A new nodule-associated bacterium, Cupriavidus consociatus sp. nov. Isolated from the root nodules of Leucaena sp. and Arachis sp. growing in a cacao field in Chiapas, Mexico

Erika-Yanet Tapia-García; Belén Chávez-Ramírez; Violeta Larios-Serrato; Ivan Arroyo-Herrera; J. Antonio Ibarra; Paulina Estrada-de los Santos

doi:10.1371/journal.pone.0324390

Abstract

Cupriavidus is a genus of bacteria that inhabit diverse ecological niches, including plant-associated and nodulating species. A previous survey of legume plants in the south of Mexico resulted in the isolation of several bacteria. This present study describes two Cupriavidus strains isolated from the nodules of Leucaena sp. and Arachis sp. plants growing in a cacao field in Chiapas, Mexico. Both strains (LEh25^T and LEh21) shared identical 16S rRNA gene sequences and 98.4% identity with Cupriavidus oxalaticus Ox1^T. However, the in silico average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values (99.99 and 99.90% similarity, respectively) indicated that they belonged to different genomic species when compared to type strains of Cupriavidus species (ANI ~ 93.2 and dDDH ~ 50% similarity). Phylogenomic analysis indicated that the novel species would be placed in the genus Cupriavidus next to C. oxalaticus Ox1^T. Neither strain could fix nitrogen in a semisolid medium, and the interactions of the type strain with Phaseolus vulgaris, and Leucaena sp. revealed the formation of nodules, although these were ineffective. The genomic analysis demonstrated the presence of nitrogen fixation and nodulation genes with the same organization as in other strains of Cupriavidus and Paraburkholderia, although lacking NodB. To complement the study of the novel species, the strains were phenotypically and chemotaxonomically analyzed, with the results indicating differences with C. oxalaticus Ox1^T and other similar type strains of Cupriavidus species. From these results, we propose the novel species Cupriavidus consociatus sp. nov. with the type strain LEh25^T=TSD-314^T = CDBB B-2085^T.

Citation: Tapia-García E-Y, Chávez-Ramírez B, Larios-Serrato V, Arroyo-Herrera I, Ibarra JA, Estrada-de los Santos P (2025) A new nodule-associated bacterium, Cupriavidus consociatus sp. nov. Isolated from the root nodules of Leucaena sp. and Arachis sp. growing in a cacao field in Chiapas, Mexico. PLoS One 20(5): e0324390. https://doi.org/10.1371/journal.pone.0324390

Editor: Ying Ma, Universidade de Coimbra, PORTUGAL

Received: May 16, 2024; Accepted: April 23, 2025; Published: May 27, 2025

Copyright: © 2025 Tapia-García 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 within the manuscript and its Supporting Information files.

Funding: This work was funded by Secretaría de Investigación y Posgrado-IPN grant numbers 2022-0660 and 2023-0831. The funders had no role in study design, data collection, and analysis, the decision to publish, or preparation of the manuscript.

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

Introduction

Cupriavidus is a genus of bacteria belonging to the class Betaproteobacteria. Currently, the genus contains 23 species, with three names not yet validly published (“C. eutrophus”, “C. malaysiensis” and “C. neocaledonicus”) according to the International Code of Nomenclature of Prokaryotes (ICNP) in the List of Prokaryotic names with Standing in Nomenclature (https://www.bacterio.net/). Cupriavidus species have been isolated from clinical specimens, plant rhizosphere samples, soil, water, and legume nodules. The genus occurs worldwide. Some strains of various Cupriavidus species are tolerant to heavy metals. Cupriavidus, which means lover of copper (cuprum = copper and avidus = eager for, loving), includes the species C. metallidurans, a bacterium with exceptional metal tolerance. This species can grow in the presence of silver, copper, zinc, lead, and other metals [1]. C. metallidurans has been proposed for the use in the bioremediation of mercury-polluted agricultural soils [2]. Other strains belonging to different Cupriavidus species with resistance to several metals are C. necator, C. alkaliphilus, C. plantarum, C. agavae, C. nantongensis, C. campinensis, C. pauculus, C. gilardii, C. taiwanensis, C. basilensis, and “C. neocaledonicus” [3–11]. However, an essential characteristic of several species is their involvement in human infections; besides C. metallidurans, the species C. gilardii, C. pauculus, C. respiraculi, C. cauae, C. basilensis, and C. taiwanensis occur in clinical settings [12,13]. Another feature of Cupriavidus is that several strains belonging to different species are plant-associated bacteria. Members of the class Alphaproteobacteria were once thought to be the only bacteria to nodulate legumes until C. taiwanensis, belonging to the Betaproteobacteria, was demonstrated to possess this activity [14,15]. Currently, Cupriavidus necator and “Cupriavidus neocaledonicus” and many other strains of Cupriavidus (not yet officially described) can nodulate legumes, especially plants from the Mimoseae tribe [16]. A recent analysis of legume nodules from the south of Mexico revealed the presence of many bacterial genera, including the genus Cupriavidus [17]. In the present study, we employed phenotypic, chemotaxonomic, and genomic analysis of two bacterial strains, identifying them as a novel Cupriavidus species with the ability to nodulate legumes but ineffective.

Materials and methods

Bacterial strains

Cupriavidus strain LEh25^T was isolated from nodules of Arachis sp. and strain LEh21 was from nodules of Leucaena sp.; both legumes were growing in a cacao field in Chiapas, Mexico (N 14° 52’ 30.18” W 92° 21’ 24.768”) [17]. The owner of the cacao field granted permission for the sample collection; therefore, no official documents were required. The bacteria were isolated by sampling five randomly chosen legume nodules selected from five wild plants. The nodules were washed three times with sterile water, then immersed in 100% ethanol at 96° for 30 s, 10% sodium hypochlorite for 10 min, and then rinsed five times with sterile water. The water from the final washing was placed in LB medium plates (BD Bioxon) to verify surface disinfection. The nodules were crushed with a plastic pestle in 40 mL of sterile water. The whole nodule suspensions were inoculated onto plates with yeast extract mannitol (YM) medium containing 5 g/L of mannitol. The plates were incubated at 30°C for 3–5 days. The bacterial isolates were stored in 35% glycerol at -70°C until further analysis.

16S rRNA gene sequence analysis

The 16S rRNA gene sequences from strain LEh25^T (MN830085) and LEh21 (MN830086) were updated from a previous study, as the latter were too short to obtain appropriate information [17]. The 16S rRNA gene fragments were amplified with primers 27F/1492R [18] and sequenced by Macrogen Inc. (https://dna.macrogen.com). The sequences were edited and assembled with ChromasPro 2.1.5 (Technelysium Pty Ltd) and compared with sequences on the EzBioCloud website to determine the closest species of Cupriavidus. A phylogenetic analysis was performed with all type strains of Cupriavidus species. The alignment was carried out by Clustal Omega [19]. The aligned sequences were used for the estimation of the evolutionary model with IQ-TREE 2 v 2.3.6 [20]. The phylogeny was obtained using the Bayesian inference method with the Beast software v 2.5 [21], and the GTR + I + G model. A total of 10⁷ generations were performed, after which 25% of the trees were discarded [22]. Ralstonia solanacearum LMG 2299^T was included as an outgroup. The software PhyML 3.1 was used for tree construction. The phylogenetic tree was displayed with FigTree v 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Whole genome sequencing

The strains LEh25^T and LEh21 were grown in 40 mL LB broth and incubated overnight (120 rpm) at 30°C. The total DNA was isolated using Moore and Dowhan’s method [23]. The genome sequence was obtained by Novogene (https://en.novogene.com/) using the Illumina Platform PE150 with libraries paired-sequenced (2 x 350 bp). The quality of the raw sequencing data was evaluated using FastQC v0.11.9 [24]. Adapter screening and quality filtering of reads were performed with Trimmomatig 0.39 [25]. De novo genome assemblies were constructed with the SPAdes 3.14 program [26]. Metrics such as N50 and misassembles were obtained with QUAST v5.0.2 [27]. Annotation was performed using the standard operating procedure of IMG Annotation Pipeline v.5.1.0 from the Joint Genome Institute. A second annotation was performed via NCBI using the NCBI Prokaryotic Genome Annotation Pipeline.

Measurement of genomic relatedness and comparative analysis

The strains were first analyzed using the web server Type (Strain) Genome Server (TYGS) (tygs.dsmz.de), which compares genome sequences with an extensive and continuously updated database of bacterial genome sequences. The genome-to-genome comparison with TYGS uses the formula d4, which is independent of genome length and is robust to incomplete draft genomes [28]. The genome sequences belonging to all type strains of Cupriavidus species and Cupriavidus sp. strains (i.e., Cupriavidus strains not assigned to any described species) were downloaded from the NCBI for genomic comparisons. The digital DNA-DNA hybridization (dDDH) values were estimated using formula 2 of the Genome-to-Genome Distance Calculator (GGDC, http://ggdc.dsmz.de/ggdc.php#9) with 70% as the level of similarity used for species definition [29]. The average nucleotide identity (ANI) values were established using JSpeciesWS online service [30], with values of 95–96% for species definition (29). Protein sequenced-based genome analysis was carried out with the OrthoVenn3 program using the ClusterVenn tool [31] to compare the orthologous clusters of genes among the genomes of strains LEh25^T, LEh21, and the closest species C. oxalaticus Ox1^T.

Phylogenomic analysis

A phylogenomic analysis was performed using the up-to-date bacteria core genes (UBCG pipeline) [32] by the maximum likelihood method. Gene support indices (GSI) were used to support the branches. The tree was inferred from 91251 nucleotide positions belonging to 92 core genes. The analysis involved the genome sequence of all Cupriavidus type species, the strains LEh25^T and LEh21, and the species Paraburkholderia unamae MTl-641^T and Paraburkholderia tropica Ppe8^T as outgroups. The tree was displayed with MEGA version 11 [33].

BOX-PCR

To determine the similarities and differences between the two strains belonging to Cupriavidus consociatus sp. nov. and type strains of closely related Cupriavidus species, the strains were analyzed using the BOX element (BOXA1), according to a previously described method [34].

Biochemical and phenotypic tests

Strains LEh25^T, LEh21, C. oxalaticus Ox1^T, C. necator N-1^T, and C. taiwanensis LMG 19424^T were characterized according to different phenotypic features. Colony morphology was determined after two days of culture on LB plates at 30°C. Temperature-dependent growth was evaluated on LB, YM, and MacConkey agar plates at 20, 25, 30, 37, and 42°C after two days. Salt tolerance in modified LB (without NaCl) was examined by adding 0.5, 1, 2, 3, 4, and 5% NaCl; the plates were incubated for five days at 30°C. The effect of pH on bacterial growth was established in LB broth adjusted with the following 1X buffers: a glycine-HCl buffer for ranges of pH 1.0–3.0; an acetate-based buffer for pH 4.0–5.0; a citric acid-phosphate buffer for pH 6.0–7.0; a Tris-HCl buffer for pH 8–9; a glycine-NaOH buffer for pH 10.0–12.0, and a KCl-NaOH buffer for pH 13.0 [35]. The liquid cultures were incubated (120 rpm) for five days at 30°C. Biochemical tests were performed with the VITEK2 System using the VITEK2 GN card according to the manufacturer’s instruction (BIOMÉRIUX).

Chemotaxonomic characterization

Whole-cell proteins for strains LEh25^T, LEh21, C. oxalaticus Ox1^T, C. alkaliphilus ASC-732^T, C. necator N-1^T, and C. taiwanensis TVV75^T were analyzed as described previously by using SDS-PAGE [34]. Briefly, the bacteria were grown in Jain and Patriquin [36] medium with reciprocal shaking (200 rpm) for 15 h at 29°C, and 1.0 mL samples were harvested by centrifugation at 12,300 × g for 10 min at 25°C. The pellet was resuspended in 70 μL of 0.125 M Tris-HCl, 4% SDS, 20% glycerol, and 10% mercaptoethanol at pH 6.8. Aliquots of 10 μL were used for SDS-PAGE. Polar lipids were analyzed for strains LEh25^T, LEh21, and C. oxalaticus Ox1^T. The strains were grown in LB liquid media for 16 h at 29°C, and the polar lipids were extracted following the Bligh and Dyer technique [37]. The chloroform phase was used for lipid analysis by two-dimensional separation on TLC plates [38]. Total polar lipids were imaged by spraying with ANS reagent (8-anilino-1-naphtalenesulfonic acid) [39] and iodine vapor [40]. The polar lipids were identified based on their migration and specific staining. Lipids containing amino groups and glycolipids were determined using the ninhydrin and periodate-Schiff techniques, respectively, as described previously [41,42].

Ubiquinone analysis in silico

The in silico analysis of ubiquinones was performed by extracting the amino acid sequences corresponding to UbiA, UbiB, UbiD, UbiE, UbiF, UbiG, UbiH, UbiI, UbiJ, and UbiX from the genome sequences of all type strains of Cupriavidus species. The sequences for each protein were individually aligned with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and then concatenated using the software Mesquite v3.81 (http://www.mesquiteproject.org/?HistoryPanel=open). The phylogenetic analysis used the maximum likelihood method and the aminoacidic model BLOSUM62. The tree was displayed with MEGA version 11 [33], and Paraburkholderia unamae MTl-641^T and Paraburkholderia tropica Pp8^T were used as outgroups.

Nitrogen fixation and nodulation analysis

Mimosa pudica seeds were treated with H₂SO₄ (95–97%) for 5 min, and Acacia sp. and Leucaena sp. seeds were treated for 10 min, then washed five times with sterile water. Next, all seeds, including those of Phaseolus vulgaris seeds, were disinfected with 10% sodium hypochlorite for 10 min and washed five times with sterile water. The seeds were placed in 15% agar-water plates and incubated at 30°C for 72 h in the dark. The germinated seeds were sown in sterile vermiculite in 200 mL pots and inoculated with 2 mL of a bacterial suspension containing approximately 1 × 10⁸ cells per milliliter. The pots were kept for 45 days in a greenhouse at 30°C with a 14 h light – 10 h dark photoperiod and watered with Fahraeus solution [43]. The experiment was performed with five plants per treatment, with plants grown in individual pots. The treatments were: a) control inoculation with water, b) inoculation with strains LEh25^T and LEh21 and c) inoculation with Paraburkholderia mimosarum PAS44^T, Paraburkholderia tuberum STM 678^T, and Paraburkholderia phymatum STM 815^T as a positive control. After incubation, five nodules were randomly selected from each plant and washed three times with sterile water. The nodule surface was sterilized as described above, and the water from the final rinse was used to verify the disinfection. Finally, the legume nodules were crushed with a plastic pestle in 40 μl of water, and the nodule suspension was inoculated (15 μl) onto plates with YM medium. The plates were incubated at 30°C for 3–5 days. Isolates were identified by amplifying and sequencing the 16S rRNA, as previously analyzed [4], to verify Koch’s postulates. Before the bacterial isolation from nodules, the roots with the nodules were washed with sterile water and placed in 100 mL vials. The vials were sealed with rubber seals, and 5% of the total volume of air was extracted and replaced with acetylene [34]. The roots were then left at room temperature for 8 h. Next, nitrogen fixation was indirectly measured with a Clarus 580 gas chromatographer (PerkinElmer) by the reduction of acetylene to ethylene [34]. In addition, the strains were individually tested for nitrogen fixation by growing the bacteria in 10 mL vials containing 5 mL of semisolid (2.3 g/L) YM medium free of nitrogen for three days. The cotton plug was replaced with a rubber seal, and 10% of the air was replaced with acetylene. The vials were incubated overnight at 30°C, and nitrogen fixation was measured as described above. The free nitrogen fixation included Paraburkholderia tropica Ppe8^T, Azospirillum brasilense sp. 7^T, Paraburkholderia caballeronis TNe-862^T, and Burkholderia orbicola TAtl-371^T.

Analysis and genetic organization of N-fixation and nodulation genes

The genome sequences of strains LEh25^T and LEh21 were explored for nitrogen fixation and nodulation genes. For nitrogen fixation, the nifHDK genes were screened in both genomes using the nifHDK sequence from C. taiwanensis TVV75^T that was obtained from the Joint Genome Institute with the GOLD Study ID Gs00111891. A phylogenetic analysis of the amino acid NifH sequence was performed with several nitrogen-fixing bacteria, primarily from the genera Paraburkholderia, Cupriavidus, and Trinickia, and the species Rhizobium etli CFN42^T and Bradyrhizobium japonicum ATCC 10324^T were used as the outgroups. The phylogenetic analysis used the maximum likelihood method and the amino acid substitution model BLOSUM62. The tree was displayed with MEGA version 11 [33]. Bootstrap analysis was performed with 1000 replications. For the analysis of nodulation genes, the organization of nodBDCIJHASU genes was searched in both genomes using the genes from C. taiwanensis TVV75^T, also gathered from the JGI database. A phylogenetic analysis of the amino acid NodC sequence was carried out with the strains of the novel species and other strains from species of Cupriavidus, Paraburkholderia, Trinickia, Rhizobium, and Mesorhizobium. The phylogenetic tree was performed similarly to the one for NifH.

Results and discussion

Phylogenetic analysis based on the 16S rRNA gene sequence

The newly obtained 16S rRNA gene sequences from both strains LEh25^T and LEh21 were identical. The sequences were compared to those of all type strains of Cupriavidus species; the results showed 98.5% similarity to C. oxalaticus Ox1^T. For bacteria, 98.7% is the accepted threshold for species delineation [44]. Therefore, the results indicate that strains LEh25^T and LEh21 belong to a new species of Cupriavidus. Moreover, the 16S rRNA sequences from strains LEh25^T and LEh21 were identical to the sequence obtained from the genome. The phylogenetic analysis grouped both strains with C. oxalaticus Ox1^T and the neighboring C. taiwanensis TVV75^T (Fig 1). The analysis corroborated the identification of the strains LEh25^T and LEh21 as members of the genus Cupriavidus.

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Fig 1. Phylogenetic analysis of Cupriavidus species is based on a comparison of 16S rRNA gene sequences.

The study was performed with 1550 nucleotides using the Bayesian inference method with the Beast software v 2.5. The analysis included the GTR + I + G model of nucleotide substitution. The novel species Cupriavidus consociatus sp. nov. is shown in red. The numbers in parentheses correspond to the accession numbers in the NCBI database. The numbers at branch points indicate bootstrap support. The bar represents substitutions per nucleotide position. Ralstonia solanacearum LMG 2299^T was used as the outgroup.

https://doi.org/10.1371/journal.pone.0324390.g001

General features of the genome

The genome sequences of strains LEh25^T and LEh21 were assembled in 237 (total length 8,337,954 bp) and 191 (8,247,320 bp) contigs, respectively. The G + C content was 65.19% for strain LEh25^T and 65.26% for strain LEh21. The content of ribosomal genes was similar between the two strains of the new species, although the number was low. This may have been due to the genome assembly since different strains from C. oxalaticus, the closest species, also contained a variable number of ribosomal genes, from zero to nine (data not shown). More information concerning the genomes for strains LEh25^T and LEh21 is summarized in the S1 Table.

Measurements of genomic relatedness

The evaluation of the genome sequence from the two Cupriavidus strains on the TYGS website showed that the strains represented a new genomic species closely related to C. oxalaticus Ox1^T (~ 50% similarity). Both strains belonged to the same genomic species (99.99% similarity). The results of the analysis with dDDH and ANI revealed that the similarity to C. oxalaticus Ox1^T was 50.1–50.2% and 93.22–93.23% for strains LEh25^T and LEh21, respectively (Table 1) values lower than the criteria for the species cut-off. Moreover, the comparison with Cupriavidus sp. (i.e., Cupriavidus strains not assigned to any described species) also showed values lower than the cut-off.

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Table 1. Comparative genomics between the novel species Cupriavidus consociatus sp. nov. and other type strains of Cupriavidus species.

https://doi.org/10.1371/journal.pone.0324390.t001

The ANI and dDDH values between the strains LEh25^T and LEh21 are 99.99% and 99.90%, respectively.

The phylogenomic analysis using the genome sequence from all type strains of Cupriavidus species indicated that the novel genomic species formed a single cluster near C. oxalaticus Ox1^T (Fig 2), demonstrating that strains LEh25^T and LEh21 belong to a new genomic species within the genus Cupriavidus.

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Fig 2. Phylogenomic analysis of Cupriavidus species using the up-to-date bacterial concatenated alignment of 92 core genes (UBCG).

A total of 91251 nucleotide positions were used. The phylogenomic tree was inferred using the maximum likelihood method. Gene support indices (GSI) are given at the branching points. Bar = 0.04 substitutions per position. Paraburkholderia unamae MTl-641^T and Paraburkholderia tropica Ppe8^T were used as the outgroups.

https://doi.org/10.1371/journal.pone.0324390.g002

Comparative genomics

The whole-genome orthologous gene comparison (Fig 3) showed that strains LEh25^T, LEh21, and Ox1^T formed 7362 clusters of genes, 7287 that were typical for the strains of the novel species, a value that was higher than the 4709 between strain LEh25^T and C. oxalaticus Ox1^T or 4716 between strain LEh21 and C. oxalaticus Ox1^T. This illustrated the differences between the novel species and its closest relative. There were 1113 singletons or proteins not in any cluster, with 1010 belonging only to C. oxalaticus Ox1^T. The three strains shared 4709 clusters, with these being associated with molecular function (126), hydrolase activity (92), and oxidoreductase activity (90).

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Fig 3. Venn diagram of the common and unique orthologous genes among Cupriavidus consociatus sp. nov.

LEh25^T, and LEh21, with Cupriavidus oxalaticus Ox1^T, using the software OrthoVenn2 [45].

https://doi.org/10.1371/journal.pone.0324390.g003

BOX-PCR

Fingerprinting obtained with BOX-PCR can help to discriminate bacterial strains at the species level [46]. The comparative analysis showed identical patterns between strains LEh25^T and LEh21 and completely different patterns with the type strains of C. oxalaticus, C. taiwanensis, and C. necator (Fig 4).

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Fig 4. BOXA1 patterns among Cupriavidus consociatus sp. nov. and several type strains of Cupriavidus species.

M, molecular marker. 1, Cupriavidus consociatus sp. nov. LEh25^T. 2, Cupriavidus consociatus sp. nov. LEh21. 3, Cupriavidus oxalaticus Ox1^T. 4, Cupriavidus taiwanensis LMG 19424^T. 5, Cupriavidus necator N-1^T.

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Biochemical and phenotypic analysis

The bacterial cells stained Gram-negative. The colonies grown in LB after 48 h were circular, convex, and whitish-colored. The strains grew in LB, YM, and MacConkey agar at 20, 25, 30, 37, and 42°C after 48 h. The strains were able to grow in LB up to 2% NaCl. The pH range for growth was 5–9. The alkalinization of L-lactate and succinate was positive. Both had activity of gamma-glutamyl-transferase, L-proline-arylamidase, and tyrosine arylamidase. The strains assimilated sodium citrate, malonate, L-malate, Ellman, and L-lactate. The complete results from biochemical and phenotypic analysis are displayed in S2 Table, and differential features with the closest species are listed in Table 2.

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Table 2. Differential phenotypic features between Cupriavidus consociatus sp. nov. and closest and relevant Cupriavidus type species.

https://doi.org/10.1371/journal.pone.0324390.t002

Chemotaxonomic characterization

Whole-cell protein patterns were analyzed in strains LEh25^T, LEh21, C. oxalaticus Ox1^T, C. alkaliphilus ASC-732^T, C. necator N-1^T, and C. taiwanensis LMG 19424^T. The analysis of the total cellular protein electrophoretic patterns provides discriminative information at or below the species level [47]. The protein patterns in the strains LEh25^T and LEh21 were identical but different from those of the other type strains of Cupriavidus species tested (Fig 5), thus illustrating the similarity in the novel species and the differences with other type strains of Cupriavidus species.

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Fig 5. Protein electropherograms (SDS-PAGE) of Cupriavidus consociatus sp. nov. and closely related and relevant type strains of Cupriavidus species.

Strains: 1, LEh25^T; 2, LEh21; 3, C. oxalaticus Ox1^T; 4, C. taiwanensis TVV75^T; 5, C. alkaliphilus ASC-732^T; 6, C. necator N1^T. MW, PageRuler molecular marker. The asterisk marks differences between strains LEh25^T and LEh21, with C. oxalaticus Ox1^T.

https://doi.org/10.1371/journal.pone.0324390.g005

The analysis of polar lipids was performed four times in independent experiments. In each of these experiments, strain LEh25^T resulted in faint spots when stained with ninhydrin, although phosphatidylethanolamine (PE) was identified (Fig 6). This was a difference between strains LEh25^T and LEh21; the latter, besides PE, also contained two unknown amino lipids. Other polar lipids consisted of phosphatidylglycerol (PG), cardiolipin (CL), and an unknown glycolipid (UG).

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Fig 6. Membrane lipid profiles of Cupriavidus consociatus sp. nov. and Cupriavidus oxalaticus Ox1^T.

The first TLC was stained with ninhydrin (a, b, and c), presenting PE (phosphatidylethanolamine) and UAL (an unknown amino lipid). The second TLC was stained with Shiff’s reagent (d, e, and f), showing PG (phosphatidylglycerol), CL (cardiolipin), and UG (an unknown glycolipid).

https://doi.org/10.1371/journal.pone.0324390.g006

Ubiquinones analysis in silico

The amino acid sequences corresponding to UbiA, UbiB, UbiD, UbiE, UbiF, UbiG, UbiH, UbiI, UbiJ, and UbiX from all type strains of Cupriavidus species were used in a phylogenetic analysis. The sequences were concatenated, producing a total of 3914 positions. The phylogenetic analysis showed that the novel species grouped with C. oxalaticus Ox1^T (Fig 7), consistent with the genome sequence comparison analysis. The major isoprenoid quinone reported in nine Cupriavidus species is ubiquinone Q-8. Thus, the association of the novel species with all type strains of Cupriavidus species in the phylogenetic analysis of ubiquinone amino acid sequences suggests that the novel species may contain ubiquinone Q-8.

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Fig 7. Phylogenetic analysis of concatenated ubiquinone amino acid sequences from type strains of Cupriavidus species.

The phylogenetic analysis used the maximum likelihood method and the amino acid substitution model BLOSUM62. The numbers at the branch points represent bootstrap support generated from 1000 replications. Bar = 0.2 nucleotide substitutions per position.

https://doi.org/10.1371/journal.pone.0324390.g007

Nitrogen fixation and nodulation analysis in vitro, in vivo and in silico

The ability of the novel species to fix nitrogen in a nitrogen-free culture medium was assessed, and the results were negative for both the novel species and B. orbicola. The positive controls P. tropica, A. brasilense and P. caballeronis fixed nitrogen in the semisolid medium. The capacity to fix nitrogen in culture media has not been thoroughly explored in the genus Cupriavidus, for example, C. necator was unable to grow in Burk’s N-free medium supplemented with glucose or sucrose, and it displayed limited growth with fructose that was not sustained after several passes in an N-free medium [48]. An experimental analysis employing the inoculation of the novel species to legume plants indicated that the bacteria could associate with P. vulgaris and Leucaena sp. in the case of strain LEh25^T and only P. vulgaris by strain LEh21 (Fig 8). In both cases, the nodules were white and ineffective regarding nitrogen fixation. Neither M. pudica nor Acacia sp. showed nodules when the plants were inoculated with the strains LEh25^T and LEh21. Nodulation of P. vulgaris and M. pudica was carried out by the control species P. mimosarum, P. tuberum and P. phymatum.

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Fig 8. Nodulation activity of Cupriavidus consociatus sp. nov.

A) Leucaena sp. and B) Phaseolus vulgaris by Cupriavidus consociatus sp. nov. LEh25^T. C) P. vulgaris by Cupriavidus consociatus sp. nov. LEh21. The arrows indicate white, ineffective nodules.

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The search for nitrogen fixation genes in the genome (genes nifHDK) of strains LEh25^T and LEh21 found a single copy with an identical arrangement to those of other Cupriavidus and Paraburkholderia strains. The NifHDK amino acid sequence identity between strains LEh25^T and LEh21 was 100% and ranged from 95 to 100% with other Cupriavidus and Paraburkholderia species. The phylogenetic analysis using NifH amino acid sequences with mostly type strains indicated the position of the novel species among Paraburkholderia, with P. diazotrophica JPY461^T as the closest species (95% identity). The nod genes nodDCIJHASU were identically organized between strains LE25^T and LEh21 (100% identity) as with other Paraburkholderia and Cupriavidus strains. However, NodB was absent in the genome of strains LEh25^T and LEh21. NodB is an oligosaccharide deacetylase that plays a central role in the Nod-factor biosynthesis and the symbiotic nodulation in plants. Possibly, this could be why neither strain could form nitrogen-fixing nodules. The phylogenetic analysis of NodC sequences showed that the novel species formed a closed group with other type strains of Cupriavidus species, different from Paraburkholderia and Trinickia strains (Fig 9).

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Fig 9. Phylogenetic analysis of NifH and NodD amino acid sequences from Cupriavidus consociatus sp. nov. and other nitrogen-fixing and nodulating bacteria

(A) NifH corresponds to nitrogen fixation activity. (B) NodC corresponds to nodulation activity. The phylogenetic analysis used the maximum likelihood method and the amino acid substitution model BLOSUM62. The numbers at branch points represent bootstrap support generated from 1000 replications. Bar = number of nucleotide substitutions per position.

https://doi.org/10.1371/journal.pone.0324390.g009

Conclusions

In this study, two Cupriavidus strains, LEh25^T and LEh21, were isolated from two legume plants growing in the wild in the south of Mexico. A comparative genomic analysis and phenotypic exploration revealed that both strains belonged to a novel species. Thus, we conclude that the two strains should be assigned to a new species in the genus Cupriavidus, and we propose the name Cupriavidus consociatus sp. nov. with LEh25^T (=TSC-314^T = CDBB B-2085^T) as the type strain.

Description of Cupriavidus consociatus sp. nov.

Cupriavidus consociatus (con.so.ci.a’tus. L. masc. part. adj. consociatus, associated with, in this case, the root nodules of legume plants).

The cells are Gram-staining-negative, aerobic. The colonies growing in LB are circular, convex, and whitish-colored. The bacteria are able to grow in LB, YM, and MacConkey agar at 20, 25, 30, 37, and 42°C, in up to 2% NaCl, and in the range of pH values of 5–9. There is no production of H₂S, but there is alkalinization of L-lactate and succinate. There are activities of gamma-glutamyl-transferase, L-proline-arylamidase, and tyrosine arylamidase, but no activity of Ala-Fe-Pro-arylamidase, L-pyrrolydonyl-arylamidase, beta-galactosidase, beta-N-acetyl-glucosaminidase, glutamyl arylamidase pNA, beta-glucosidase, beta-xylosidase, beta-alanine arylamidase pNA, lipase, palatinose, urease, alpha-glucosidase, beta-N-acetyl-galactominidase, alpha-galactosidase, phosphatase, glycine arylamidase, ornithine decarboxylase, lysine decarboxylase, beta-glucuronidase, or Glu-Gly-Arg-arylamidase. The species can assimilate sodium citrate, malonate, L-malate, Ellman, and L-lactate but not adonitol, L-arabitol, D-cellobiose, D-glucose, D-maltose, D-mannitol, D-mannose, D-sorbitol, saccharose, D-tagatose, D-trehalose, 5-keto-D-gluconate, L-histidine, and coumarate. Test for glucose fermentation and O/129 resistance were negative. Total polar lipids consisted of phosphatidylethanolamine (PE), an unknown amino lipid (UAL), phosphatidylglycerol (PG), cardiolipin (CL), and an unknown glycolipid (UG). Both strains have nif and nod genes but were unable to fix nitrogen and strain LEh25^T produced ineffective nodules.

The type strain LEh25^T=TSD-314^T = CDBB B-2085^T was isolated from root nodules from Arachis sp. growing in a cacao field in Chiapas, México.

Nucleotide sequence accession number

The GenBank accession numbers for the 16S rRNA gene sequences of strains LEh25^T and LEh21 are MN830085 and MN830086, respectively. The GenBank accession numbers for the genome sequences of strains LEh25^T and LEh21 are JAGIQA000000000 and JASMMW000000000.1, respectively.

Supporting information

S1 Table. Genomic features of Cupriavidus consociatus sp. nov. LEh25^T and LEh21.

https://doi.org/10.1371/journal.pone.0324390.s001

(DOCX)

S2 Table. Phenotypic features between Cupriavidu consociatus sp. nov. and close and relevant Cupriavidus type species.

https://doi.org/10.1371/journal.pone.0324390.s002

(DOCX)

S3 Table. Genome and ubiquinone protein accession numbers.

https://doi.org/10.1371/journal.pone.0324390.s003

(XLSX)

S4 Table. Genome and NifH protein accession numbers.

https://doi.org/10.1371/journal.pone.0324390.s004

(XLSX)

S5 Table. Genome and NodC protein accession numbers.

https://doi.org/10.1371/journal.pone.0324390.s005

(XLSX)

Acknowledgments

We thank Edwin Vázquez Guerrero for technical support. EYTG, BCR, VLS, IAH, JAI, and PES, thanks to CONAHCyT’s support. JAI and PES also thank COFAA and EDI.

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Figures

Abstract

Introduction

Materials and methods

Bacterial strains

16S rRNA gene sequence analysis

Whole genome sequencing

Measurement of genomic relatedness and comparative analysis

Phylogenomic analysis

BOX-PCR

Biochemical and phenotypic tests

Chemotaxonomic characterization

Ubiquinone analysis in silico

Nitrogen fixation and nodulation analysis

Analysis and genetic organization of N-fixation and nodulation genes

Results and discussion

Phylogenetic analysis based on the 16S rRNA gene sequence

General features of the genome

Measurements of genomic relatedness

Comparative genomics

BOX-PCR

Biochemical and phenotypic analysis

Chemotaxonomic characterization

Ubiquinones analysis in silico

Nitrogen fixation and nodulation analysis in vitro, in vivo and in silico

Conclusions

Description of Cupriavidus consociatus sp. nov.

Nucleotide sequence accession number

Supporting information

S1 Table. Genomic features of Cupriavidus consociatus sp. nov. LEh25T and LEh21.

S2 Table. Phenotypic features between Cupriavidu consociatus sp. nov. and close and relevant Cupriavidus type species.

S3 Table. Genome and ubiquinone protein accession numbers.

S4 Table. Genome and NifH protein accession numbers.

S5 Table. Genome and NodC protein accession numbers.

Acknowledgments

References

S1 Table. Genomic features of Cupriavidus consociatus sp. nov. LEh25^T and LEh21.