# traG Gene Is Conserved acrossMesorhizobium spp. Able to Nodulate the Same Host Plant and Expressed in Response to Root Exudates

Authors: A. Paço; J. R. da-Silva; F. Eliziário; C. Brígido; S. Oliveira; A. Alexandre
Journal: BioMed Research International
 (2019)

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## Abstract

Evidences for an involvement of the bacterial type IV secretion system (T4SS) in the symbiotic relationship between rhizobia and legumes have been pointed out by several recent studies. However, information regarding this secretion system inMesorhizobiumis still very scarce. The aim of the present study was to investigate the phylogeny and expression of thetraG gene, which encodes a substrate receptor of the T4SS. In addition, the occurrence and genomic context of this and other T4SS genes, namely, genes fromtra/trbandvirB/virD4 complexes, were also analyzed in order to unveil the structural and functional organization of T4SS in mesorhizobia. The location of the T4SS genes in the symbiotic region of the analyzed rhizobial genomes, along with thetraG phylogeny, suggests that T4SS genes could be horizontally transferred together with the symbiosis genes. Regarding the T4SS structural organization inMesorhizobium, thevirB/virD4 genes were absent in all chickpea (Cicer arietinum L.) microsymbionts and in theLotus symbiontMesorhizobium japonicum MAFF303099T. Interestingly, the presence of genes belonging to another secretion system (T3SS) was restricted to these strains lacking thevirB/virD4 genes. ThetraG gene expression was detected inM. mediterraneum Ca36T andM. ciceri LMS-1 strains when exposed to chickpea root exudates and also in the early nodules formed byM. mediterraneum Ca36T, but not in older nodules. This study contributes to a better understanding of the importance of T4SS in mutualistic symbiotic bacteria.


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## Body

## 1. Introduction

Rhizobia are able to fix atmospheric nitrogen when in symbiosis with legumes, providing ammonia to these plants. The rhizobial symbiosis genes include two main classes of genes, namely, nodulation and nitrogen fixation genes [1]. Nodulation genes (e.g.,nodABC) are implicated in biosynthesis and secretion of Nod factors, molecules involved in root infection, and nodule development. Nitrogen fixation genes include genes involved in the synthesis, processing, and assembly of nitrogenase complex (e.g.,nifHDK, fixGH), responsible for N2-fixation [2–4]. The expression of these sets of genes is regulated by two key transcriptional regulators, namely, NodD for nodulation genes and NifA for nitrogen fixation genes [3, 5]. Nevertheless, the involvement of other bacterial genes in legume-rhizobia symbioses has been described. Some studies have shown that genes commonly found among prokaryotes and involved in a diversity of cellular mechanisms have also a role in legume-Rhizobium symbiosis, as, for example, stress response genes [6–8], quorum sensing, or secretion system genes [9–12].Although several studies have shown the involvement of the rhizobial secretion systems in the symbiotic rhizobia-legume relationship [11–13], their precise role is far from being fully understood. Such transfer systems are ancestrally related to virulence (transmission of antibiotic resistance genes and virulence genes) and to the mating pair formation complexes in pathogenic bacteria [14]. However, with the increasing number of available rhizobia genomes, the occurrence of these systems has also been described in symbiotic bacteria [12]. These systems allow DNA uptake or release, i.e., the translocation of genetic material between bacterial strains or between bacteria and eukaryotic cells [15, 16]. In addition, types III (T3SS), IV (T4SS), and VI (T6SS) systems have the ability to directly translocate bacterial proteins into the cells of eukaryotic susceptible hosts [17, 18]. Therefore, several studies suggest a role for this translocated bacterial material in the primary interplay between bacteria and their hosts for the establishment of a mutualistic symbiosis [10, 13].In rhizobia, the studies regarding the T4SS are restricted toMesorhizobium japonicum [19, 20],Rhizobium etli [16],Ensifer meliloti, andEnsifer medicae[12, 21]. However, it is considered that, in general, the rhizobial T4SS protein apparatus is similar to the bacterial pathogenAgrobacterium tumefaciens. In theA. tumefaciens strain C58 three types of T4SS genes were described:vir,avh, andtrb [21]. The Tra/Trb and VirB/VirD4 protein complexes are the most studied. The first one is responsible for the conjugative transfer of the Ti plasmid between bacteria and the second one for the delivery T-DNA and effectors proteins to plant cells [22]. In these complexes, the TraG and VirD4 proteins (coupling proteins) have a role as substrate receptors, corresponding to the first T4SS components that contact the substrate (effector proteins or DNA), recruiting specific substrates to the translocation channel [17, 18, 22, 23]. On the other hand, the Trb and VirB proteins are involved in the assembly of the translocation channels [22].One of the evidences that points out the symbiotic importance of T4SS is the genomic location of the T4SS genes. InM. japonicum R7A strain, the genes coding for the Tra/Trb and VirB/VirD4 complexes were found to be located in the symbiosis island [20]. Besides that, the expression of T4SSvirB/virD4 genes seems to be regulated by NodD, which is the main regulator ofnod genes expression [19, 21]. Therefore, the T4SSvirB/virD4 genes expression could be temporally coordinated with Nod Factors production, suggesting a function of T4SS VirB/VirD4 proteins in the early steps of the legume-rhizobia symbioses [9, 11, 20]. Nevertheless, this finding of coordinated expression between T4SS andnod genes [20] did not include the analysis of genes coding for the T4SS Tra/Trb complex. Up to now, it is unknown if any relation exists between the expression oftra/trb genes and some crucial step for the establishment of symbiotic rhizobia-legume relationship.In addition to the findings mentioned above, an interesting aspect observed in someLotussymbionts is that strains harboring the T4SSvirB/virD4 genes do not present T3SS genes andvice versa[19]. This suggests that the symbiotic role of T4SS could be analogous to the symbiotic role of T3SS, even more because in some rhizobia the expression of T3SS genes is also regulated by NodD, and this secretion system is located in the symbiotic island [9].The present study aimed to contribute to our understanding of the structural and functional organization of T4SS in mesorhizobia, using mesorhizobia strains able to nodulate chickpea (Cicer arietinumL.) as a study case. The phylogeny and expression of the T4SStraG gene, which belongs to the T4SStra/trb complex, were investigated. Moreover, the occurrence and genomic context of this and other T4SS genes from thetra/trb andvirB/virD4 complexes were also analyzed in several mesorhizobia strains. Interestingly, the T4SStra/trb complex is present in all the studiedMesorhizobium strains, contrarily to the T4SSvirB/virD4 complex, which was not detected in theCicer arietinummesorhizobia analyzed.


## 2. Materials and Methods

### 2.1. Bacterial Strains, Growth Conditions, and DNA Extraction

The bacterial strains used in this work are listed in Table1. From the 35 rhizobia strains analysed, 21 areCicer arietinum symbionts and, from these, 16 are native isolates obtained from a collection of PortugueseCicer arietinum mesorhizobia previously characterized [25–27]. The remaining 14 rhizobia strains are able to nodulate a diverse range of plant hosts (Table 1).Table 1
Rhizobia strains used in the present study.
Species Strains/ Isolates Host plant NCBI Accession number JGI Bioproject ID NCBI Genome nodD nifA traG 16S rRNA Bradyrhizobium elkanii WSM2783 Lotononis carinata  PRJNA163061 Bradyrhizobium elkanii USDA94 Glycine max  PRJNA165317 Ensifer meliloti 5A14 Medicago sativa  PRJNA167593 Ensifer meliloti C0438LL Medicago sativa  PRJNA47287 Mesorhizobium australicum WSM2073T Biserrula pelecinus  PRJNA47287 Mesorhizobium ciceri bv. biserrulae WSM1271 Biserrula pelecinus  PRJNA48991 Mesorhizobium ciceri CMG6 Cicer arietinum  PRJNA182744 Mesorhizobium ciceri UPM-Ca7 Cicer arietinum KT966794 KT966812 KT966830 DQ444456 NA Mesorhizobium ciceri WSM4083 Bituminaria bituminosa  PRJNA78191 Mesorhizobium ciceri LMS-1 Cicer arietinum KT966809 KT966811 KT966829 JQ033929 PRJNA507072 Mesorhizobium ciceri CC1192 Cicer arietinum  PRJNA317272 Mesorhizobium japonicum MAFF303099T Lotusspp.  PRJNA18 Mesorhizobium japonicum R7A Lotusspp.  PRJNA74389 Mesorhizobium loti CJ3sym Lotusspp.  PRJNA165305 Mesorhizobium loti NZP2037 Lotusspp.  PRJNA81803 Mesorhizobium loti R88b Lotusspp.  PRJNA76961 Mesorhizobium mediterraneum UPM-Ca36T Cicer arietinum KT966810 KT966813 KT966831 L38825 NZ_NPKI00000000 Mesorhizobium metallidurans STM 2683T Anthyllis vulneraria  PRJEB1501 Mesorhizobium muleiense CGMCC 1.11022T Cicer arietinum  PRJNA329780 Mesorhizobium opportunistum WSM2075T Biserrula pelecinus  PRJNA33861 Mesorhizobiumsp. 29 Cicer arietinum KT966796 KT966814 KT966832 AY225384 NA Mesorhizobiumsp. 64b Cicer arietinum KT966797 KT966815 KT966833 AY225385 NA Mesorhizobiumsp. BR-15 Cicer arietinum KT966798 KT966816 KT966834 EU652125 NA Mesorhizobiumsp. EE-7 Cicer arietinum KT966799 KT966817 KT966835 AY225397 NA Mesorhizobiumsp. EE-12 Cicer arietinum KT966800 KT966818 KT966836 AY225398 NA Mesorhizobiumsp. G-55 Cicer arietinum KT966801 KT966819 KT966837 EU652149 NA Mesorhizobiumsp. PII-3 Cicer arietinum KT966802 KT966820 KT966838 EU652106 NA Mesorhizobiumsp. PMI-6 Cicer arietinum KT966795 KT966821 KT966839 EU652121 NA Mesorhizobiumsp. S-1 Cicer arietinum KT966803 KT966822 KT966840 EU652169 NA Mesorhizobiumsp. S-8 Cicer arietinum KT966793 KT966823 KT966841 EU652116 NA Mesorhizobiumsp. SL-9 Cicer arietinum KT966804 KT966824 KT966842 JN191663 NA Mesorhizobiumsp. ST-2 Cicer arietinum KT966805 KT966825 KT966843 AY225401 NA Mesorhizobiumsp. STR-2 Cicer arietinum KT966806 KT966826 KT966844 EU652117 NA Mesorhizobiumsp. STR-14 Cicer arietinum KT966807 KT966827 KT966845 EU652118 NA Mesorhizobiumsp. V-15b Cicer arietinum KT966808 KT966828 KT966846 EF504315 NA Bioproject ID was retrieved from JGI database [24] and NCBI accession numbers for sequences resulting from this study are shown in bold. NA, not available.The total DNA of 18Cicer arietinum mesorhizobia strains grown in TY medium during 16 hours at 28°C was extracted using the E.Z.N.A. bacterial DNA kit (Omega Bio-Tek), according to the manufacturer’s instructions.


### 2.2. Amplification oftraG, nod, andnifA Genes

ThetraG gene was amplified using the primers traGF 5′-ATGCTGACCTACCAGACGCC-3′ and traGRint 5′-CGGAAACTCGTCCAGCATCA-3′, designed to target conserved regions of this gene inMesorhizobium, which allow the amplification of an internal region of ~ 1155 bp. ThenodD andnifAgenes were amplified using primers NodDF 5′-ATGCGTTTCAAAGGACTTG-3′, NodDR 5′-TCACAGCGGGGCAGCCATCC-3′, NifAF 5′-ATGGGCTGCCAAATGGAACG-3′, and NifAR 5′-TCAGAGACGCTTGATCTCGA-3′. These primers allow the amplification of nearly complete sequences of these two genes (918 bp fornodD and 1059 bp fornifA,). The PCR reactions were performed in a final volume of 50 μL, using 20 ng of total DNA, 1× reaction buffer, 0.2 mM of each dNTP, 1.5mM of MgSO4(traGandnifA) or 1 mM of MgSO4 (nodD), 15 pmol of each primer, and 0.02 U/μL of KOD Hot Start DNA polymerase (Merck Millipore). The amplification programs were 2 min of initial denaturation at 95°C and 30 cycles of 20 s at 95°C, followed by 10 s of annealing at 58°C (traG andnodD) or 62°C (nifA) and an extension step of 15 s (nodD), 17 s (nifA), or 23 s (traG) at 70°C. PCR products were purified using the GFX DNA purification kit (GE Healthcare) or MinElute Gel Extraction kit (QIAGEN) following the manufacturer’s instructions. Sequencing reactions were performed by Macrogen Europe (Amsterdam, Netherlands).The sequences ofnodD, nifA, andtraGgenes from theCicer arietinum mesorhizobia strains have been deposited in the GenBank database under the accession numbers KT966793 to KT966810, KT966811 to KT966828 and KT966829 to KT966846, respectively (Table 1).


### 2.3. Phylogenetic Analysis

ThenodD,nifA,traG, and 16S rRNA nucleotide sequences from several rhizobia strains able to nodulate different plant hosts (Anthyllis vulneraria,Biserrula pelecinus,Bituminaria bituminosa,Cicer arietinum,Glycine max,Lotononis carinata,Lotusspp., andMedicago sativa) were either obtained in this study or retrieved from NCBI or JGI IMG database [24, 28] (Table 1). Sequences were analyzed and aligned using BIOEDIT (version 7.0.4.1) [29]. Molecular phylogenies fornodD,nifA,traG, and 16S rRNA nucleotide sequences were generated with MEGA6 version 6.0.6 [30], using the Maximum Likelihood method, with the distance correction calculated by Tamura 3-parameter model, with rate among sites gamma distributed fornodD phylogeny and rate among sites gamma distributed with invariant sites fornifA,traG, and 16S rRNA gene phylogenies.The phylogenetic trees were rooted using theBradyrhizobium elkanii andE. meliloti species as outgroup. Robustness of tree nodes was evaluated using bootstrap analyses, with 1000 replicates.


### 2.4. Analysis of the Genomic Regions Containing the T4SS Genes, Symbiosis Genes, and T3SS Genes

The occurrence and genome context of T4SS genes, symbiosis genes, and two T3SS genes were analyzed in the genomes ofCicer arietinum symbionts (Mesorhizobium mediterraneum UPM-Ca36T,M. muleienseCGMCC 1.11022T, M. ciceri LMS-1,M. ciceri CMG6, andM. ciceri CC1192),Biserrula pelecinus symbionts (M. ciceri bv.biserrulae WSM1271,M. australicum WSM2073T, andM. opportunistum WSM2075T), andLotus spp. symbionts (M. loti NZP2037,M. japonicum R7A, andM. japonicum MAFF303099T) (formerM. loti strains, reclassification according to [31]) using local BLAST tool from BIOEDIT (version 7.0.4.1) [29]. All these genomes are available in JGI IMG or NCBI genome database. TheM. ciceri LMS-1 genome sequencing data were obtained in our lab (Bioproject accession PRJNA507072) and the contigs used in this work were submitted to NCBI (accession numbers MK226192 to MK226197).In total, the localization of 38 genes was analyzed, namely,nod genes (nodA,nodB,nodC, andnodD),nif genes (nifA,nifH,nifD,nifK,nifE, andnifN),fix genes (fixG,fixH),vir genes (virA,virG,virB1,virB2,virB3,virB4,virB5,virB6,virB7,virB8,virB9,virB10,virB11, andvirD4),tra/trb genes (traG,trbB,trbC,trbD,trbE,trbJ,trbL,trbF,trbG, andtrbI), and the T3SS genesrhcJ andrhcN (named as in [32]). This analysis was based on the comparison of these mesorhizobial genomes with the well-characterized symbiosis island ofM. japonicum R7A, in which the T4SS genes are located [33].


### 2.5. Analysis oftraGandnodAGenes Expression inM. ciceri LMS-1 andM. mediterraneum UPM-Ca36T

To evaluate thetraG andnodA genes expression in theCicer arietinum-nodulatingM. mediterraneum UPM-Ca36T andM. ciceri LMS-1, the total RNA from those strains was extracted from free-living cell cultures with and without exposure toCicer arietinum root exudates and also from the bacteroids at two different time points.The root exudates were obtained as described by [34], with slight modifications, namely using minimal medium described by [35]. For expression analysis in free-living conditions, with and without root exudates, cell cultures were grown in five mL of liquid TY medium at 28°C until exponential-phase. After centrifugation at 8000 g during five minutes, cells were resuspended in five mL of root exudates and incubated for 24 hours at 28°C. Five mL of minimal medium [35] was used to resuspend cells not exposed to root exudates and these were incubated under the same conditions as previously mentioned.For expression analysis in bacteroids,Cicer arietinum plants were grown and inoculated as described by [36], being then used to collect root nodules at 15 and 25 days after rhizobial inoculation (dpi). The nodules were treated for posterior RNA extraction as described by [37]. Total RNA of free-living bacteria and bacteroids was extracted using the GeneJET™ RNA Purification Kit (ThermoFisher Scientific). DNA contamination was removed by digestion with DNase I (Roche Diagnostics), followed by RNA cleanup using the same RNA Purification Kit mentioned before. Approximately, 1 μg of total RNA was subjected to reverse transcription for cDNA synthesis, using the RevertAid First Strand cDNA Synthesis kit (ThermoFisher Scientific).ThetraG andnodA genes expression was analyzed by semiquantitative RT-PCR analyses as described in [38]. The cDNA previously obtained was used for PCR amplification of partial sequences of thetraG andnodAgenes (primers traGIntF 5′- GGCCAATCTACAAGCCGTGG -3′ and traGIntR3 5′- GCCCACCGTGAAGACCCATA -3′ fortraG; primers NodAIntF 5′- ccgaatgtcgagtggaagtt -3′ and NodAIntR3 5′- ctcgccaactttgatgaagc -3′ fornodA), which generates a fragment of 193 and 234 bp, respectively. These PCR reactions were performed in a final volume of 50 μL, using 2 μL of cDNA (~40 ng), 1× reaction Green GoTaq® Flexi buffer, 0.2 mM of each dNTP, 1.5 mM MgCl2,15 pmol of each primer and 0.025U/μL of GoTaq® G2 Flexi DNA Polymerase (Promega, Fitchburg, U.S.A). The amplification program was 2 min of initial denaturation at 95°C, 30 cycles of 60 s at 95°C, 60 s at 59°C (traG) or 54°C (nodA), 12 s (traG), or 14 s (nodA) at 72°C and a final extension of 5 min at 72°C.The amplification of the 16S rRNA gene was used to normalize the relativetraGandnodAtranscript abundance. Primers IntF and IntR [25] were used to generate a fragment of 199 bp. This PCR reaction was performed in a final volume of 50 μL, using 2 μL of cDNA (~40 ng), 1× reaction Green GoTaq® Flexi buffer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 15 pmol of each primer, and 0.025U/μL of GoTaq® G2 Flexi DNA Polymerase (Promega, Fitchburg, USA). The amplification program was 2 min of initial denaturation at 95°C, 30 cycles of 60 s at 95°C, 60 s at 56°C, 12 s at 72°C, and a final extension of 5 min at 72°C. Densitometric analyses of ethidium bromide-stained agarose gels were performed using Kodak Digital Science 1D version 2.0.3 (Eastman Kodak Company). Positive controls with total DNA ofM. ciceri LMS-1 andM. mediterraneum UPM-Ca36T as template and negative controls without reverse transcriptase enzyme were performed. Three biological replicates were used for the expression analysis of the genes mentioned above.The data obtained from the RT-PCR analyses were compared using Student’st-test (differences were considered statistically significant atP<0.05, representing the 95% confidence interval).


## 2.1. Bacterial Strains, Growth Conditions, and DNA Extraction

The bacterial strains used in this work are listed in Table1. From the 35 rhizobia strains analysed, 21 areCicer arietinum symbionts and, from these, 16 are native isolates obtained from a collection of PortugueseCicer arietinum mesorhizobia previously characterized [25–27]. The remaining 14 rhizobia strains are able to nodulate a diverse range of plant hosts (Table 1).Table 1
Rhizobia strains used in the present study.
Species Strains/ Isolates Host plant NCBI Accession number JGI Bioproject ID NCBI Genome nodD nifA traG 16S rRNA Bradyrhizobium elkanii WSM2783 Lotononis carinata  PRJNA163061 Bradyrhizobium elkanii USDA94 Glycine max  PRJNA165317 Ensifer meliloti 5A14 Medicago sativa  PRJNA167593 Ensifer meliloti C0438LL Medicago sativa  PRJNA47287 Mesorhizobium australicum WSM2073T Biserrula pelecinus  PRJNA47287 Mesorhizobium ciceri bv. biserrulae WSM1271 Biserrula pelecinus  PRJNA48991 Mesorhizobium ciceri CMG6 Cicer arietinum  PRJNA182744 Mesorhizobium ciceri UPM-Ca7 Cicer arietinum KT966794 KT966812 KT966830 DQ444456 NA Mesorhizobium ciceri WSM4083 Bituminaria bituminosa  PRJNA78191 Mesorhizobium ciceri LMS-1 Cicer arietinum KT966809 KT966811 KT966829 JQ033929 PRJNA507072 Mesorhizobium ciceri CC1192 Cicer arietinum  PRJNA317272 Mesorhizobium japonicum MAFF303099T Lotusspp.  PRJNA18 Mesorhizobium japonicum R7A Lotusspp.  PRJNA74389 Mesorhizobium loti CJ3sym Lotusspp.  PRJNA165305 Mesorhizobium loti NZP2037 Lotusspp.  PRJNA81803 Mesorhizobium loti R88b Lotusspp.  PRJNA76961 Mesorhizobium mediterraneum UPM-Ca36T Cicer arietinum KT966810 KT966813 KT966831 L38825 NZ_NPKI00000000 Mesorhizobium metallidurans STM 2683T Anthyllis vulneraria  PRJEB1501 Mesorhizobium muleiense CGMCC 1.11022T Cicer arietinum  PRJNA329780 Mesorhizobium opportunistum WSM2075T Biserrula pelecinus  PRJNA33861 Mesorhizobiumsp. 29 Cicer arietinum KT966796 KT966814 KT966832 AY225384 NA Mesorhizobiumsp. 64b Cicer arietinum KT966797 KT966815 KT966833 AY225385 NA Mesorhizobiumsp. BR-15 Cicer arietinum KT966798 KT966816 KT966834 EU652125 NA Mesorhizobiumsp. EE-7 Cicer arietinum KT966799 KT966817 KT966835 AY225397 NA Mesorhizobiumsp. EE-12 Cicer arietinum KT966800 KT966818 KT966836 AY225398 NA Mesorhizobiumsp. G-55 Cicer arietinum KT966801 KT966819 KT966837 EU652149 NA Mesorhizobiumsp. PII-3 Cicer arietinum KT966802 KT966820 KT966838 EU652106 NA Mesorhizobiumsp. PMI-6 Cicer arietinum KT966795 KT966821 KT966839 EU652121 NA Mesorhizobiumsp. S-1 Cicer arietinum KT966803 KT966822 KT966840 EU652169 NA Mesorhizobiumsp. S-8 Cicer arietinum KT966793 KT966823 KT966841 EU652116 NA Mesorhizobiumsp. SL-9 Cicer arietinum KT966804 KT966824 KT966842 JN191663 NA Mesorhizobiumsp. ST-2 Cicer arietinum KT966805 KT966825 KT966843 AY225401 NA Mesorhizobiumsp. STR-2 Cicer arietinum KT966806 KT966826 KT966844 EU652117 NA Mesorhizobiumsp. STR-14 Cicer arietinum KT966807 KT966827 KT966845 EU652118 NA Mesorhizobiumsp. V-15b Cicer arietinum KT966808 KT966828 KT966846 EF504315 NA Bioproject ID was retrieved from JGI database [24] and NCBI accession numbers for sequences resulting from this study are shown in bold. NA, not available.The total DNA of 18Cicer arietinum mesorhizobia strains grown in TY medium during 16 hours at 28°C was extracted using the E.Z.N.A. bacterial DNA kit (Omega Bio-Tek), according to the manufacturer’s instructions.


## 2.2. Amplification oftraG, nod, andnifA Genes

ThetraG gene was amplified using the primers traGF 5′-ATGCTGACCTACCAGACGCC-3′ and traGRint 5′-CGGAAACTCGTCCAGCATCA-3′, designed to target conserved regions of this gene inMesorhizobium, which allow the amplification of an internal region of ~ 1155 bp. ThenodD andnifAgenes were amplified using primers NodDF 5′-ATGCGTTTCAAAGGACTTG-3′, NodDR 5′-TCACAGCGGGGCAGCCATCC-3′, NifAF 5′-ATGGGCTGCCAAATGGAACG-3′, and NifAR 5′-TCAGAGACGCTTGATCTCGA-3′. These primers allow the amplification of nearly complete sequences of these two genes (918 bp fornodD and 1059 bp fornifA,). The PCR reactions were performed in a final volume of 50 μL, using 20 ng of total DNA, 1× reaction buffer, 0.2 mM of each dNTP, 1.5mM of MgSO4(traGandnifA) or 1 mM of MgSO4 (nodD), 15 pmol of each primer, and 0.02 U/μL of KOD Hot Start DNA polymerase (Merck Millipore). The amplification programs were 2 min of initial denaturation at 95°C and 30 cycles of 20 s at 95°C, followed by 10 s of annealing at 58°C (traG andnodD) or 62°C (nifA) and an extension step of 15 s (nodD), 17 s (nifA), or 23 s (traG) at 70°C. PCR products were purified using the GFX DNA purification kit (GE Healthcare) or MinElute Gel Extraction kit (QIAGEN) following the manufacturer’s instructions. Sequencing reactions were performed by Macrogen Europe (Amsterdam, Netherlands).The sequences ofnodD, nifA, andtraGgenes from theCicer arietinum mesorhizobia strains have been deposited in the GenBank database under the accession numbers KT966793 to KT966810, KT966811 to KT966828 and KT966829 to KT966846, respectively (Table 1).


## 2.3. Phylogenetic Analysis

ThenodD,nifA,traG, and 16S rRNA nucleotide sequences from several rhizobia strains able to nodulate different plant hosts (Anthyllis vulneraria,Biserrula pelecinus,Bituminaria bituminosa,Cicer arietinum,Glycine max,Lotononis carinata,Lotusspp., andMedicago sativa) were either obtained in this study or retrieved from NCBI or JGI IMG database [24, 28] (Table 1). Sequences were analyzed and aligned using BIOEDIT (version 7.0.4.1) [29]. Molecular phylogenies fornodD,nifA,traG, and 16S rRNA nucleotide sequences were generated with MEGA6 version 6.0.6 [30], using the Maximum Likelihood method, with the distance correction calculated by Tamura 3-parameter model, with rate among sites gamma distributed fornodD phylogeny and rate among sites gamma distributed with invariant sites fornifA,traG, and 16S rRNA gene phylogenies.The phylogenetic trees were rooted using theBradyrhizobium elkanii andE. meliloti species as outgroup. Robustness of tree nodes was evaluated using bootstrap analyses, with 1000 replicates.


## 2.4. Analysis of the Genomic Regions Containing the T4SS Genes, Symbiosis Genes, and T3SS Genes

The occurrence and genome context of T4SS genes, symbiosis genes, and two T3SS genes were analyzed in the genomes ofCicer arietinum symbionts (Mesorhizobium mediterraneum UPM-Ca36T,M. muleienseCGMCC 1.11022T, M. ciceri LMS-1,M. ciceri CMG6, andM. ciceri CC1192),Biserrula pelecinus symbionts (M. ciceri bv.biserrulae WSM1271,M. australicum WSM2073T, andM. opportunistum WSM2075T), andLotus spp. symbionts (M. loti NZP2037,M. japonicum R7A, andM. japonicum MAFF303099T) (formerM. loti strains, reclassification according to [31]) using local BLAST tool from BIOEDIT (version 7.0.4.1) [29]. All these genomes are available in JGI IMG or NCBI genome database. TheM. ciceri LMS-1 genome sequencing data were obtained in our lab (Bioproject accession PRJNA507072) and the contigs used in this work were submitted to NCBI (accession numbers MK226192 to MK226197).In total, the localization of 38 genes was analyzed, namely,nod genes (nodA,nodB,nodC, andnodD),nif genes (nifA,nifH,nifD,nifK,nifE, andnifN),fix genes (fixG,fixH),vir genes (virA,virG,virB1,virB2,virB3,virB4,virB5,virB6,virB7,virB8,virB9,virB10,virB11, andvirD4),tra/trb genes (traG,trbB,trbC,trbD,trbE,trbJ,trbL,trbF,trbG, andtrbI), and the T3SS genesrhcJ andrhcN (named as in [32]). This analysis was based on the comparison of these mesorhizobial genomes with the well-characterized symbiosis island ofM. japonicum R7A, in which the T4SS genes are located [33].


## 2.5. Analysis oftraGandnodAGenes Expression inM. ciceri LMS-1 andM. mediterraneum UPM-Ca36T

To evaluate thetraG andnodA genes expression in theCicer arietinum-nodulatingM. mediterraneum UPM-Ca36T andM. ciceri LMS-1, the total RNA from those strains was extracted from free-living cell cultures with and without exposure toCicer arietinum root exudates and also from the bacteroids at two different time points.The root exudates were obtained as described by [34], with slight modifications, namely using minimal medium described by [35]. For expression analysis in free-living conditions, with and without root exudates, cell cultures were grown in five mL of liquid TY medium at 28°C until exponential-phase. After centrifugation at 8000 g during five minutes, cells were resuspended in five mL of root exudates and incubated for 24 hours at 28°C. Five mL of minimal medium [35] was used to resuspend cells not exposed to root exudates and these were incubated under the same conditions as previously mentioned.For expression analysis in bacteroids,Cicer arietinum plants were grown and inoculated as described by [36], being then used to collect root nodules at 15 and 25 days after rhizobial inoculation (dpi). The nodules were treated for posterior RNA extraction as described by [37]. Total RNA of free-living bacteria and bacteroids was extracted using the GeneJET™ RNA Purification Kit (ThermoFisher Scientific). DNA contamination was removed by digestion with DNase I (Roche Diagnostics), followed by RNA cleanup using the same RNA Purification Kit mentioned before. Approximately, 1 μg of total RNA was subjected to reverse transcription for cDNA synthesis, using the RevertAid First Strand cDNA Synthesis kit (ThermoFisher Scientific).ThetraG andnodA genes expression was analyzed by semiquantitative RT-PCR analyses as described in [38]. The cDNA previously obtained was used for PCR amplification of partial sequences of thetraG andnodAgenes (primers traGIntF 5′- GGCCAATCTACAAGCCGTGG -3′ and traGIntR3 5′- GCCCACCGTGAAGACCCATA -3′ fortraG; primers NodAIntF 5′- ccgaatgtcgagtggaagtt -3′ and NodAIntR3 5′- ctcgccaactttgatgaagc -3′ fornodA), which generates a fragment of 193 and 234 bp, respectively. These PCR reactions were performed in a final volume of 50 μL, using 2 μL of cDNA (~40 ng), 1× reaction Green GoTaq® Flexi buffer, 0.2 mM of each dNTP, 1.5 mM MgCl2,15 pmol of each primer and 0.025U/μL of GoTaq® G2 Flexi DNA Polymerase (Promega, Fitchburg, U.S.A). The amplification program was 2 min of initial denaturation at 95°C, 30 cycles of 60 s at 95°C, 60 s at 59°C (traG) or 54°C (nodA), 12 s (traG), or 14 s (nodA) at 72°C and a final extension of 5 min at 72°C.The amplification of the 16S rRNA gene was used to normalize the relativetraGandnodAtranscript abundance. Primers IntF and IntR [25] were used to generate a fragment of 199 bp. This PCR reaction was performed in a final volume of 50 μL, using 2 μL of cDNA (~40 ng), 1× reaction Green GoTaq® Flexi buffer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 15 pmol of each primer, and 0.025U/μL of GoTaq® G2 Flexi DNA Polymerase (Promega, Fitchburg, USA). The amplification program was 2 min of initial denaturation at 95°C, 30 cycles of 60 s at 95°C, 60 s at 56°C, 12 s at 72°C, and a final extension of 5 min at 72°C. Densitometric analyses of ethidium bromide-stained agarose gels were performed using Kodak Digital Science 1D version 2.0.3 (Eastman Kodak Company). Positive controls with total DNA ofM. ciceri LMS-1 andM. mediterraneum UPM-Ca36T as template and negative controls without reverse transcriptase enzyme were performed. Three biological replicates were used for the expression analysis of the genes mentioned above.The data obtained from the RT-PCR analyses were compared using Student’st-test (differences were considered statistically significant atP<0.05, representing the 95% confidence interval).


## 3. Results

### 3.1. Phylogenetic Analysis

With the purpose of comparing the phylogeny of thetraG gene with that of symbiosis genes, thenodD, nifA, andtraGnucleotide sequences of these genes were analyzed for 33 mesorhizobia strains able to nodulate different plant hosts (Anthyllis vulneraria, Biserrula pelecinus, Bituminaria bituminosa, Cicer arietinum, Glycine max, Lotononis carinata, Lotusspp.,andMedicago sativa) (Figures 1, 2, and 3, respectively). In order to also compare thetraG phylogeny with the species tree, a phylogeny based on the taxonomic marker 16S rRNA gene was also generated using the same set of strains (Figure 4).Figure 1
Maximum Likelihood tree based onnodDgene sequences (alignment length of 731 bp). Tamura 3-parameter model with rate among sites gammas distributed was used. Percentage of bootstrap support (1000 replicates) is indicated on internal branches. Scale bar indicates 0.05 substitutionsper site.Figure 2
Maximum Likelihood tree based onnifAgene sequences (alignment length of 1014 bp). Tamura 3-parameter model with rate among sites gamma distributed with invariant sites was used. Percentage of bootstrap support (1000 replicates) is indicated on internal branches. Scale bar indicates 0.1 substitutionsper site.Figure 3
Maximum Likelihood tree based ontraGgene sequences (alignment length of 1004 bp). Tamura 3-parameter model with rate among sites gamma distributed with invariant sites was used. Percentage of bootstrap support (1000 replicates) is indicated on internal branches. Scale bar indicates 0.2 substitutionsper site.Figure 4
Maximum Likelihood tree based on 16S rRNA gene sequences (alignment length of 1278 bp). Tamura 3-parameter model with rate among sites gamma distributed with invariant sites was used. Percentage of bootstrap support (1000 replicates) is indicated on internal branches. Scale bar indicates 0.01 substitutionsper site.As expected, the phylogenetic trees based on sequences of the symbiosis genesnodD (Figure 1) andnifA (Figure 2) showed similar topologies. Well defined clusters that correspond to the different host plants were identified, reflecting the high level of sequence conservation among strains that nodulate the same host legume. Three main clusters of mesorhizobia strains could be distinguished: mesorhizobia able to nodulateCicer arietinum; strains that were isolated fromBiserrula pelecinus grouping closer with a strain nodulatingAnthyllis vulneraria; and mesorhizobia able to nodulate severalLotusspecies.M. ciceri WSM 4083, a symbiont ofBituminaria bituminosa, grouped apart from these clusters (Figures 1 and 2). Contrary to thenodD phylogenetic analysis, thenifA phylogeny reflects the fact thatLotussymbionts share a highernifA sequence similarity withBradyrhizobiumstrains than with other mesorhizobia, which nodulateCicer arietinumandBiserrula pelecinus (Figure 2).Similarly to the phylogenies based on symbiosis genes (nodD andnifA), thetraG based-phylogeny also showed rhizobia strains clustering according to their host range (Figure 3), rather than species affiliation. For instance, all symbionts ofCicer arietinum, Biserrula pelecinus, orLotusspp. formed separated clusters. ThetraG gene is conserved among theMesorhizobium species able to nodulate the same host plant, which suggests that this gene was prone to horizontal gene transfer events.As expected, the 16S rRNA-based phylogeny presented a different topology from those based on thenodD ornifA genes. This phylogeny comprises three main clusters, corresponding to different genera, namely,Mesorhizobium,Ensifer,andBradyrhizobium (Figure 4).Cicer arietinum mesorhizobia native isolates are found scattered along theMesorhizobium cluster.


### 3.2. Genomic Localization and Organization of T4SS Genes, T3SS Genes, and Symbiosis Genes

In silico analyses for genomic localization of the T4SS genes (both thetra/trb andvirB/virD4 complexes), symbiosis genes, and two T3SS genes were performed using genomic data from several mesorhizobia strains (Figure 5). Symbionts ofCicer arietinum, Biserrula pelecinus, andLotusspp.were included in these analyses. Nevertheless, we need to consider that the genomes of majority of theCicer arietinum symbionts analyzed are in draftstatus, with the exception of theM. ciceri CC1192 genome, which is complete [39].Figure 5
Genomic context analysis of T4SS, T3SS and symbiosis genes in the symbiotic islands/regions of the genomes of several mesorhizobia strains. This analysis includes the genomes of symbionts of three different host plants, namelyCicer arietinum,Biserrula pelecinus, and differentLotusspecies. Thetra/trbandvirB/virD4 complexes identified in the mesorhizobia strains and studied in this work present similarities with the T4SSe-like and T4SSb/c-like ofEnsifer strains, according to [21]. Slash sign (/) indicates a random genetic distance.In all the genomes analyzed, the T4SStraG/trb genes complex was always found near nitrogen fixation genes, namely,fixG andfixH(Figure 5). In addition, BLAST analyses suggest that none of the fiveCicer arietinummicrosymbionts (M. mediterraneumUPM-Ca36T,M. muleiense CGMCC 1.11022T,M. ciceri LMS-1,M. ciceriCMG6, andM. ciceri CC1192) include the T4SSvirB/virD4 gene complex, homologous to thevirB/virD4 of theM. japonicum R7A. Nevertheless, all the analyzedCicer arietinummesorhizobia strains encode genes assigned to the T3SS, namely,rhcJ andrhcN, which were not found in theM. japonicum R7A andM. loti NZP2037 genomes or in the genomes ofBiserrula pelecinus nodulating strains. For twoCicer arietinum mesorhizobia species the genomic data available shows the localization of these T3SS genes near thetra/trb complex and consequently close to thefixGandfixH genes. Similarly to theCicer arietinum-nodulating mesorhizobia strains, theM. japonicum MAFF303099T, aLotusspp. symbiont, does not encode thevirB/virD4 genes and also shows the T3SSrhcJ andrhcN genes located close to thetra/trb complex (Figure 5).Interestingly, in strainsM. japonicumR7A andM. loti NZP2037, thenod genes are localized nearby thevirB/virD4T4SS genes and not close to thenif genes as in symbionts ofBiserrula pelecinus (M. ciceri bv.biserrulae WSM1271,M. australicum WSM2073T, andM. opportunistum WSM2075T). InM. japonicum MAFF303099T, thenod cluster is neither close to thenif genes nor close to the T4SS or T3SS analysed genes. For most ofCicer arietinum mesorhizobia genomes analysed, thenod genes were detected in a different scaffold from that ofnif, T4SS, or T3SS genes. Nevertheless, these data are consistent with an organization similar to the one found in the complete genome ofM. ciceri CC1192, aCicer arietinumsymbiont (Figure 5).


### 3.3. Analyses oftraGandnodAGenes Expression by Semiquantitative RT-PCR

To understand the timing of the T4SStraG gene expression, the expression of this gene and of thenodA gene was evaluated by semiquantitative RT-PCR in free-living bacteria grown in the presence and absence ofCicer arietinum root exudates and also in bacteroids from root nodules collected at two different time points (15 and 21 days after inoculation) (Figure 6). ThetraG andnodA gene expression analyses were performed forM. mediterraneumUPM-Ca36T andM. ciceri LMS-1 strains, bothCicer arietinumsymbionts. In free-living conditions, thetraG gene expression was only detected when bacteria were exposed toCicer arietinumroot exudates (Figures 6(a) and 6(c)). In bacteroids, thetraG gene expression was only detected in developing nodules (collected at 15 dpi) and exclusively for nodules induced byM. mediterraneumUPM-Ca36T. In older nodules (21 dpi) thetraG transcripts were no longer detected. For the strain UPM-Ca36T, approximately the same levels oftraG transcripts were detected for bacteria exposed to exudates and bacteroids within 15 dpi nodules. As expected, in both strains, thenodA gene expression (regulated by NodD) was only observed in free-living bacteria when exposed toCicer arietinum root exudates and in early stage of the nodulation process (15 dpi nodules) (Figures 6(b) and 6(d)). Nevertheless, the abundance ofnodA transcripts in those nodules was significantly lower compared to the levels of transcription detected for this gene when free-living bacteria were exposed toCicer arietinum root exudates.Figure 6
Analysis oftraG andnodA gene expression by semiquantitative RT-PCR inM. mediterraneum UPM-Ca36T andM. ciceri LMS-1 strains. These genes expression level were evaluated in bacteria (1; no exudates) nonexposed toCicer arietinum exudates (2; exudates) exposed toCicer arietinum root exudates and in bacteroids within nodules collected (3; nodules 15 dpi) 15 days after inoculation and (4; nodules 21 dpi) bacteroids within nodules of plants collected 21 days after inoculation. (a)M. mediterraneum UPM-Ca36TtraG expression. (b)M. mediterraneum UPM-Ca36TnodA gene expression. (c)M. ciceri LMS-1traG expression. (d)M. ciceri LMS-1nodA expression. Relative abundance oftraG andnodA transcripts was normalized against 16S rRNA gene expression. Data are presented as the mean and standard deviation values of three independent biological replicates. Different letters (A, B) correspond to statistical significant differences (P < 0.05).
(a) (b) (c) (d)


## 3.1. Phylogenetic Analysis

With the purpose of comparing the phylogeny of thetraG gene with that of symbiosis genes, thenodD, nifA, andtraGnucleotide sequences of these genes were analyzed for 33 mesorhizobia strains able to nodulate different plant hosts (Anthyllis vulneraria, Biserrula pelecinus, Bituminaria bituminosa, Cicer arietinum, Glycine max, Lotononis carinata, Lotusspp.,andMedicago sativa) (Figures 1, 2, and 3, respectively). In order to also compare thetraG phylogeny with the species tree, a phylogeny based on the taxonomic marker 16S rRNA gene was also generated using the same set of strains (Figure 4).Figure 1
Maximum Likelihood tree based onnodDgene sequences (alignment length of 731 bp). Tamura 3-parameter model with rate among sites gammas distributed was used. Percentage of bootstrap support (1000 replicates) is indicated on internal branches. Scale bar indicates 0.05 substitutionsper site.Figure 2
Maximum Likelihood tree based onnifAgene sequences (alignment length of 1014 bp). Tamura 3-parameter model with rate among sites gamma distributed with invariant sites was used. Percentage of bootstrap support (1000 replicates) is indicated on internal branches. Scale bar indicates 0.1 substitutionsper site.Figure 3
Maximum Likelihood tree based ontraGgene sequences (alignment length of 1004 bp). Tamura 3-parameter model with rate among sites gamma distributed with invariant sites was used. Percentage of bootstrap support (1000 replicates) is indicated on internal branches. Scale bar indicates 0.2 substitutionsper site.Figure 4
Maximum Likelihood tree based on 16S rRNA gene sequences (alignment length of 1278 bp). Tamura 3-parameter model with rate among sites gamma distributed with invariant sites was used. Percentage of bootstrap support (1000 replicates) is indicated on internal branches. Scale bar indicates 0.01 substitutionsper site.As expected, the phylogenetic trees based on sequences of the symbiosis genesnodD (Figure 1) andnifA (Figure 2) showed similar topologies. Well defined clusters that correspond to the different host plants were identified, reflecting the high level of sequence conservation among strains that nodulate the same host legume. Three main clusters of mesorhizobia strains could be distinguished: mesorhizobia able to nodulateCicer arietinum; strains that were isolated fromBiserrula pelecinus grouping closer with a strain nodulatingAnthyllis vulneraria; and mesorhizobia able to nodulate severalLotusspecies.M. ciceri WSM 4083, a symbiont ofBituminaria bituminosa, grouped apart from these clusters (Figures 1 and 2). Contrary to thenodD phylogenetic analysis, thenifA phylogeny reflects the fact thatLotussymbionts share a highernifA sequence similarity withBradyrhizobiumstrains than with other mesorhizobia, which nodulateCicer arietinumandBiserrula pelecinus (Figure 2).Similarly to the phylogenies based on symbiosis genes (nodD andnifA), thetraG based-phylogeny also showed rhizobia strains clustering according to their host range (Figure 3), rather than species affiliation. For instance, all symbionts ofCicer arietinum, Biserrula pelecinus, orLotusspp. formed separated clusters. ThetraG gene is conserved among theMesorhizobium species able to nodulate the same host plant, which suggests that this gene was prone to horizontal gene transfer events.As expected, the 16S rRNA-based phylogeny presented a different topology from those based on thenodD ornifA genes. This phylogeny comprises three main clusters, corresponding to different genera, namely,Mesorhizobium,Ensifer,andBradyrhizobium (Figure 4).Cicer arietinum mesorhizobia native isolates are found scattered along theMesorhizobium cluster.


## 3.2. Genomic Localization and Organization of T4SS Genes, T3SS Genes, and Symbiosis Genes

In silico analyses for genomic localization of the T4SS genes (both thetra/trb andvirB/virD4 complexes), symbiosis genes, and two T3SS genes were performed using genomic data from several mesorhizobia strains (Figure 5). Symbionts ofCicer arietinum, Biserrula pelecinus, andLotusspp.were included in these analyses. Nevertheless, we need to consider that the genomes of majority of theCicer arietinum symbionts analyzed are in draftstatus, with the exception of theM. ciceri CC1192 genome, which is complete [39].Figure 5
Genomic context analysis of T4SS, T3SS and symbiosis genes in the symbiotic islands/regions of the genomes of several mesorhizobia strains. This analysis includes the genomes of symbionts of three different host plants, namelyCicer arietinum,Biserrula pelecinus, and differentLotusspecies. Thetra/trbandvirB/virD4 complexes identified in the mesorhizobia strains and studied in this work present similarities with the T4SSe-like and T4SSb/c-like ofEnsifer strains, according to [21]. Slash sign (/) indicates a random genetic distance.In all the genomes analyzed, the T4SStraG/trb genes complex was always found near nitrogen fixation genes, namely,fixG andfixH(Figure 5). In addition, BLAST analyses suggest that none of the fiveCicer arietinummicrosymbionts (M. mediterraneumUPM-Ca36T,M. muleiense CGMCC 1.11022T,M. ciceri LMS-1,M. ciceriCMG6, andM. ciceri CC1192) include the T4SSvirB/virD4 gene complex, homologous to thevirB/virD4 of theM. japonicum R7A. Nevertheless, all the analyzedCicer arietinummesorhizobia strains encode genes assigned to the T3SS, namely,rhcJ andrhcN, which were not found in theM. japonicum R7A andM. loti NZP2037 genomes or in the genomes ofBiserrula pelecinus nodulating strains. For twoCicer arietinum mesorhizobia species the genomic data available shows the localization of these T3SS genes near thetra/trb complex and consequently close to thefixGandfixH genes. Similarly to theCicer arietinum-nodulating mesorhizobia strains, theM. japonicum MAFF303099T, aLotusspp. symbiont, does not encode thevirB/virD4 genes and also shows the T3SSrhcJ andrhcN genes located close to thetra/trb complex (Figure 5).Interestingly, in strainsM. japonicumR7A andM. loti NZP2037, thenod genes are localized nearby thevirB/virD4T4SS genes and not close to thenif genes as in symbionts ofBiserrula pelecinus (M. ciceri bv.biserrulae WSM1271,M. australicum WSM2073T, andM. opportunistum WSM2075T). InM. japonicum MAFF303099T, thenod cluster is neither close to thenif genes nor close to the T4SS or T3SS analysed genes. For most ofCicer arietinum mesorhizobia genomes analysed, thenod genes were detected in a different scaffold from that ofnif, T4SS, or T3SS genes. Nevertheless, these data are consistent with an organization similar to the one found in the complete genome ofM. ciceri CC1192, aCicer arietinumsymbiont (Figure 5).


## 3.3. Analyses oftraGandnodAGenes Expression by Semiquantitative RT-PCR

To understand the timing of the T4SStraG gene expression, the expression of this gene and of thenodA gene was evaluated by semiquantitative RT-PCR in free-living bacteria grown in the presence and absence ofCicer arietinum root exudates and also in bacteroids from root nodules collected at two different time points (15 and 21 days after inoculation) (Figure 6). ThetraG andnodA gene expression analyses were performed forM. mediterraneumUPM-Ca36T andM. ciceri LMS-1 strains, bothCicer arietinumsymbionts. In free-living conditions, thetraG gene expression was only detected when bacteria were exposed toCicer arietinumroot exudates (Figures 6(a) and 6(c)). In bacteroids, thetraG gene expression was only detected in developing nodules (collected at 15 dpi) and exclusively for nodules induced byM. mediterraneumUPM-Ca36T. In older nodules (21 dpi) thetraG transcripts were no longer detected. For the strain UPM-Ca36T, approximately the same levels oftraG transcripts were detected for bacteria exposed to exudates and bacteroids within 15 dpi nodules. As expected, in both strains, thenodA gene expression (regulated by NodD) was only observed in free-living bacteria when exposed toCicer arietinum root exudates and in early stage of the nodulation process (15 dpi nodules) (Figures 6(b) and 6(d)). Nevertheless, the abundance ofnodA transcripts in those nodules was significantly lower compared to the levels of transcription detected for this gene when free-living bacteria were exposed toCicer arietinum root exudates.Figure 6
Analysis oftraG andnodA gene expression by semiquantitative RT-PCR inM. mediterraneum UPM-Ca36T andM. ciceri LMS-1 strains. These genes expression level were evaluated in bacteria (1; no exudates) nonexposed toCicer arietinum exudates (2; exudates) exposed toCicer arietinum root exudates and in bacteroids within nodules collected (3; nodules 15 dpi) 15 days after inoculation and (4; nodules 21 dpi) bacteroids within nodules of plants collected 21 days after inoculation. (a)M. mediterraneum UPM-Ca36TtraG expression. (b)M. mediterraneum UPM-Ca36TnodA gene expression. (c)M. ciceri LMS-1traG expression. (d)M. ciceri LMS-1nodA expression. Relative abundance oftraG andnodA transcripts was normalized against 16S rRNA gene expression. Data are presented as the mean and standard deviation values of three independent biological replicates. Different letters (A, B) correspond to statistical significant differences (P < 0.05).
(a) (b) (c) (d)


## 4. Discussion

Although some studies have already analyzed rhizobia nodulatingLotus andMedicago species [12, 19–21], little is known about the T4SS inCicer arietinum mesorhizobia. Herein, the mesorhizobiatraG phylogeny and expression of this gene inCicer arietinummesorhizobia strains, as well as the occurrence and genomic context of this and other T4SS genes, were investigated.The phylogenetic analysis performed using the T4SStraG gene sequences from nativeCicer arietinum mesorhizobia isolates, together withtraG sequences from other mesorhizobia with genomes completely or partially sequenced, shows that mesorhizobia strains nodulating the same host plant group in the same cluster, regardless of their species affiliation. ThetraG-based phylogeny is similar to the ones obtained by the phylogenetic analysis of the symbiosis genesnodD andnifAand consistent with previous studies reporting phylogenies of symbiosis genes [36, 40]. This suggests that thetraG gene may be prone to horizontal gene transfer together with the symbiosis genes. This is further supported by the genomic context analysis performed in this work for 11 mesorhizobia, which shows proximity in terms of localization between thetraG/trbgenes and the symbiosis genesfixG andfixH,involved in bacterial nitrogen fixation [3]. Moreover, our analysis also verified that thetraGgene is localized within the previously identified symbiosis island ofBiserrula pelecinus andLotus spp. symbionts, namely, forM. ciceri bv.biserrulae WSM1271 [41],M. australicum WSM2073T [42],M. opportunistum WSM2075T [43],M. japonicum R7A [33],Mesorhizobium loti NZP2037 [44], andM. japonicum MAFF303099T [45].Horizontal transfer of symbiosis genes between different species on the soil would allow a rhizobia strain to acquire the ability to nodulate a new host, when receiving a specific set of symbiosis genes [40, 46–52]. A well-known example of this event was reported for the strainsM. australicumWSM2073T andM. opportunistumWSM2075T, which seem to have received the complete symbiotic island from the inoculant strainM. ciceribv.biserrulaeWSM1271 and therefore gained the ability to nodulate the introduced legumeBiserrula pelecinus [53, 54]. The present work suggests that the nonsymbiotic genetraG may have been transferred horizontally between strains that nodulate the same host. In fact, the TraG protein could be involved in the effective transference of the symbiotic region in these mesorhizobia strains, since this protein has been described as having a crucial function in bacterial conjugation [55] and was shown to be required for horizontal gene transfer of the symbiosis island fromAzorhizobium caulinodans to other rhizobia [56].Analysis of theM. japonicum R7A genome [33, 57] suggested at least two types of T4SS genes clusters inMesorhizobium genomes, namely, thetra/trb and thevirB/virD4genes. Similar organization of these genes clusters was reported forEnsifer strains, namely, the T4SSb/c (virB/virD4) and T4SSe (tra/trb) clusters [21]. Our analysis shows that this is true for other mesorhizobia strains, namely,M. ciceri bv. biserrulae WSM1271 [40, 57],M. australicum WSM2073T [42],M. opportunistum WSM2075T [43], andM. loti NZP2037 [44, 58]. However, the work of Hubber and collaborators [19] shows that the structural organization of the T4SS in mesorhizobia is not always composed by bothtra/trb andvirB/virD4 gene clusters. In theLotus symbiontM. japonicum MAFF303099T, the absence of the T4SSvirB/virD4 complex was reported [19]. These authors proposed that inM. japonicum MAFF303099T the absence of these genes is somehow compensated by the presence of T3SS genes, which are not encoded in theM. japonicum R7A genome. The present analysis ofCicer arietinumsymbionts contributes to our understanding about this topic, supporting the idea that the absence of the T4SSvirB/virD4 gene cluster could be more common in mesorhizobia genomes than initially assumed, since in theseCicer arietinummesorhizobia strains the T4SSvirB/virD4 complex seems to be absent, while the T3SS genes were detected in all the strains. Altogether, these data could also suggest that, in mesorhizobia, the role(s) of the T4SS VirB/VirD4 proteins could be at least partially substituted by the role(s) of the T3SS proteins. Further studies are required to verify this putative functional redundancy.The analysis of thetraG gene expression in twoCicer arietinumsymbionts,M. mediterraneum UPM-Ca36T, andM. ciceri LMS-1 shows that this gene is expressed in free-living bacteria when exposed toCicer arietinumroot exudates and may also be expressed in recently formed root nodules. A very similar expression profile is observed for the nodulation genenodA. Although these findings are consistent with a putative regulation of thetraG gene by NodD, which would be similar to what was previously reported for T4SSvirB/virD4genes as well as T3SS genes [21], nonod-box was detected upstream thetraG gene in the mesorhizobia genomes analyzed. The expression oftraG during nodulation was also supported by another study reporting high-resolution transcriptome analyses in bacteroids ofEnsifersp. NGR234, which detected T4SS gene expression (at very low levels), inVigna unguiculata andLeucaena leucocephala nodules [59]. Ling and collaborators [56], using a gene knockout approach, showed that TraG is required for conjugative DNA transfer in rhizobia. A role of TraG in horizontal transfer events of symbiosis islands is consistent with the timing of expression detected for this gene, which in this work was seen to be activated by legume exudates and even in developing nodules.


## 5. Conclusions

The location of T4SS genes clusters in the symbiotic regions of rhizobia genomes, as well as the timing of thetraG gene expression, supports the previously reported role of TraG in the horizontal transfer events of symbiosis genes. Moreover, the phylogeny oftraG is similar to thenodD andnifA phylogenies, which suggests thattraGis horizontally transferred together with these symbiosis genes. This study addressing several aspects of thetraG gene in mesorhizobia contributes to a better understanding of the structural and functional organization of this secretion system in mutualistic symbiotic bacteria.



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*Source: 3715271-2019-01-30.xml*