan, L. angustifolius; Llu, L. luteus; Lco, L. cosentinii; Lmi, L. micranthus; Lcor, Lotus corniculatus; Rsp, Retama sphaerocarpa; Mat, Macroptilium atropurpureum. View Large DISCUSSION NopE proteins are T3SS-dependent effectors that contain two unusual autocatalytic domains called MIIA domains first described for B. diazoefficiens USDA 110 (Wenzel et al.2010; Schirrmeister et al.2013). In this work, a new group of proteins, MdcE, with a single MIIA domain has been identified. Proteins with a single MIIA domain were detected only in B. elkanii WSM 1741 and in Bradyrhizobium sp. strains isolated from the endemic plant in Eastern Spain, Lupinus mariae-josephae. Out of 23 Bradyrhizobium strains studied, 12 encoded two proteins containing MIIA domains (Fig. 1b). In phytopathogenic bacteria, the presence of multiple genes for the same effector family can be frequently observed (Ma et al.2006; Lewis et al.2011). Also in rhizobia, gene duplication and diversification of T3SS-dependent effectors have been described (Tampakaki 2014). The genome of Sinorhizobium fredii HH103 codes for two NopM effector proteins and for NopP that shares 45% sequence identity with the deduced effector NopI (Jiménez-Guerrero et al.2017). In B. diazoefficiens USDA 110 two genes for NopT and NopP, respectively, have been identified (Zehner et al.2008; Fotiadis et al.2012). The homologous effector proteins NopT1 and NopT2 show in vitro cysteine protease activity, but only NopT1 elicits hypersensitive-like responses in plant cells indicating a functional diversification of the effectors (Fotiadis et al.2012). In rhizobia, the presence of genes encoding proteins that harbor MIIA domains is restricted to strains belonging to the genus Bradyrhizobium. Interestingly, these genes are likely subjected to T3SS-dependent regulation since all possess a tts box in their putative promoter region. In the genomes of B. japonicum strains analyzed in this study, the genes nopE1 and nopE2 are organized similarly to those in B. diazoefficiens (Süβ et al.2006; Zehner et al.2008). The Bradyrhizobium strains LmjM3, LmjG2, LmjTa10 and WSM 1741 contain two genes encoding proteins with MIIA domain that are localized within conserved gene clusters of T3SSs. The data suggest the presence of two different T3SS gene clusters in the genome of these Bradyrhizobium strains. So far, only in Sinorhizobium sp. was the presence of multiple T3SSs reported (Schmeisser et al.2009; Sugawara et al.2013; Vinardell et al.2015). In the strains B. sp. th_b2, B. sp. ORS285, B. sp. LmjTa10 and B. japonicum USDA 4, USDA 38, USDA 122, USDA 123, USDA 124 and USDA 135, the available sequence information is not sufficient to describe the genomic region around both MIIA domain coding genes. Furthermore, most genome sequences used in this study are still unfinished drafts and additional MIIA domain coding genes might be hidden. Outside rhizobia, proteins containing MIIA domains have been described in a small set of α-, β-, γ-, and δ-proteobacteria (Schirrmeister et al.2011, 2013). Interestingly, all these deduced proteins contain only one MIIA domain. The encoding genes were found mostly in conjunction with a predicted T3SS gene cluster (Schirrmeister et al.2011, 2013). The similarity of these proteins with the identified MdcE proteins in bradyrhizobia is limited to the length of the MIIA domain although the MIIA domains in these proteins are clearly different from those in the bradyrhizobia. This novel MIIA domain described here for MdcE of strain LmjC contains a functional autocleavage site, which was inactivated by site-directed mutagenesis as was previously observed for the MIIA domains of NopE1 from B. diazoefficiens USDA 110 and VIC_0 01052 from Vibrio coralliilyticus ATCC-BAA450 (Wenzel et al.2010; Schirrmeister et al.2013). Some other proteins possessing metal ion-induced autocleavage activity have been described. The RTX protein FrpC from Neisseria meningitidis does not share significant sequence similarity with MIIA domains, but it shows a similar autocleavage activity that can be induced by calcium ions (Osička et al.2004). The induction of the cleavage of FrpC was proposed upon binding of calcium to predicted EF-hand motifs (Osička et al.2004). A calcium binding site similar to the one found in EF-hand motifs was predicted also for NopE1 of B. diazoefficiens USDA 110 (Schirrmeister et al.2011). In contrast, no obvious calcium binding motif was detected in the sequence of MdcELmjC. The autocatalytic cleavage of MdcELmjC can also be induced with Cd2+, Mn2+ and Cu2+ ions. These findings are similar to the studies on the MIIA domain of VIC_001052 of V. coralliilyticus (Schirrmeister et al.2013). Metal ion-induced cleavage could be observed in this MIIAVc domain with Cd2+, Mn2+ and Co2+, but not with Cu2+ (Schirrmeister et al.2013; Ibe, Schirrmeister and Zehner 2015). The reason why MIIA domains differ in their autocleavage activity with the tested metal ions is unknown, and may be elucidated when information on the structure of the proteins bearing the MIIA domains and the metal ion-binding sites are available. The loss of nodulation associated to L. angustifolius and M. atropurpureum observed by the inoculation with the LmjC mdcE− mutant would be consistent with a role of this protein in the definition of the symbiotic host range as has been demonstrated for NopE1 and NopE2 from B. diazoefficiens in the interaction with different legumes (Schirrmeister et al.2011). However, we cannot rule out that the mdcE mutant may be affecting the downstream gene, LmjC_7343, separated by 104 bp. The interposon in mdcE could interrupt LmjC_7343 transcription, although there might be some promoter activity in the intergenic region. The distance from Lmj_7343 to the next downstream gene, LmjC_7342, is 241 bp and it likely belongs to a different transcriptional unit. The existence of proteins bearing single and double MIIA domains increases the diversity of this family of proteins. However, the physiological function(s) of NopE and MdcE proteins are still unknown and further studies are needed to elucidate putative targets of these proteins. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. Acknowledgements We thank Ana Isabel Bautista and Alvaro Salinero for technical assistance. FUNDING This work was supported by the German Academic Exchange Service [5705041 to LR and SZ] and Ministerio de Economía Industria y Competitividad [BIO2013-43040 to JP and CSD2009-00006 and CGL2011-26932 to JI]. Conflict of interest. None declared. REFERENCES Dai WJ, Zeng Y, Xie ZP et al. Symbiosis-promoting and deleterious effects of NopT, a novel type 3 effector of Rhizobium sp strain NGR234. J Bacteriol 2008; 190: 5101– 10. Google Scholar CrossRef Search ADS PubMed Deakin WJ, Broughton WJ. Symbiotic use of pathogenic strategies: rhizobial protein secretion systems. Nat Rev Microbiol 2009; 7: 312– 20. Google Scholar PubMed Deng W, Marshall NC, Rowland JL et al. Assembly, structure, function and regulation of type III secretion systems. Nat Rev Microbiol 2017; 15: 323– 37. Google Scholar CrossRef Search ADS PubMed Downie JA. The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol Rev 2010; 34: 150– 70. Google Scholar CrossRef Search ADS PubMed Eichinger V, Nussbaumer T, Platzer A et al. EffectiveDB—updates and novel features for a better annotation of bacterial secreted proteins and Type III, IV, VI secretion systems. Nucleic Acids Res 2015; 44: D669– 74. Google Scholar CrossRef Search ADS PubMed Fauvart M, Michiels J. Rhizobial secreted proteins as determinants of host specificity in the rhizobium-legume symbiosis. FEMS Microbiol Lett 2008; 285: 1– 9. Google Scholar CrossRef Search ADS PubMed Fotiadis CT, Dimou M, Georgakopoulos DG et al. Functional characterization of NopT1 and NopT2, two type III effectors of Bradyrhizobium japonicum. FEMS Microbiol Lett 2012; 327: 66– 77. Google Scholar CrossRef Search ADS PubMed Green ER, Mecsas J. Bacterial secretion systems: an overview. Microbiol Spectr 2016; 4, DOI: 10.1128/microbiolspec.VMBF-0012-2015. Hempel J, Zehner S, Göttfert M et al. Analysis of the secretome of the soybean symbiont Bradyrhizobium japonicum. J Biotechnol 2009; 140: 51– 8. Google Scholar CrossRef Search ADS PubMed Ibe S, Schirrmeister J, Zehner S. Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal. J Biotechnol 2015; 208: 22– 7. Google Scholar CrossRef Search ADS PubMed Jiménez-Guerrero I, Pérez-Montaño F, Medina C et al. The Sinorhizobium (Ensifer) fredii HH103 nodulation outer protein NopI is a determinant for efficient nodulation of soybean and cowpea plants. Appl Environ Microbiol 2017; 83: e02770– 16. Google Scholar CrossRef Search ADS PubMed Jiménez-Guerrero I, Pérez-Montaño F, Monreal JA et al. The Sinorhizobium (Ensifer) fredii HH103 Type 3 secretion system suppresses early defense responses to effectively nodulate soybean. Mol Plant Microbe Interact 2015; 28: 790– 99. Google Scholar CrossRef Search ADS PubMed Johnson M, Zaretskaya I, Raytselis Y et al. NCBI BLAST: a better web interface. Nucleic Acids Res 2008; 36: W5– 9. Google Scholar CrossRef Search ADS PubMed Jones KM, Walker GC. Responses of the model legume Medicago truncatula to the rhizobial exopolysaccharide succinoglycan. Plant Signal Behav 2008; 3: 888– 90. Google Scholar CrossRef Search ADS PubMed Kimbrel JA, Thomas WJ, Jiang Y et al. Mutualistic co-evolution of type III effector genes in Sinorhizobium fredii and Bradyrhizobium japonicum. PLoS Pathog 2013; 9: e1003204. Google Scholar CrossRef Search ADS PubMed Krause A, Doerfel A, Göttfert M. Mutational and transcriptional analysis of the type III secretion system of Bradyrhizobium japonicum. Mol Plant Microbe Interact 2002; 15: 1228– 35. Google Scholar CrossRef Search ADS PubMed Krishnan HB, Lorio J, Kim WS et al. Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257. Mol Plant Microbe Interact 2003; 16: 617– 25. Google Scholar CrossRef Search ADS PubMed Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 2016; 33: 1870– 74. Google Scholar CrossRef Search ADS PubMed Larkin MA, Blackshields G, Brown NP et al. ClustalW and ClustalX version 2. Bioinformatics 2007; 23: 2947– 8. Google Scholar CrossRef Search ADS PubMed Lewis JD, Lee A, Ma W et al. The YopJ superfamily in plant-associated bacteria. Mol Plant Pathol 2011; 12: 928– 37. Google Scholar CrossRef Search ADS PubMed López-Baena FJ, Vinardell JM, Pérez-Montaño F et al. Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103. Microbiology 2008; 154: 1825– 36. Google Scholar CrossRef Search ADS PubMed Ma W, Dong FF, Stavrinides J et al. Type III effector diversification via both pathoadaptation and horizontal transfer in response to a coevolutionary arms race. PLoS Genet 2006; 2: e209. Google Scholar CrossRef Search ADS PubMed Margaret I, Lucas MM, Acosta-Jurado S et al. The Sinorhizobium fredii HH103 lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules. PLoS One 2013; 8: e74717. Google Scholar CrossRef Search ADS PubMed Marie C, Deakin WJ, Ojanen-Reuhs T et al. TtsI, a key regulator of Rhizobium species NGR234 is required for type III-dependent protein secretion and synthesis of rhamnose-rich polysaccharides. Mol Plant Microbe Interact 2004; 17: 958– 66. Google Scholar CrossRef Search ADS PubMed Marie C, Deakin WJ, Viprey V et al. Characterization of Nops, nodulation outer proteins, secreted via the type III secretion system of NGR234. Mol Plant Microbe Interact 2003; 16: 743– 51. Google Scholar CrossRef Search ADS PubMed Meinhardt LW, Krishnan HB, Balatti PA et al. Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257. Mol Microbiol 1993; 9: 17– 29. Google Scholar CrossRef Search ADS PubMed Nei M, Kumar S. Molecular Evolution and Phylogenetics . New York: Oxford University Press, 2000. Nelson MS, Sadowsky MJ. Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes. Front Plant Sci 2015; 6: 491 Google Scholar CrossRef Search ADS PubMed Okazaki S, Kaneko T, Sato S et al. Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system. Proc Natl Acad Sci U S A 2013; 110: 17131– 6. Google Scholar CrossRef Search ADS PubMed Okazaki S, Okabe S, Higashi M et al. Identification and functional analysis of type III effector proteins in Mesorhizobium loti. Mol Plant Microbe Interact 2010; 23: 223– 34. Google Scholar CrossRef Search ADS PubMed Oldroyd GE. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 2013; 11: 252– 63. Google Scholar CrossRef Search ADS PubMed Osička R, Procházková K, Šulc M et al. A novel “clip-and-link” activity of repeat in toxin (RTX) proteins from Gram-negative pathogens. Covalent protein cross-linking by an Asp-Lys isopeptide bond upon calcium-dependent processing at an Asp-Pro bond. J Biol Chem 2004; 279: 24944– 56. Google Scholar CrossRef Search ADS PubMed Pagni M, Ioannidis V, Cerutti L et al. MyHits: improvements to an interactive resource for analyzing protein sequences. Nucleic Acids Res 2007; 35: W433– 37. Google Scholar CrossRef Search ADS PubMed Perret X, Freiberg C, Rosenthal A et al. High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234. Mol Microbiol 1999; 32: 415– 25. Google Scholar CrossRef Search ADS PubMed Prentki P, Krisch HM. In vitro insertional mutagenesis with a selectable DNA fragment. Gene 1984; 29: 303– 13. Google Scholar CrossRef Search ADS PubMed Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 2000; 16: 276– 77. Google Scholar CrossRef Search ADS PubMed Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987; 4: 406– 25. Google Scholar PubMed Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual . Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989. Sánchez C, Iannino F, Deakin WJ et al. Characterization of the Mesorhizobium loti MAFF303099 type-three protein secretion system. Mol Plant Microbe Interact 2009; 22: 519– 28. Google Scholar CrossRef Search ADS PubMed Sánchez-Cañizares C, Rey L, Durán D et al. Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus. Syst Appl Microbiol 2011; 34: 207– 15. Google Scholar CrossRef Search ADS PubMed Schäfer A, Tauch A, Jäger W et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 1994; 145: 69– 73. Google Scholar CrossRef Search ADS PubMed Schirrmeister J, Friedrich L, Wenzel M et al. Characterization of the self-cleaving effector protein NopE1 of Bradyrhizobium japonicum. J Bacteriol 2011; 193: 3733– 9. Google Scholar CrossRef Search ADS PubMed Schirrmeister J, Zocher S, Flor L et al. The domain of unknown function DUF1521 exhibits metal ion-inducible autocleavage activity—a novel example from a putative effector protein of Vibrio coralliilyticus ATCC BAA-450. FEMS Microbiol Lett 2013; 343: 177– 82. Google Scholar CrossRef Search ADS PubMed Schmeisser C, Liesegang H, Krysciak D et al. Rhizobium sp. strain NGR234 possesses a remarkable number of secretion systems. Appl Environ Microbiol 2009; 75: 4035– 45. Google Scholar CrossRef Search ADS PubMed Skorpil P, Saad MM, Boukli NM et al. NopP, a phosphorylated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii. Mol Microbiol 2005; 57: 1304– 17. Google Scholar CrossRef Search ADS PubMed Staehelin C, Krishnan HB. Nodulation outer proteins: double-edged swords of symbiotic rhizobia. Biochem J 2015; 470: 263– 74. Google Scholar CrossRef Search ADS PubMed Süβ C, Hempel J, Zehner S et al. Identification of genistein-inducible and type III-secreted proteins of Bradyrhizobium japonicum. J Biotechnol 2006; 126: 69– 77. Google Scholar CrossRef Search ADS PubMed Sugawara M, Epstein B, Badgley BD et al. Comparative genomics of the core and accessory genomes of 48 Sinorhizobium strains comprising five genospecies. Genome Biol 2013; 14: R17. Google Scholar CrossRef Search ADS PubMed Tampakaki AP. Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria. Front Plant Sci 2014; 5: 114. Google Scholar CrossRef Search ADS PubMed Thompson JD, Gibson TJ, Higgins DG. Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics 2002; Chapter 2:Unit 2.3, DOI: https://doi.org/10.1002/0471250953.bi0203s00. Vinardell JM, Acosta-Jurado S, Zehner S et al. The Sinorhizobium fredii HH103 genome: a comparative analysis with S. fredii strains differing in their symbiotic behavior with soybean. Mol Plant Microbe Interact 2015; 8: 811– 24. Google Scholar CrossRef Search ADS Vincent JM. A Manual for the Practical Study of the Root-Nodule Bacteria . Oxford: Blackwell, 1970. Viprey V, Del Greco A, Golinowski W et al. Symbiotic implications of type III protein secretion machinery in Rhizobium. Mol Microbiol 1998; 28: 1381– 9. Google Scholar CrossRef Search ADS PubMed Wang Y, Zhang Q, Sun M et al. High-accuracy prediction of bacterial type III secreted effectors based on position-specific amino acid composition profiles. Bioinformatics 2011; 27: 777– 84. Google Scholar CrossRef Search ADS PubMed Wassem R, Kobayashi H, Kambara K et al. TtsI regulates symbiotic genes in Rhizobium species NGR234 by binding to tts boxes. Mol Microbiol 2008; 68: 736– 48. Google Scholar CrossRef Search ADS PubMed Wenzel M, Friedrich L, Göttfert M et al. The type III-secreted protein NopE1 affects symbiosis and exhibits a calcium-dependent autocleavage activity. Mol Plant Microbe Interact 2010; 23: 124– 9. Google Scholar CrossRef Search ADS PubMed Yang Y, Zhao J, Morgan RL et al. Computational prediction of type III secreted proteins from gram-negative bacteria. BMC Bioinformatics 2010; 11 Suppl. 1: S47. Google Scholar CrossRef Search ADS PubMed Yu NY, Wagner JR, Laird MR et al. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 2010; 13: 1608– 15. Google Scholar CrossRef Search ADS Zehner S, Schober G, Wenzel M et al. Expression of the Bradyrhizobium japonicum type III secretion system in legume nodules and analysis of the associated tts box promoter. Mol Plant Microbe Interact 2008; 21: 1087– 93. Google Scholar CrossRef Search ADS PubMed © FEMS 2017. All rights reserved. For permissions, please e-mail: email@example.com
FEMS Microbiology Letters – Oxford University Press
Published: Mar 1, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera