NrcR, a New Transcriptional Regulator of Rhizobium tropici CIAT 899 Involved in the Legume Root-Nodule Symbiosis

Pablo del Cerro; Amanda A. P. Rolla-Santos; Rocío Valderrama-Fernández; Antonio Gil-Serrano; Ramón A. Bellogín; Douglas Fabiano Gomes; Francisco Pérez-Montaño; Manuel Megías; Mariangela Hungría; Francisco Javier Ollero

doi:10.1371/journal.pone.0154029

Abstract

The establishment of nitrogen-fixing rhizobium-legume symbioses requires a highly complex cascade of events. In this molecular dialogue the bacterial NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, are responsible for the biosynthesis and secretion of Nod factors which are key molecules for successful nodulation. Other transcriptional regulators related to the symbiotic process have been identified in rhizobial genomes, including negative regulators such as NolR. Rhizobium tropici CIAT 899 is an important symbiont of common bean (Phaseolus vulgaris L.), and its genome encompasses intriguing features such as five copies of nodD genes, as well as other possible transcriptional regulators including the NolR protein. Here we describe and characterize a new regulatory gene located in the non-symbiotic plasmid pRtrCIAT899c, that shows homology (46% identity) with the nolR gene located in the chromosome of CIAT 899. The mutation of this gene, named nrcR (nolR-like plasmid c Regulator), enhanced motility and exopolysaccharide production in comparison to the wild-type strain. Interestingly, the number and decoration of Nod Factors produced by this mutant were higher than those detected in the wild-type strain, especially under salinity stress. The nrcR mutant showed delayed nodulation and reduced competitiveness with P. vulgaris, and reduction in nodule number and shoot dry weight in both P. vulgaris and Leucaena leucocephala. Moreover, the mutant exhibited reduced capacity to induce the nodC gene in comparison to the wild-type CIAT 899. The finding of a new nod-gene regulator located in a non-symbiotic plasmid may reveal the existence of even more complex mechanisms of regulation of nodulation genes in R. tropici CIAT 899 that may be applicable to other rhizobial species.

Citation: del Cerro P, Rolla-Santos AAP, Valderrama-Fernández R, Gil-Serrano A, Bellogín RA, Gomes DF, et al. (2016) NrcR, a New Transcriptional Regulator of Rhizobium tropici CIAT 899 Involved in the Legume Root-Nodule Symbiosis. PLoS ONE 11(4): e0154029. https://doi.org/10.1371/journal.pone.0154029

Editor: Francisco Martinez-Abarca, Estacion Experimental del Zaidin—CSIC, SPAIN

Received: February 23, 2016; Accepted: April 7, 2016; Published: April 20, 2016

Copyright: © 2016 del Cerro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are in the body of the manuscript and in the supporting information.

Funding: This work was supported by the Project AGL2012-38831 of the Ministerio de Economía y Competitividad (Spanish Government, www.mineco.gob.es), CNPq (National Council for Scientific and Technological Development, http://cnpq.br/, Science without Borders 400205/2012-5). AAPR-S acknowledges a postdoctoral fellowship from CNPq (National Council for Scientific and Technological Development, http://cnpq.br/, Science without Borders 400205/2012-5). MH a research fellow from CNPq (NPq (National Council for Scientific and Technological Development, http://cnpq.br/). FPM a postdoctoral fellowship from VPP (University of Seville, https://investigacion.us.es/secretariado-investigacion/plan-propio). PdC a FPU fellowship from Ministerio de Economía y Competitividad (FPU14/00160, Spanish Government, www.mineco.gob.es). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The establishment of the symbiosis between rhizobia and their specific host legumes involves highly complex events that culminate in the formation of nodules and in the establishment of the nitrogen fixation process. Nodulation requires a molecular dialogue between the bacteria and the host plants. The dialogue begins with the exudation of molecules from the legume roots, mostly flavonoids, which are recognized by the specific rhizobium, inducing the transcription of a set of nodulation genes [1, 2]. These genes are responsible for the biosynthesis and secretion of Nod factors (NFs), identified as lipochitooligosaccharides, required for launching the nodulation and the nitrogen-fixation process [3–7].

A large number of bacterial transcriptional regulators actuate nodulation, the most important of which are the NodD proteins, which belong to the LysR-type transcriptional-regulator family. The nodD genes are constitutively expressed and their cognate proteins are responsible for the recognition of suitable flavonoids, which start the transcription of the nodulation genes [8, 9]. Another important group of transcriptional regulators is the family of metal-sensing regulatory proteins. In this case, a specific metal-ion union regulates the protein’s allosteric conformation and modulates the expression of several target genes. In bacteria, the arsenic repressor family (ArsR) represents one of the most important regulators of the family of metal-sensing proteins. In this family, there are positive and negative transcriptional regulators, which have been described for many bacteria, including several rhizobial species [10–12]. A well-characterized member of this family is the NolR protein, originally described as a general repressor of the activator nodD1 and the common nodABC operon in Sinorhizobium meliloti [13–15]. Two models to explain the repression through the NolR protein have been proposed: i) in promoters with overlapping transcription initiation and operator sites, NolR binding prevents gene expression enabling RNA polymerase interaction; and ii) in promoter regions containing upstream nod box (NB) sequences for NodD proteins, NolR binds this promoter region and alters NodD association resulting in inactivation of the gene expression [16]. In S. fredii HH103, a nolR mutant provoked changes in NF decoration and exopolysaccharide production [17]. However, S. meliloti and S. medicae nolR mutants increased the nodC expression, and, in the case of S. medicae, improved the competitiveness and nodulation efficiency on Medicago truncatula and Medicago sativa [18].

Rhizobium tropici strain CIAT 899 (hereafter CIAT 899) is a successful microsymbiont of common bean (P. vulgaris L.) in tropical acid soils. The main features of this broad host-range strain include its high tolerance of environmental stresses such as high temperature, acidity and salinity [19–22]. An intriguing feature of CIAT 899 relies on its capacity of producing a large variety of NFs even in the absence of flavonoids when grown under abiotic stresses such as acid or saline conditions [23–30]. Genome sequencing of strain CIAT 899 displayed a large number of genes that code for transcriptional-regulator families, including five nodD genes (all of them located in the symbiotic plasmid; pRtrCIAT899b) and one nolR gene (located in the chromosome). Moreover, another gene located in the megaplasmid pRtrCIAT899c and encoding an undescribed protein which shares homology with the canonical NolR protein has also been identified [31]. In this study, we performed experiments to shed light on the role of this new, undescribed nolR-like transcriptional regulator gene—which we named nrcR—during the symbiotic process.

Materials and Methods

Bacteria growth conditions, plasmids, mutant obtention

Strains and plasmids used in this study are listed in Table 1. R. tropici CIAT 899 strains were grown at 28°C on tryptone yeast (TY) medium [32], B^- minimal medium [33] or yeast extract mannitol (YM) medium [34], supplemented when necessary with apigenin 3.7 μM or with NaCl 300 mM. Escherichia coli strains were cultured on LB medium [35] at 37°C. When required, the media were supplemented with the appropriate antibiotics as previously described [36]. A similar number of colony forming units (cfu/ml) was obtained for both strains by plating bacteria on addecuated solid medium (data not shown).

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Table 1. Bacterial strains and plasmids employed in this study.

https://doi.org/10.1371/journal.pone.0154029.t001

Primers nolR-like-F (5’–TAG CAG AGC GAT GTC AGA) and nolR-like-R (5’–CGA TGC CAA TTT CCG GAA) were used for amplifying the nrcR gene. The 980-bp PCR product was cloned in pGEM®-T Easy (PROMEGA) (Amp^R 100 μg mL^-1) and the resulting plasmid was digested with the enzyme HindIII, which cuts the nrcR gene in one site, and ligated to a 2 kb fragment containg the Ω interposon (Spc^R 100 μg mL^-1) (S1 Fig). Lastly, this plasmid was digested with EcoRI and the 2.98-kb fragment containing the nrcR::Ω was cloned in the rhizobial suicide vector pK18mob [37], that confers resistance to kanamycin (km^R 30 μg mL^-1), equally restricted with EcoRI. The generated plasmid pK18mob containing the nrcR::Ω fragment was transferred from E. coli strain DH5α to R. tropici CIAT 899 strain by conjugation as described by Simon [38], using plasmid pRK2013 as helper [39], and used for the homogenization of the mutated version of nrcR in CIAT 899 by using the methodology previously described [40]. The deletion event was confirmed by PCR and hybridization.

To complement in trans the nrcR mutation, the pGEM®-T Easy plasmid harboring the 980-bp PCR product that contains the whole nrcR gene was digested by EcoRI and cloned into the expression vector pBBR1-MCS-5 (Gm^R 10 μg mL^-1) [41], equally digested with the same restriction enzyme, to generate the plasmid pMUS1333. This plasmid was transferred to ncrR::Ω mutant strain by conjugation, obtaining the ncrR::Ω (pMUS1333) strain. The in cis complementation of the nrcR mutant was carried out cloning the EcoRI fragment containing the entire nrcR gene into the vector pK18mob, generating plasmid pMUS1353. This plasmid was transferred by conjugation into the ncrR::Ω strain and the kanamycin resistant transconjugant was selected. The ncrR::Ω (pMUS1353) strain harbors the plasmid inserted into the pRtrCIAT899c by simple recombination, thus containing both wild-type and mutated nrcR gene.

The parental and derivative strains were deposited at the culture collection of the Department of Biology of the Universidad de Sevilla and at the Diazotrophic and Plant Growth Promoting Bacteria Culture Collection of Embrapa Soja (WFCC Collection # 1213, WDCM Collection # 1054).

Bioinformatics

The Phyre2 web portal (http://www.sbg.bio.ic.ac.uk/phyre2) was used to analyze and predict the function of the NrcR protein [42]. To confirm the prediction obtained, comparisons and similarity analysis among NrcR and the set of protein were carry out using the Basic Local Alignment Search tool (BLAST) suite of NCBI [43].

Specific protein alignments were performed using the ClustalW online platform, [44]. The multiple sequence alignments were edited by using Boxshade at EMBnet.

Motility assays

Swimming and surface motility phenotypes were assayed on TY and B^- minimal medium, supplemented when necessary with NaCl 300 mM or apigenin 3.7 μM. The CIAT 899 and derivative strains were grown in 5 ml of TY medium on an orbital shaker (180 rpm) for 96 h at 28°C. Aliquots of 2 μL of culture suspensions were sink-inoculated in swimming assays (0.28% agar) or drop-inoculated in surface motility assays (0.4% agar) onto Petri dishes and air-dried. Bacterial growth was determined measuring the optical density (O.D.) at 600 nm. All plates were wrapped with parafilm and incubated at 28°C in an upright position and the halo diameters were measure each 24 h. Each experiment was performed three times with three replicates each time.

Biofilm formation assay

The biofilm formation assay on polystyrene surfaces was performed as previously described [45]. CIAT 899 and derivative strains were grown on TY medium, supplemented with NaCl 300 mM or apigenin 3.7 μM when required, for 7 days with gentle rocking at 28°C. Each experiment was performed three times with eight replicates per time.

Quantitative and qualitative analyses of external polysaccharides

The anthrone-H₂SO₄ method, which measures the total reducing sugar content in a given sample [46], was used to determine the total carbohydrate amounts of EPS contained in supernatants of the bacterial cultures. For this purpose, CIAT 899 and derivative strains were grown in 5 mL of liquid YM medium on an orbital shaker (180 rpm) for 96 h at 28°C. When required, the media were supplemented with NaCl (300 mM) or apigenin (3.7 μM). Samples of 1 mL were centrifuged to remove cells. Cell-free culture supernatants were assayed for EPS content via sulfuric acid hydrolysis in the presence of the colorimetric indicator anthrone. Every experiment was performed three times with three replicates each time.

For the isolation of EPS, CIAT 899 and derivatives strains were cultured for five days at 28°C (late stationary phase) in 45 mL of YM medium and concentrated to about 20% of the initial volume on a rotary evaporator; following, three volumes of cold ethanol were added and the solution was maintained at 4°C for 24 h. Following, the solution was centrifuged and the resulting precipitate was dissolved in water and purified by dialysis against distilled water at 4°C, and then freeze-dried. Monosaccharides were identified on Gas-Liquid Chromatography (GLC Agilent 7809A) coupled with mass spectrometry (MS Agilent 5975 mass detector) separation of their per-O-trimethylsilylated methyl glycosides as previously described [29]. Finally, Nuclear Magnetic Resonance (NMR) was carried out. Samples were deuterium-exchanged twice by freeze-drying from D2O and then examined in solution (around 5 mg 750 μL^-1) in 99.9% D2O. ¹H-NMR spectra were recorded at 353 K on a Bruker AV500 spectrometer operating at 500.20 MHz (1H). The HDO signal (4.22 ppm at 353 K) was used as reference.

Lipopolysaccharide (LPS) extraction, separation on SDS-PAGE, and silver staining were performed as previously described using the same bacteria, medium and conditions [29].

Identification of NFs

Purification and LC-MS/MS analyses of NFs produced by the wild-type and nrcR mutant strains growth in B^- minimal medium, supplemented when required with NaCl 300 mM or apigenin 3.7 μM, were performed as described previously [27].

RNA isolation, cDNA synthesis and qRT-PCR

The wild-type and nrcR mutant strains were pre-cultured in 10 mL aliquots of TY medium at 100 rpm and 28°C in the dark. After 48 h, the strains pre-inoculated were transferred to fresh media supplemented when necessary with 300 mM of NaCl or 3.7 μM of apigenin. These cultures were grown under the same conditions than pre-cultures until to reach an OD 600 nm of 0.5 to 0.6.

Total RNA was extracted using Trizol® reagent (Invitrogen/Life Technologies) as previously described [47]. The total RNA concentration was estimated in a NanoDrop ND 1000 spectrophotometer (NanoDrop-Technologies, Inc.) and the integrity was assessed by gel electrophoresis. Extracted RNA samples were treated with DNAse I (Invitrogen/Life Technologies) and the first stand of cDNA was synthesized using SuperscriptIII^TM reverse transcriptase (Invitrogen/Life Technologies), according to manufacturer’s protocol.

Primers for the qRT-PCR assays (genes nodC, exoA, and exoX), based on R. tropici CIAT 899 genome, were designed using Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/), to obtain amplicons of 50–150 bp. A pair of primers for 16S rRNA was also obtained and applied to normalize the relative expression of the targets. To avoid unspecific alignments, the primers sequences were searched against the R. tropici CIAT 899 genome (Accession numbers NC_020059, NC_020060, NC_020061, NC_020062, for chromosome, pRtrCIAT899a, pRtrCIAT899b and pRtrCIAT899c, respectively).

qRT-PCR reactions were performed as described before [29]. The fold changes of three biological samples with three technical replicates of each condition were obtained.

Nodulation assays

For the evaluation of the symbiotic phenotypes, wild-type and nrcR mutant strains of R. tropici CIAT 899 were grown in YM medium till the concentration of 10⁹ cells mL^-1. Surface-sterilized seeds of common bean (P. vulgaris) and leucaena (L. leucocephala.) were pre-germinated for 2 days at 28°C and placed on sterilized pouch bags or Leonard jars containing Fåhraeus N-free solution [34]. Germinated seeds were then inoculated with 1 mL of bacterial culture or with 1 mL of a mix (1/1, v/v) of two bacterial cultures for competitiveness assays. Growth conditions were 16 h at 26°C in the light and 8 h and 18°C in the dark, with 70% of humidity.

Nodulation parameters were evaluated after 15 days for early nodulation test, and after 30 or 50 days in late nodulation assays for common bean or leucaena, respectively. In all cases, shoots were dried at 70°C for 48 h and weighed.

To evaluate nodule occupancy in the competitiveness assays, 200 nodules from eight plants (25 nodules per plant), were analyzed. Nodules were surface-sterilized [34] and placed on TY plates to confirm lack of surface contamination. These nodules were smashed, streaked in TY with actidione (100 μg/mL), and grown at 28°C for 2–3 days. Isolated colonies were independently picked on TY and TY supplemented with spectinomicyn to discrimine the wild-type and nrcR::Ω mutant strain (Spc^R).

Isolation of the nrcR::Ω, nrcR::Ω (pMUS1333) or nrcR::Ω (pMUS1353) strains from nodules was performed growing nodule extracts on TY plates supplemented with suitable antibiotics.

Nodulation experiments were performed three times.

Results

Gene characterization

The sequence of the R. tropici CIAT 899 genome revealed the presence of an undescribed transcriptional regulator member of the ArsR family (locus tag RTCIAT899_PC05430) located in the megaplasmid pRtrCIAT899c (2.08 Mb) [31]. Protein blast of this transcriptional regulator showed 46% identity and 65% of positive residues with the peptide sequence of the chromosomal R. tropici CIAT 899 NolR protein (locus tag RTCIAT899_CH13035). Interestingly, the highest homology between the NrcR and NolR proteins was found within the DNA-binding domain (64% of identity and 72% of positive residues), indicating that the proteins share some regulatory DNA targets (S2 Fig). Besides, NrcR also showed high identities with several undescribed ArsR-type proteins (69%–93%) of different soil bacteria and with various rhizobia NolR proteins (41%–48%) (S1 Table). Lastly, the Phyre2 web portal for protein modeling and functioning revealed that this new protein featured the highest values of confidence (99.9) and identity with the crystal structure of S. fredii NolR protein.

Taking into consideration these data, we decided to name this gene nrcR, meaning “nolR-like plasmid c Regulator.” The nrcR gene was mutated by insertion of the Ω interposon and complemented in cis and in trans with the whole nrcR gene copy (See Materials and Methods).

Studies of the swimming and surface motilities, biofilm formation, EPS production and LPS profiles in the nrcR mutant

Phenotypes related to the symbiotic process, such as swimming and on surface motility, biofilm formation, EPS production and LPS profiles, were evaluated in the wild-type and in the nrcR mutant strains in the presence or in the absence of apigenin and salt.

No changes were observed in swimming motility, biofilm formation or LPS profile between the wild-type and the nrcR mutant strains (data not shown).

Surface motility was assayed on TY (rich) and B^- (minimal) media. Only in the case of the rich-medium assays (TY), were significant differences in surface motility found (Fig 1). The nrcR::Ω mutant was about 40% more mobile than the parental strain in TY medium and TY supplemented with apigenin (Fig 1A and 1B). Moreover, in the case of the mutant strain, increased mucosity was observed when bacteria were drop-inoculated on the surface of TY + 0.4% agar medium supplemented or not with apigenin (S3 Fig). The presence of salt resulted in drastic reduction of this motility in all assayed strains. Nevertheless, while the wild-type strain was practically immobile under saline condition, the nrcR mutant was mobile (Fig 1C). As expected, these increments in motility were lost in the complemented strain nrcR::Ω (pMUS1333) in all conditions assayed.

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Fig 1. Surface motility phenotype of R. tropici CIAT 899 and the nrcR::Ω mutant strain.

Quantified motile ring diameters of wild-type strain (black continuous line), nrcR::Ω mutant strain (dark gray discontinuous line) and the nrcR::Ω (pMUS1333) complemented strain (light gray continuous line). Values represent the averages of three different experiments per strain. The nrcR::Ω and nrcR::Ω (pMUS1333) strains parameters were individually compared with their correspondent CIAT 899 strain in each condition by using the Mann-Whitney non-parametric test. Values tagged by asterisks (*) are significantly different (α = 5%). (A) TY medium. (B) TY medium supplemented with 3.7 μM of apigenin. (C) TY medium supplemented with 300 mM of NaCl.

https://doi.org/10.1371/journal.pone.0154029.g001

EPS production was evaluated by the anthrone method. The nrcR::Ω strain produced a significantly higher quantity of EPS than with CIAT 899 strain in all conditions assayed (Fig 2). This phenotype was reversed in the complemented nrcR::Ω (pMUS1333) strain. Moreover, in the presence of apigenin, the EPS production seemed to slightly decrease in both wild-type and nrcR mutant strains (in comparison to the EPS production in YM or YM supplemented with salt). In contrast, qRT-PCR experiments were performed to measure the expression of the exoA gene, involved in biosynthesis of EPS, and the exoX gene, an inhibitor of EPS production, in the mutant and in the wild-type strains. No significant differences in the relative expressions of exoA and exoX were detected in all assay conditions (data not shown). Finally, we evaluated the EPS composition by monosaccharide analysis and nuclear magnetic resonance (NMR). The sugar composition and 1H-NMR spectra obtained were almost identical in both CIAT 899 and nrcR::Ω strains (data not shown). Therefore, results suggest that the NrcR transcriptional regulator inhibits, directly or indirectly, the production of EPS, but with no effect on its qualitative composition.

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Fig 2. EPS production of R. tropici CIAT 899 (black bars), the nrcR::Ω (dark gray bars) and the nrcR::Ω (pMUS1333) (light gray bars) strains.

Values were relative to CIAT 899 under control condition. Each experiment was performed three times. Both nrcR::Ω and nrcR::Ω (pMUS1333) strain parameters were individually compared with the parental strain without induction by using the Mann-Whitney non-parametric test. Values tagged by asterisks (*) are significantly different at the level of α = 5%.

https://doi.org/10.1371/journal.pone.0154029.g002

NF-production patterns and nod-gene expression of the nrcR mutant

In order to determine if the nrcR mutation affects the production and decoration of these molecules, the NF profiles of bacteria grown in B^- supplemented or not with apigenin or salt were determined (Tables 2–4) (see Materials and Methods). Under control conditions, both wild-type and nrcR mutant strains synthesized 11 and 15 different NFs, respectively. From these NFs, eight were produced by both strains, three were exclusively produced by the parental strain and seven only by the mutant strain (Table 2). Furthermore, in the nrcR::Ω strain, four NFs presenting five subunits of GlcNAc (V) were sulphated (S), and two of them were also N-methylated (NMe) [V (C_18:1, S), V (C_18:0, S), V (C_18:1, NMe, S) and V (C_18:0, NMe, S)]. In contrast, the CIAT 899 strain synthesized only one NF sulphated and N-methylated [V (C_18:1, NMe, S].

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Table 2. Nod Factor structures biosynthesized under control conditions (B^- medium) by the wild-type strain CIAT 899 and the nrcR:: Ω mutant.

https://doi.org/10.1371/journal.pone.0154029.t002

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Table 3. Nod Factor structures biosynthesized in the presence of apigenin (3.7 μM) by the wild-type strain CIAT 899 and the nrcR:: Ω mutant.

https://doi.org/10.1371/journal.pone.0154029.t003

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Table 4. Nod Factor structures biosynthesized in the presence of NaCl 300 mM by the wild-type strain CIAT 899 and the nrcR:: Ω mutant.

https://doi.org/10.1371/journal.pone.0154029.t004

On the other hand, in the presence of apigenin, both strains synthesized 29 NFs (Table 3), 23 of which were common to both strains, and 6 were exclusive to each strain. Interestingly, only the wild-type strain synthesized NF deacetylated at the glucosamine residue [V (C_18:1, dNAc) and V (C_18:1, NMe, dNAc)], while the nrcR::Ω strain produced NFs with five residues of N-acetyl glucosamine and fatty acid of 20 atoms of carbon [V (C_20:1), V (C_20:1, NMe) and V (C_20:1, NMe, S)], which were not synthesized by the parental strain.

Finally, in the presence of salt, the nrcR mutant strain synthesized 47 NFs versus 36 produced by the parental strain (Table 4). The CIAT 899 strain produced only 3 NFs with a fatty acid comprising 20 atoms of carbon [V (C_20:0, NMe), V (C_20:1, NMe), and V (C_20:0, NMe, S)], while the nrcR::Ω strain synthesized 7 NFs with fatty acids of 20 atoms of carbon, including a backbone with 3, 4, and 5 residues of N-acetylglucosamine (Table 4).

In summary, the NFs produced by the nrcR mutant strain presented more sulphated substitutions in the reducing N-acetylglucosamine residue than those produced by the wild-type strain. In fact, the parental strain produced, in salt conditions, only a sulphated NF, V (C_18:1, S), which was not produced by the mutant nrcR::Ω strain (Table 4). Moreover, in relation to the fatty acids of 20 atoms of carbon, the wild-type strain synthesized only 4 of these NFs, one in the presence of apigenin and 3 in salt conditions, while the mutant strain synthesized 15 NFs harbouring C₂₀ fatty acid, 4 in the presence of apigenin and 12 in the presence of salt (Tables 3 and 4). Therefore, the study of NF-production patterns indicated that mutation in the nrcR gene results in differences in number and decoration of the NFs, especially under salinity stress.

To verify the potential role of the nrcR gene in NFs synthesis, the relative expression of nodC gene was evaluated. This gene is highly transcribed in the presence both of apigenin and of salt in R. tropici CIAT 899. In fact, this gene is part of the main set of genes responsible for the synthesis of NFs, the nodABCSUIJHPQ operon. qRT-PCR experiments showed that the nodC relative expression was significantly lower in the presence both of apigenin and of salt in the nrcR::Ω mutant strain in comparison with the expression of this gene in the wild-type strain (Fig 3). In the mutant strain, the expression of the nodC gene decreased about 35-fold to 9-fold in the presence of salt and about 40-fold to 9.5-fold in the presence of apigenin.

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Fig 3. qRT-PCR analysis of the expression of nodC gene from R. tropici CIAT 899 and the nrcR::Ω strain grown in the absence or presence of apigenin (3.7 μM) or NaCl (300 mM).

Expression data were individually compared with the expression without inducing molecules of the wild-type strain by using the Mann-Whitney non-parametrical test. The asterisks (*) indicate a significant different at the level α = 5%. Black bars represent wild-type strain and dark gray bars represent the nrcR::Ω mutant strain.

https://doi.org/10.1371/journal.pone.0154029.g003

Symbiotic performance

The nodulation phenotype in common bean was firstly evaluated in plants grown in pouch bags for 15 days. Although plants inoculated with CIAT 899 developed nodules 15 days after inoculation, none were observed on plants inoculated with the nrcR mutant strain (Fig 4). Furthermore, symbiotic performance was also evaluated in Leonard jars using common bean and leucaena as host plants. At 30 days after inoculation in common bean, the nrcR mutant significantly decreased nodule number (by 36%) in comparison to the parental strain. Furthermore, the decrease in nodulation was reflected in lower shoot dry weight (25% less) of plants inoculated with the mutant strain (Table 5).

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Fig 4. Early nodulation phenotype in common bean inoculated with CIAT 899 and nrcR::Ω strains assayed in pouch bags.

1 mL of a culture containg 10⁹ cells mL^-1 was used as inoculum. Experiment performed under controlled conditions of growth chamber and plants harvested at 15 days after inoculation. (A) wild-type strain. (B) Uninoculated control. (C) nrcR::Ω strain. (D) Roots inoculated with the CIAT 899 strain. (E) Roots inoculated with the nrcR::Ω strain.

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Table 5. Plant responses to inoculation of common bean and leucaena with R. tropici CIAT 899 and derivative strains.

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When leucaena was inoculated with the nrcR mutant, significant differences in nodulation were also observed at 50 days, i.e. decreases of 41% in nodule number and of 20% in shoot dry weight in comparison to the wild-type strain. The in trans complementation [nrcR::Ω (pMUS1333)] of the nrcR mutant did not restore the nodulation phenotypes (Table 5). In fact, the rhizobia isolated from nodules induced by this strain in bean and leucaena plants were Spc^r Gm^s, indicating loss of the pMUS1333 plasmid. For this reason, an in cis nrcR-complemented strain [nrcR::Ω (pMUS1353)] was constructed. In this case, the complementation restored symbiotic characteristics in both plants (Table 5).

Moreover, the competitiveness assays showed that the mutant strain was less competitive for nodulation on common bean plants than the wild-type strain (Fig 5). Indeed, after 45 days of inoculation, the nrcR mutant occupied only 10% of the nodules.

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Fig 5. Competitiveness assay of CIAT 899 and nrcR::Ω strains in common bean.

0,5 mL of each bacterial culture containing 10⁹ cells mL^-1 was used as inoculum. Percentages of nodule occupancy were evaluated for both strains in common bean plants after 45 days of inoculation. Values are the averages of three experiments. The nrcR::Ω strain values were individually compared with their correspondent wild-type strain CIAT 899 by using the Mann-Whitney non-parametric test. Value tagged by asterisk (*) is significantly different at the level α = 5%.

https://doi.org/10.1371/journal.pone.0154029.g005

Discussion

The participation of NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, is required for nod-gene expression in rhizobia, which represents the beginning of the nodulation process. The absence of the transcriptional regulator repressor NolR provokes changes in NF decoration as well as decreases in nodulation efficiency in other rhizobial species and strains such as S. fredii and S. meliloti, indicating that a fine-tuning of nod-gene expression is required for optimal nodulation of the plant host [13, 17]. A structural and biochemical analysis of the NolR protein revealed that a DNA-protein interaction domain is essential to the full understanding of its transcriptional repression role in several target genes [18]. In this study, we investigated a new potential symbiotic regulator belonging to the ArsR family of proteins located in the non-symbiotic plasmid pRtrCIAT899c of R. tropici CIAT 899. Here, we report that this transcriptional regulator, named NrcR, shares homology in the sequence, especially in the DNA-binding domain, with NolR. We have demonstrated that the mutation of the nrcR gene affects several steps of the symbiosis of CIAT 899 with host legumes, revealing a novel level of regulation.

We found that the NrcR transcriptional regulator inhibits surface motility, as the mutant strain consistently showed higher mobility than the parental strain (Fig 1). Nowadays, five types of motility are ascribed to bacteria [48] and it remains to be discovered which one is altered by the absence of NrcR. For this reason, we have generically defined this phenotype as a surface motility type.

The EPS produced by rhizobia has different structural functions, such as a protective barrier against environmental stresses [49, 50]. In CIAT 899, the EPS is an octasaccharide formed by D-glucose, D-galactose, pyruvic and acetic acids, with a ratio of 3:1 between glucose and galactose [51]. Interestingly, the nrcR::Ω strain produced a significantly higher amount of EPS than the parental CIAT 899 (Fig 2), but with no difference in the proportion of sugar detected in the NMR analysis. In addition to the protective role of the CIAT 899 EPS, it is worth mentioning that an increase in the EPS production by S. fredii HH103 promotes surface motility [52]. Thus, it is possible that the over-production of EPS by the nrcR mutant could contribute to its increased motility. Remarkably, nrcR gene is located in the CIAT 899 megaplasmid that also carries the exo genes, which are implied in the biosynthesis of EPS [53]. Thus, the relative position of all these genes is another clue that could be relating EPS production with the NrcR protein. However, the higher production of EPS by the nrcR mutant was not related to an increased expression of the exoA gene (involved in the biosynthesis of EPS), nor to a repression of the exoX gene (a negative regulator of the EPS biosynthesis in rhizobia). Therefore, it is possible that the nrcR mutant shows higher capacity of exportation of EPS than the parental strain.

Moreover, a mutation in the nrcR gene provoked a clear change in the number and decoration of the NFs synthesized, especially under saline conditions (Tables 2–4). It has been established that CIAT 899 is a host-promiscuous strain that shows high tolerance of environmental stresses [19–22]. Interestingly, the synthesis of a large variety of NFs from R. tropici CIAT 899 has been related to both features [23–30]. In contrast, qRT-PCR experiments showed decreases in the relative expression of the nodC gene in the nrcR mutant in comparison to that of the parental strain in the presence both of apigenin and of salt (Fig 3). Nevertheless, nodC expression in the mutant strain supplemented with apigenin or salt were 9.5-fold and 9-fold respectively higher than in the parental CIAT 899 strain grown without inducers, confirming that the nrcR mutant is able to synthesize NFs. Consequently, the nrcR::Ω strain could decrease the quantity of NFs but increasing their diversity in comparison to those produced by the parental strain.

The role of the NrcR transcriptional regulator in the symbiosis was also confirmed in nodulation tests with common bean and leucaena. The main effect was observed in the early steps of nodulation, with absence of nodules at 15 days of inoculation with the mutant strain (Fig 4). In addition, when co-inoculated with the wild-type strain, nodule occupancy by the mutant was far lower, possibly related to a lower root-colonization capacity (Fig 5). Competitiveness is critical under field conditions to the success or failure of introduced inoculant strains [54–59]. Furthermore, certain symbiosis-related phenotypes, such as motility or EPS production, can also affect bacterial competitiveness [60, 61]. It has been described that the mutation on the exoA gene, decreasing EPS production, increases the competitiveness of S. fredii HH103 for soybean nodulation [52]. Therefore, we may suppose that decreased competitiveness of the nrcR mutant strain is related to the EPS over-production caused by the mutation. Lastly, the nrcR in trans complementation [nrcR::Ω (pMUS1333)] did not restore the capacity of nodulating bean and leucaena, because the strain lost the pMUS1333 plasmid, as demonstrated after the re-isolation of bacteria from nodules. However, the in cis complementation of the mutant strain [nrcR::Ω (pMUS1353)] restored the symbiotic phenotype (Table 5).

In summary, NolR is a global regulatory protein that responds to environmental factors and regulates the expression of intracellular metabolic genes [62, 63]. We hypothesize that the high tolerance of R. tropici CIAT 899 to environmental stresses is coordinated by a complex mechanism involving a broad range of transcriptional regulators. Evolution would have selected some of these regulators, such as the NrcR protein, to regulate nodulation and other features such as EPS production and motility even when nod-gene inducing molecules are limited. Therefore, the NrcR transcriptional regulator may represent an important strategy of CIAT 899 to succeed as a symbiont in stressing tropical conditions. In this context, the higher EPS production and motility detected in the nrcR mutant may represent an additional protective mechanism for tolerating abiotic stresses. Moreover, symbiotic behavior of the mutant could also represent an indirect effect of these protective mechanisms, affecting probably the early steps of nodulation. The activation of this transcriptional regulator seems to be dependent on flavonoids or salt. Therefore, our results suggest that NrcR is a significant component in the regulatory network present in R. tropici CIAT 899, which is necessary for orchestration of the symbiotic machinery and other important processes such as tolerance of abiotic stresses.

Supporting Information

S1 Fig.

(A) Gene neighborhood of nrcR gene in the pRtrCIAT899c of R. tropici CIAT 899 genome. (B) HindIII endonuclease point and primers nolR-like-F and nolR-like-R location (C) Mutant interposon insertion.

https://doi.org/10.1371/journal.pone.0154029.s001

(TIF)

S2 Fig. Alignment of NolR, ArsR and NrcR amino acid sequences using the ClustalW online platform and manipulated with Boxshade at EMBnet.

Black and gray boxes indicate identical and similar amino acids, respectively. (A) Complete sequence alignment of NolR of S. fredii HH103, and NolR, ArsR and NrcR proteins of R. tropici CIAT 899. (B) Putative DNA-binding domain of NolR of S. fredii HH103, and NolR and NrcR of R. tropici CIAT 899.

https://doi.org/10.1371/journal.pone.0154029.s002

(TIF)

S3 Fig. Mucous halo observed in surface motility of R. tropici CIAT 899 assays on TY supplemented with 0.4% agar.

(A) CIAT 899. (B) nrcR::Ω mutant.

https://doi.org/10.1371/journal.pone.0154029.s003

(TIF)

S1 Table.

https://doi.org/10.1371/journal.pone.0154029.s004

(PDF)

Acknowledgments

We thank the Servicio de Espectrometría de Masas, Servicio de Resonancia Magnética Nuclear and Servicio de Biología of the Centro de Investigación Tecnología e Innovacción (CITIUS) of the University of Seville for technical support.

Author Contributions

Conceived and designed the experiments: MM MH FJO. Performed the experiments: PdC AAPR-S RV-F DFG FPM RAB AG-S. Analyzed the data: PdC AAPR-S RV-F DFG FPM MM MH RAB AG-S FJO. Contributed reagents/materials/analysis tools: MM MH FJO. Wrote the paper: PdC FPM AG-S MM MH FJO.

References

1. Hungria M, Johnston AWB, Phillips DA. Effects of flavonoids released naturally from bean (Phaseolus vulgaris) on nodD-regulated gene transcription in Rhizobium leguminosarum bv. phaseoli. Mol Plant-Microbe Interact. 1992;5: 199–203. pmid:1421508
- View Article
- PubMed/NCBI
- Google Scholar
2. Hungria M, Phillips DA. Effects of a seed color mutation on rhizobial nod-gene-inducing flavonoids and nodulation in common bean. Mol Plant Microbe Interact. 1993;6: 418–422.
- View Article
- Google Scholar
3. Denarié J, Debellé F, Promé JC. Rhizobium lipo-chitinoligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Ann Rev Biochem. 1996;65: 503–535. pmid:8811188
- View Article
- PubMed/NCBI
- Google Scholar
4. Schlaman HRM, Phillips DA, Kondorosi E. Genetic organization and transcriptional regulation of rhizobial nodulation genes. In Spaink HP, Kondorosi A, Hooykaas PJJ, editors. The Rhizobiaceae. Kluwer Academic Publishers; 1998. pp. 361–386.
5. Geurts R, Bisseling T. Rhizobium Nod factor perception and signalling. Plant Cell. Supplement 2002; S239–S249.
- View Article
- Google Scholar
6. Brencic A, Winans SC. Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria. Microbiol Mol Biol Rev. 2005; 69: 155–194. pmid:15755957
- View Article
- PubMed/NCBI
- Google Scholar
7. Ormeño-Orrillo E, Hungria M, Martínez-Romero E. Dinitrogen-fixing prokaryotes. In Rosenberg E, DeLong EF, Stackebrandt E, Lory S, Thompson F, editors. The Prokaryotes: prokaryotic physiology and biochemistry. Berlin Heidelberg: Springer-Verlag; 2013. pp. 427–451.
8. Spaink HP. Root nodulation and infection factors produced by rhizobial bacteria. Annu Rev Microbiol. 2000;54: 257–288. pmid:11018130
- View Article
- PubMed/NCBI
- Google Scholar
9. Oldroyd GE. Speak, friend, and enter: signaling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol. 2013;11: 252–263. pmid:23493145
- View Article
- PubMed/NCBI
- Google Scholar
10. Shi W, Wu J, Rosen BP. Identification of a putative metal binding site in a new family of metalloregulatory proteins. J Biol Chem. 1994;269: 19826–19829. pmid:8051064
- View Article
- PubMed/NCBI
- Google Scholar
11. Kiss E, Mergaert P, Olàh B, Kereszt A, Staehelin C, Davies AE, et al. Conservation of nolR in the Sinorhizobium and Rhizobium genera of the Rhizobiaceae family. Mol Plant Microbe Interact. 1998;11: 1186–1195.
- View Article
- Google Scholar
12. Osman D, Cavet JS. Bacterial metal-sensing proteins exemplified by ArsR-SmtB family repressors. Nat Prod Rep. 2010;27: 668–680. pmid:20442958
- View Article
- PubMed/NCBI
- Google Scholar
13. Kondorosi E, Gyuris J, Schmidt J, John M, Duda E, Hoffmann B, et al. Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation. EMBO J. 1989;8: 1331–1340. pmid:16453883
- View Article
- PubMed/NCBI
- Google Scholar
14. Kondorosi E, Pierre M, Cren M, Haumann U, Buire M, Hoffmann B, et al. Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti. J Mol Biol. 1991;222: 885–896. pmid:1840615
- View Article
- PubMed/NCBI
- Google Scholar
15. Cren M, Kondorosi A, Kondorosi E. NolR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis. Mol Microbiol. 1995;15: 733–747. pmid:7783644
- View Article
- PubMed/NCBI
- Google Scholar
16. Lee SG, Krishnan HB, Jez JM. Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR. Proc Natl Acad Sci USA. 2014;111: 6509–6514. pmid:24733893
- View Article
- PubMed/NCBI
- Google Scholar
17. Vinardell JM, Ollero FJ, Hidalgo A, López-Baena FJ, Medina C, Ivanov-Vangelov K, et al. NolR regulates diverse symbiotic signals of Sinorhizobium fredii HH103. Mol Plant Microbe Interact. 2004;17: 676–685. http://dx.doi.org/10.1094/MPMI.2004.17.6.676. pmid:15195950
- View Article
- PubMed/NCBI
- Google Scholar
18. Sugawara M, Sadowsky MJ. Enhanced nodulation and nodule development by nolR mutants of Sinorhizobium medicae on specific Medicago host genotypes. Mol Plant Microbe Interact. 2014;27: 328–335. pmid:24283939
- View Article
- PubMed/NCBI
- Google Scholar
19. Martínez-Romero E, Segovia L, Mercante FM, Franco AA, Graham P, Pardo MA. Rhizobium tropici, a novel species nodulating Phaseolus vulgaris L. beans and Leucaena sp. trees. Int J Syst Bacteriol. 1991;41: 417–426. pmid:1715738
- View Article
- PubMed/NCBI
- Google Scholar
20. Hernández-Lucas I, Segovia L, Martínez-Romero E, Pueppke SG. Phylogenetic relationships and host range of Rhizobium spp. that nodulate Phaseolus vulgaris L. Appl Environ Microbiol. 1995;61: 2775–2779. pmid:7618891
- View Article
- PubMed/NCBI
- Google Scholar
21. Pinto FGS, Hungria M, Mercante FM. Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N₂ with common bean (Phaseolus vulgaris L.). Soil Biol Biochem. 2007;39: 1851–1864.
- View Article
- Google Scholar
22. Hungria M, Andrade DS, Chueire LMO, Probanza A, Gutiérrez-Mañero FJ, Megías M. Isolation and characterization of new efficient and competitive bean (Phaseolus vulgaris L.) rhizobia from Brazil. Soil Biol Biochem. 2000; 21:1515–1528.
- View Article
- Google Scholar
23. Poupot R, Martínez-Romero E, Promé JC. Nodulation factors from Rhizobium tropici are sulfated or nonsulfated chitopentasaccharides containing an N-methyl-N-acylglucosaminyl terminus. Biochemistry. 1993;32: 10430–10435. pmid:8399187
- View Article
- PubMed/NCBI
- Google Scholar
24. Marks BB, Megías M, Ollero FJ, Nogueira MA, Araujo RS, Hungria M. Maize growth promotion by inoculation with Azospirillum brasilense and metabolites of Rhizobium tropici enriched on lipo-chitooligosaccharides (LCOs). AMB Express. 2015;5: 71. pmid:26567001
- View Article
- PubMed/NCBI
- Google Scholar
25. Morón B, Soria-Díaz ME, Ault J, Verroios G, Noreen S, Rodríguez-Navarro DN, et al. Low pH changes the profile of nodulation factors produced by Rhizobium tropici CIAT 899. Chem Biol. 2005;12: 1029–1040. pmid:16183027
- View Article
- PubMed/NCBI
- Google Scholar
26. Estévez J, Soria-Díaz ME, de Córdoba FF, Morón B, Manyani H, Gil A, et al. Different and new Nod factors produced by Rhizobium tropici CIAT 899 following Na⁺ stress. FEMS Microbiol Lett. 2009;293: 220–231. http://dx.doi.org/10.1111/j.1574-6968.2009.01540.x. pmid:19260963
- View Article
- PubMed/NCBI
- Google Scholar
27. Guasch-Vidal B, Estévez J, Dardanelli MS, Soria-Díaz ME, de Córdoba FF, Balog CIA, et al. High NaCl concentrations induce the nod genes of Rhizobium tropici CIAT899 in the absence of flavonoid inducers. Mol Plant Microbe Interact. 2013;26: 451–460. pmid:23216086
- View Article
- PubMed/NCBI
- Google Scholar
28. van Hameren B, Hayashi S, Gresshoff PM, Ferguson BJ. Advances in the identification of novel factors required in soybean nodulation, a process critical to sustainable agriculture and food security. J Plant Biol Soil Health. 2013;1: 6.
- View Article
- Google Scholar
29. del Cerro P, Rolla-Santos AAP, Gomes DF, Marks B, Pérez-Montaño F, Rodríguez-Carvajal MA, et al. Regulatory nodD1 and nodD2 genes of Rhizobium tropici strain CIAT 899 and their roles in the early stages of molecular signaling and host-legume nodulation. BMC Genomics. 2015; 16: 251. pmid:25880529
- View Article
- PubMed/NCBI
- Google Scholar
30. del Cerro P, Rolla-Santos AAP, Gomes DF, Marks B, Espuny MR, Rodríguez-Carvajal MA, et al. Opening the “black box” of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899. BMC Genomics. 2015;16: 864. pmid:26502986
- View Article
- PubMed/NCBI
- Google Scholar
31. Ormeño-Orrillo E, Menna P, Gonzaga LA, Ollero FJ, Nicolas MF, Rodrigues EP, et al. Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.). BMC Genomics. 2012;13: 735. pmid:23270491
- View Article
- PubMed/NCBI
- Google Scholar
32. Beringer J E. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974;84: 188–198. pmid:4612098
- View Article
- PubMed/NCBI
- Google Scholar
33. Spaink HP, Aarts A, Stacey G, Bloemberg GV, Lugtenberg BJJ, Kennedy EP. Detection and separation of Rhizobium and Bradyrhizobium Nod metabolites using thin-layer chromatography. Mol Plant Microbe Interact. 1992;5: 72–80. pmid:1600238
- View Article
- PubMed/NCBI
- Google Scholar
34. Vincent JM. The modified Fåhraeus slide technique. In Vincent JM, editor. A manual for the practical study of root nodule bacteria. Oxford, UK. Blackwell Scientific Publications; 1970. pp. 144–145.
35. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor NY USA. 1989.
36. Lamrabet Y, Bellogín RA, Cubo T, Espuny MR, Gil-Serrano A, Krishnan HB, et al. Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans. Mol Plant Microbe Interact. 1999;12: 207–217. pmid:10065558
- View Article
- PubMed/NCBI
- Google Scholar
37. Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A. 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. pmid:8045426
- View Article
- PubMed/NCBI
- Google Scholar
38. Simon R. High frequency mobilization of gram-negative bacterial replicons by the in vivo constructed Tn5-Mob transposon. Mol Gen Genet. 1984;196: 413–420. pmid:6094969
- View Article
- PubMed/NCBI
- Google Scholar
39. Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA. 1979;76: 1648–1652. pmid:377280
- View Article
- PubMed/NCBI
- Google Scholar
40. López-Baena FJ, Vinardell JM, Pérez-Montaño F, Crespo-Rivas JC, Bellogín RA, Espuny MR, et al. Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103. Microbiology-SGM. 2008;154: 1825–1836.
- View Article
- Google Scholar
41. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene. 1995;166: 175–176. pmid:8529885
- View Article
- PubMed/NCBI
- Google Scholar
42. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. protocols. 2015;10: 845–858. pmid:25950237
- View Article
- PubMed/NCBI
- Google Scholar
43. Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. NCBI BLAST: A better web interface. Nucleic Acids Res. 2008;36: W5–9. pmid:18440982
- View Article
- PubMed/NCBI
- Google Scholar
44. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. ClustalW and ClustalX version 2. Bioinformatics. 2007;23: 2947–2948. pmid:17846036
- View Article
- PubMed/NCBI
- Google Scholar
45. Pérez-Montaño F, Jiménez-Guerrero I, Del Cerro P, Baena-Ropero I, López-Baena FJ, Ollero FJ, et al. The symbiotic biofilm of Sinorhizobium fredii SMH12, necessary for successful colonization and symbiosis of Glycine max cv Osumi, is regulated by Quorum Sensing systems and inducing flavonoids via NodD1. PLoS One. 2014;9: e105901. pmid:25166872
- View Article
- PubMed/NCBI
- Google Scholar
46. Tomlinson AD, Ramey-Hartung B, Day TV, Merrit PM, Fuqua C. Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility. Microbiology-SGM. 2010; 156: 2670–2681.
- View Article
- Google Scholar
47. Gomes DF, Batista JSS, Rolla AAP, Silva LPS, Bloch C, Galli-Terasawa LV, et al. Proteomic analysis of free-living Bradyrhizobium diazoefficiens: highlighting potential determinants of a successful symbiosis. BMC Genomics. 2014; 15: 643. pmid:25086822
- View Article
- PubMed/NCBI
- Google Scholar
48. Kearns DB. A field guide to bacterial swarming motility. Nat Rev Microbiol. 2010;8: 634–644. pmid:20694026
- View Article
- PubMed/NCBI
- Google Scholar
49. Downie JA. The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol Rev. 2010;34: 150–170. pmid:20070373
- View Article
- PubMed/NCBI
- Google Scholar
50. Janczarek M. Environmental signals and regulatory pathways that influence exopolysaccharide production in rhizobia. Int J Mol Sci. 2011;12: 7898–7933. pmid:22174640
- View Article
- PubMed/NCBI
- Google Scholar
51. Gil-Serrano A, Sánchez del Junco A, Tejero-Mateo P, Megías M, Caviedes MA. Structure of the extracellular polysaccharide secreted by Rhizobium leguminosarum var. phaseoli CIAT 899. Carbohydr Res. 1990;204: 103–107. pmid:2279241
- View Article
- PubMed/NCBI
- Google Scholar
52. Rodríguez-Navarro DN, Rodríguez-Carvajal MA, Acosta-Jurado S, Soto MJ, Margaret I, Crespo-Rivas JC, et al. Structure and Biological Roles of Sinorhizobium fredii HH103 Exopolysaccharide. PLoS One. 2014;9: e115391. pmid:25521500
- View Article
- PubMed/NCBI
- Google Scholar
53. Geniaux E, Flores M, Palacios R, Martinez E. Presence of Megaplasmids in Rhizobium tropici and Further Evidence of Differences between the Two R. tropici Subtypes. Int J Syst Bacteriol. 1995;45: 392–394.
- View Article
- Google Scholar
54. Streeter JG. Failure of inoculant rhizobia to overcome the dominance of indigenous strains for nodule formation. Can J Microbiol. 1994;40: 513–522.
- View Article
- Google Scholar
55. Toro N. Nodulation competitiveness in the Rhizobium legume symbiosis. World J Microbiol Biotechnol. 1996;12: 157–162. pmid:24415163
- View Article
- PubMed/NCBI
- Google Scholar
56. Triplett EW, Sadowsky MJ. Genetics of competition for nodulation of legumes. Annu Rev. Microbiol. 1992;46: 399–428. pmid:1444262
- View Article
- PubMed/NCBI
- Google Scholar
57. Vargas MAT, Mendes IC, Hungria M. The response of field grown Phaseolus vulgaris to Rhizobium inoculation and nitrogen fertilization in two Cerrado soils. Biol Fertil Soils. 2000;32: 228–233.
- View Article
- Google Scholar
58. Hungria M, Campo RJ, Chueire LMO, Grange L, Megías M. Symbiotic effectiveness of fast-growing rhizobial strains isolated from soybean nodules in Brazil. Biol Fertil Soils. 2001;33: 387–394.
- View Article
- Google Scholar
59. Mendes IC, Vargas MAT, Hungria M. Establishment of Bradyrhizobium japonicum and B. elkanii in a Brazilian Cerrados oxisol. Biol Fertil Soils. 2004;40: 28–35.
- View Article
- Google Scholar
60. Liu R, Tran VM, Schmidt EL. Nodulating competitiveness of a nonmotile Tn7 mutant of Bradyrhizobium japonicum in nonsterile soil. Appl Environ Microbiol. 1989;55: 1895–1900. pmid:16347986
- View Article
- PubMed/NCBI
- Google Scholar
61. McDermoti TR, Graham PH. Competitive ability and efficiency in nodule formation of strains of Bradyrhizobium japonicum. Appl Environ Microbiol. 1990;56: 3035–3039. pmid:16348311
- View Article
- PubMed/NCBI
- Google Scholar
62. Chen H, Higgins J, Kondorosi E, Kondorosi A, Djordjevic MA, Weinman JJ, et al. Identification of nolR-regulated proteins in Sinorhizobium meliloti using proteome analysis. Electrophoresis 2000;21: 3823–3832. pmid:11271500
- View Article
- PubMed/NCBI
- Google Scholar
63. Chen H, Gao K, Kondorosi E, Kondorosi A, Rolfe BG. Functional genomic analysis of global regulator NolR in Sinorhizobium meliloti. Mol Plant Microbe Interact. 2005;18: 1340–1352. pmid:16478054
- View Article
- PubMed/NCBI
- Google Scholar
64. Spaink HP. Rhizobial lipo-oligosaccharides: Answers and questions. Plant Mol Biol. 1992;20: 977–986. pmid:1463833
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Hungria M, Johnston AWB, Phillips DA. Effects of flavonoids released naturally from bean (Phaseolus vulgaris) on nodD-regulated gene transcription in Rhizobium leguminosarum bv. phaseoli. Mol Plant-Microbe Interact. 1992;5: 199–203. pmid:1421508
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Hungria M, Phillips DA. Effects of a seed color mutation on rhizobial nod-gene-inducing flavonoids and nodulation in common bean. Mol Plant Microbe Interact. 1993;6: 418–422.
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref3] 3. Denarié J, Debellé F, Promé JC. Rhizobium lipo-chitinoligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Ann Rev Biochem. 1996;65: 503–535. pmid:8811188
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Schlaman HRM, Phillips DA, Kondorosi E. Genetic organization and transcriptional regulation of rhizobial nodulation genes. In Spaink HP, Kondorosi A, Hooykaas PJJ, editors. The Rhizobiaceae. Kluwer Academic Publishers; 1998. pp. 361–386.

[ref5] 5. Geurts R, Bisseling T. Rhizobium Nod factor perception and signalling. Plant Cell. Supplement 2002; S239–S249.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref6] 6. Brencic A, Winans SC. Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria. Microbiol Mol Biol Rev. 2005; 69: 155–194. pmid:15755957
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref7] 7. Ormeño-Orrillo E, Hungria M, Martínez-Romero E. Dinitrogen-fixing prokaryotes. In Rosenberg E, DeLong EF, Stackebrandt E, Lory S, Thompson F, editors. The Prokaryotes: prokaryotic physiology and biochemistry. Berlin Heidelberg: Springer-Verlag; 2013. pp. 427–451.

[ref8] 8. Spaink HP. Root nodulation and infection factors produced by rhizobial bacteria. Annu Rev Microbiol. 2000;54: 257–288. pmid:11018130
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref9] 9. Oldroyd GE. Speak, friend, and enter: signaling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol. 2013;11: 252–263. pmid:23493145
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref10] 10. Shi W, Wu J, Rosen BP. Identification of a putative metal binding site in a new family of metalloregulatory proteins. J Biol Chem. 1994;269: 19826–19829. pmid:8051064
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref11] 11. Kiss E, Mergaert P, Olàh B, Kereszt A, Staehelin C, Davies AE, et al. Conservation of nolR in the Sinorhizobium and Rhizobium genera of the Rhizobiaceae family. Mol Plant Microbe Interact. 1998;11: 1186–1195.
View Article
Google Scholar

[34] View Article

[35] Google Scholar

[ref12] 12. Osman D, Cavet JS. Bacterial metal-sensing proteins exemplified by ArsR-SmtB family repressors. Nat Prod Rep. 2010;27: 668–680. pmid:20442958
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref13] 13. Kondorosi E, Gyuris J, Schmidt J, John M, Duda E, Hoffmann B, et al. Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation. EMBO J. 1989;8: 1331–1340. pmid:16453883
View Article
PubMed/NCBI
Google Scholar

[41] View Article

[42] PubMed/NCBI

[43] Google Scholar

[ref14] 14. Kondorosi E, Pierre M, Cren M, Haumann U, Buire M, Hoffmann B, et al. Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti. J Mol Biol. 1991;222: 885–896. pmid:1840615
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref15] 15. Cren M, Kondorosi A, Kondorosi E. NolR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis. Mol Microbiol. 1995;15: 733–747. pmid:7783644
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref16] 16. Lee SG, Krishnan HB, Jez JM. Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR. Proc Natl Acad Sci USA. 2014;111: 6509–6514. pmid:24733893
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref17] 17. Vinardell JM, Ollero FJ, Hidalgo A, López-Baena FJ, Medina C, Ivanov-Vangelov K, et al. NolR regulates diverse symbiotic signals of Sinorhizobium fredii HH103. Mol Plant Microbe Interact. 2004;17: 676–685. http://dx.doi.org/10.1094/MPMI.2004.17.6.676. pmid:15195950
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref18] 18. Sugawara M, Sadowsky MJ. Enhanced nodulation and nodule development by nolR mutants of Sinorhizobium medicae on specific Medicago host genotypes. Mol Plant Microbe Interact. 2014;27: 328–335. pmid:24283939
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref19] 19. Martínez-Romero E, Segovia L, Mercante FM, Franco AA, Graham P, Pardo MA. Rhizobium tropici, a novel species nodulating Phaseolus vulgaris L. beans and Leucaena sp. trees. Int J Syst Bacteriol. 1991;41: 417–426. pmid:1715738
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref20] 20. Hernández-Lucas I, Segovia L, Martínez-Romero E, Pueppke SG. Phylogenetic relationships and host range of Rhizobium spp. that nodulate Phaseolus vulgaris L. Appl Environ Microbiol. 1995;61: 2775–2779. pmid:7618891
View Article
PubMed/NCBI
Google Scholar

[69] View Article

[70] PubMed/NCBI

[71] Google Scholar

[ref21] 21. Pinto FGS, Hungria M, Mercante FM. Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N₂ with common bean (Phaseolus vulgaris L.). Soil Biol Biochem. 2007;39: 1851–1864.
View Article
Google Scholar

[73] View Article

[74] Google Scholar

[ref22] 22. Hungria M, Andrade DS, Chueire LMO, Probanza A, Gutiérrez-Mañero FJ, Megías M. Isolation and characterization of new efficient and competitive bean (Phaseolus vulgaris L.) rhizobia from Brazil. Soil Biol Biochem. 2000; 21:1515–1528.
View Article
Google Scholar

[76] View Article

[77] Google Scholar

[ref23] 23. Poupot R, Martínez-Romero E, Promé JC. Nodulation factors from Rhizobium tropici are sulfated or nonsulfated chitopentasaccharides containing an N-methyl-N-acylglucosaminyl terminus. Biochemistry. 1993;32: 10430–10435. pmid:8399187
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref24] 24. Marks BB, Megías M, Ollero FJ, Nogueira MA, Araujo RS, Hungria M. Maize growth promotion by inoculation with Azospirillum brasilense and metabolites of Rhizobium tropici enriched on lipo-chitooligosaccharides (LCOs). AMB Express. 2015;5: 71. pmid:26567001
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref25] 25. Morón B, Soria-Díaz ME, Ault J, Verroios G, Noreen S, Rodríguez-Navarro DN, et al. Low pH changes the profile of nodulation factors produced by Rhizobium tropici CIAT 899. Chem Biol. 2005;12: 1029–1040. pmid:16183027
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref26] 26. Estévez J, Soria-Díaz ME, de Córdoba FF, Morón B, Manyani H, Gil A, et al. Different and new Nod factors produced by Rhizobium tropici CIAT 899 following Na⁺ stress. FEMS Microbiol Lett. 2009;293: 220–231. http://dx.doi.org/10.1111/j.1574-6968.2009.01540.x. pmid:19260963
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref27] 27. Guasch-Vidal B, Estévez J, Dardanelli MS, Soria-Díaz ME, de Córdoba FF, Balog CIA, et al. High NaCl concentrations induce the nod genes of Rhizobium tropici CIAT899 in the absence of flavonoid inducers. Mol Plant Microbe Interact. 2013;26: 451–460. pmid:23216086
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref28] 28. van Hameren B, Hayashi S, Gresshoff PM, Ferguson BJ. Advances in the identification of novel factors required in soybean nodulation, a process critical to sustainable agriculture and food security. J Plant Biol Soil Health. 2013;1: 6.
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref29] 29. del Cerro P, Rolla-Santos AAP, Gomes DF, Marks B, Pérez-Montaño F, Rodríguez-Carvajal MA, et al. Regulatory nodD1 and nodD2 genes of Rhizobium tropici strain CIAT 899 and their roles in the early stages of molecular signaling and host-legume nodulation. BMC Genomics. 2015; 16: 251. pmid:25880529
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref30] 30. del Cerro P, Rolla-Santos AAP, Gomes DF, Marks B, Espuny MR, Rodríguez-Carvajal MA, et al. Opening the “black box” of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899. BMC Genomics. 2015;16: 864. pmid:26502986
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref31] 31. Ormeño-Orrillo E, Menna P, Gonzaga LA, Ollero FJ, Nicolas MF, Rodrigues EP, et al. Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.). BMC Genomics. 2012;13: 735. pmid:23270491
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref32] 32. Beringer J E. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974;84: 188–198. pmid:4612098
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref33] 33. Spaink HP, Aarts A, Stacey G, Bloemberg GV, Lugtenberg BJJ, Kennedy EP. Detection and separation of Rhizobium and Bradyrhizobium Nod metabolites using thin-layer chromatography. Mol Plant Microbe Interact. 1992;5: 72–80. pmid:1600238
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref34] 34. Vincent JM. The modified Fåhraeus slide technique. In Vincent JM, editor. A manual for the practical study of root nodule bacteria. Oxford, UK. Blackwell Scientific Publications; 1970. pp. 144–145.

[ref35] 35. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor NY USA. 1989.

[ref36] 36. Lamrabet Y, Bellogín RA, Cubo T, Espuny MR, Gil-Serrano A, Krishnan HB, et al. Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans. Mol Plant Microbe Interact. 1999;12: 207–217. pmid:10065558
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref37] 37. Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A. 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. pmid:8045426
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref38] 38. Simon R. High frequency mobilization of gram-negative bacterial replicons by the in vivo constructed Tn5-Mob transposon. Mol Gen Genet. 1984;196: 413–420. pmid:6094969
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref39] 39. Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA. 1979;76: 1648–1652. pmid:377280
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref40] 40. López-Baena FJ, Vinardell JM, Pérez-Montaño F, Crespo-Rivas JC, Bellogín RA, Espuny MR, et al. Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103. Microbiology-SGM. 2008;154: 1825–1836.
View Article
Google Scholar

[140] View Article

[141] Google Scholar

[ref41] 41. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene. 1995;166: 175–176. pmid:8529885
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref42] 42. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. protocols. 2015;10: 845–858. pmid:25950237
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref43] 43. Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. NCBI BLAST: A better web interface. Nucleic Acids Res. 2008;36: W5–9. pmid:18440982
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref44] 44. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. ClustalW and ClustalX version 2. Bioinformatics. 2007;23: 2947–2948. pmid:17846036
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref45] 45. Pérez-Montaño F, Jiménez-Guerrero I, Del Cerro P, Baena-Ropero I, López-Baena FJ, Ollero FJ, et al. The symbiotic biofilm of Sinorhizobium fredii SMH12, necessary for successful colonization and symbiosis of Glycine max cv Osumi, is regulated by Quorum Sensing systems and inducing flavonoids via NodD1. PLoS One. 2014;9: e105901. pmid:25166872
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

[ref46] 46. Tomlinson AD, Ramey-Hartung B, Day TV, Merrit PM, Fuqua C. Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility. Microbiology-SGM. 2010; 156: 2670–2681.
View Article
Google Scholar

[163] View Article

[164] Google Scholar

[ref47] 47. Gomes DF, Batista JSS, Rolla AAP, Silva LPS, Bloch C, Galli-Terasawa LV, et al. Proteomic analysis of free-living Bradyrhizobium diazoefficiens: highlighting potential determinants of a successful symbiosis. BMC Genomics. 2014; 15: 643. pmid:25086822
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref48] 48. Kearns DB. A field guide to bacterial swarming motility. Nat Rev Microbiol. 2010;8: 634–644. pmid:20694026
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref49] 49. Downie JA. The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol Rev. 2010;34: 150–170. pmid:20070373
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

[ref50] 50. Janczarek M. Environmental signals and regulatory pathways that influence exopolysaccharide production in rhizobia. Int J Mol Sci. 2011;12: 7898–7933. pmid:22174640
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

[ref51] 51. Gil-Serrano A, Sánchez del Junco A, Tejero-Mateo P, Megías M, Caviedes MA. Structure of the extracellular polysaccharide secreted by Rhizobium leguminosarum var. phaseoli CIAT 899. Carbohydr Res. 1990;204: 103–107. pmid:2279241
View Article
PubMed/NCBI
Google Scholar

[182] View Article

[183] PubMed/NCBI

[184] Google Scholar

[ref52] 52. Rodríguez-Navarro DN, Rodríguez-Carvajal MA, Acosta-Jurado S, Soto MJ, Margaret I, Crespo-Rivas JC, et al. Structure and Biological Roles of Sinorhizobium fredii HH103 Exopolysaccharide. PLoS One. 2014;9: e115391. pmid:25521500
View Article
PubMed/NCBI
Google Scholar

[186] View Article

[187] PubMed/NCBI

[188] Google Scholar

[ref53] 53. Geniaux E, Flores M, Palacios R, Martinez E. Presence of Megaplasmids in Rhizobium tropici and Further Evidence of Differences between the Two R. tropici Subtypes. Int J Syst Bacteriol. 1995;45: 392–394.
View Article
Google Scholar

[190] View Article

[191] Google Scholar

[ref54] 54. Streeter JG. Failure of inoculant rhizobia to overcome the dominance of indigenous strains for nodule formation. Can J Microbiol. 1994;40: 513–522.
View Article
Google Scholar

[193] View Article

[194] Google Scholar

[ref55] 55. Toro N. Nodulation competitiveness in the Rhizobium legume symbiosis. World J Microbiol Biotechnol. 1996;12: 157–162. pmid:24415163
View Article
PubMed/NCBI
Google Scholar

[196] View Article

[197] PubMed/NCBI

[198] Google Scholar

[ref56] 56. Triplett EW, Sadowsky MJ. Genetics of competition for nodulation of legumes. Annu Rev. Microbiol. 1992;46: 399–428. pmid:1444262
View Article
PubMed/NCBI
Google Scholar

[200] View Article

[201] PubMed/NCBI

[202] Google Scholar

[ref57] 57. Vargas MAT, Mendes IC, Hungria M. The response of field grown Phaseolus vulgaris to Rhizobium inoculation and nitrogen fertilization in two Cerrado soils. Biol Fertil Soils. 2000;32: 228–233.
View Article
Google Scholar

[204] View Article

[205] Google Scholar

[ref58] 58. Hungria M, Campo RJ, Chueire LMO, Grange L, Megías M. Symbiotic effectiveness of fast-growing rhizobial strains isolated from soybean nodules in Brazil. Biol Fertil Soils. 2001;33: 387–394.
View Article
Google Scholar

[207] View Article

[208] Google Scholar

[ref59] 59. Mendes IC, Vargas MAT, Hungria M. Establishment of Bradyrhizobium japonicum and B. elkanii in a Brazilian Cerrados oxisol. Biol Fertil Soils. 2004;40: 28–35.
View Article
Google Scholar

[210] View Article

[211] Google Scholar

[ref60] 60. Liu R, Tran VM, Schmidt EL. Nodulating competitiveness of a nonmotile Tn7 mutant of Bradyrhizobium japonicum in nonsterile soil. Appl Environ Microbiol. 1989;55: 1895–1900. pmid:16347986
View Article
PubMed/NCBI
Google Scholar

[213] View Article

[214] PubMed/NCBI

[215] Google Scholar

[ref61] 61. McDermoti TR, Graham PH. Competitive ability and efficiency in nodule formation of strains of Bradyrhizobium japonicum. Appl Environ Microbiol. 1990;56: 3035–3039. pmid:16348311
View Article
PubMed/NCBI
Google Scholar

[217] View Article

[218] PubMed/NCBI

[219] Google Scholar

[ref62] 62. Chen H, Higgins J, Kondorosi E, Kondorosi A, Djordjevic MA, Weinman JJ, et al. Identification of nolR-regulated proteins in Sinorhizobium meliloti using proteome analysis. Electrophoresis 2000;21: 3823–3832. pmid:11271500
View Article
PubMed/NCBI
Google Scholar

[221] View Article

[222] PubMed/NCBI

[223] Google Scholar

[ref63] 63. Chen H, Gao K, Kondorosi E, Kondorosi A, Rolfe BG. Functional genomic analysis of global regulator NolR in Sinorhizobium meliloti. Mol Plant Microbe Interact. 2005;18: 1340–1352. pmid:16478054
View Article
PubMed/NCBI
Google Scholar

[225] View Article

[226] PubMed/NCBI

[227] Google Scholar

[ref64] 64. Spaink HP. Rhizobial lipo-oligosaccharides: Answers and questions. Plant Mol Biol. 1992;20: 977–986. pmid:1463833
View Article
PubMed/NCBI
Google Scholar

[229] View Article

[230] PubMed/NCBI

[231] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Bacteria growth conditions, plasmids, mutant obtention

Bioinformatics

Motility assays

Biofilm formation assay

Quantitative and qualitative analyses of external polysaccharides

Identification of NFs

RNA isolation, cDNA synthesis and qRT-PCR

Nodulation assays

Results

Gene characterization

Studies of the swimming and surface motilities, biofilm formation, EPS production and LPS profiles in the nrcR mutant

NF-production patterns and nod-gene expression of the nrcR mutant

Symbiotic performance

Discussion

Supporting Information

S1 Fig.

S2 Fig. Alignment of NolR, ArsR and NrcR amino acid sequences using the ClustalW online platform and manipulated with Boxshade at EMBnet.

S3 Fig. Mucous halo observed in surface motility of R. tropici CIAT 899 assays on TY supplemented with 0.4% agar.

S1 Table.

Acknowledgments

Author Contributions

References