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. 2020 May 9;10(6):242. doi: 10.1007/s13205-020-02210-8

Draft genome of five Cupriavidus plantarum strains: agave, maize and sorghum plant-associated bacteria with resistance to metals

Ivan Arroyo-Herrera 1,#, Fernando Uriel Rojas-Rojas 1,2,#, Karla Daniela Lozano-Cervantes 1, Violeta Larios-Serrato 1, María Soledad Vásquez-Murrieta 1, William B Whtiman 3, J Antonio Ibarra 1, Paulina Estrada-de los Santos 1,✉
PMCID: PMC7211221  PMID: 32405446

Abstract

Five strains of Cupriavidus plantarum, a metal-resistant, plant-associated bacterium, were selected for genome sequencing through the Genomic Encyclopedia of Bacteria and Archaea (GEBA) Phase IV project at the Joint Genome Institute (JGI) of the U.S. Department of Energy (DOE). The genome of the strains was in the size range of 6.2–6.4 Mbp and encoded 5605–5834 proteins; 16.9–23.7% of these genes could not be assigned to a COG-associated functional category. The G + C content was 65.83–65.99%, and the genomes encoded 59–67 stable RNAs. The strains were resistant in vitro to arsenite, arsenate, cobalt, chromium, copper, nickel and zinc, and their genomes possessed the resistance genes for these metals. The genomes also encoded the biosynthesis of potential antimicrobial compounds, such as terpenes, phosphonates, bacteriocins, betalactones, nonribosomal peptides, phenazine and siderophores, as well as the biosynthesis of cellulose and enzymes such as chitinase and trehalase. The average nucleotide identity (ANI) and DNA-DNA in silico hybridization of the genomes confirmed that C. plantarum is a single species. Moreover, the strains cluster within a single group upon multilocus sequence analyses with eight genes and a phylogenomic analyses. Noteworthy, the ability of the species to tolerate high concentrations of different metals might prove useful for bioremediation of naturally contaminated environments.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02210-8) contains supplementary material, which is available to authorized users.

Keywords: Cupriavidus, Plant-associated bacteria, Metal resistance, Rhizobacteria, PGP bacteria

Introduction

Cupriavidus was formed in 2004 by reclassification of a number beta-proteobacterial species originally assigned to the genus Ralstonia (Vaneechoutte et al. 2004; Vandamme and Coenye 2004). Currently, this genus comprises 17 species according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN, bacterio.net). The species of Cupriavidus have been isolated from diverse ecological environments, such as soil, water, legume plant nodules and human clinical samples (Coenye et al. 2003; Sato et al. 2006; Cuadrado et al. 2010; da Silva et al. 2012; Estrada-de los Santos et al. 2012, 2014; Martínez-Aguilar et al. 2013). The species isolated from nosocomial infections are believed to be opportunistic pathogens (Vandamme et al. 1999; Coenye et al. 2003; Karafin et al. 2010). One interesting feature of Cupriavidus is the ability of many species to grow in the presence of heavy metals (Monchy et al. 2007; Goris et al. 2001; Estrada et al. 2012, 2014; Singh et al. 2015). During a survey for plant-growth promoting rhizobacteria in the north of Mexico, we collected numerous rhizospheric soil samples and isolated a number of Cupriavidus strains (Estrada-de los Santos et al. 2011). These strains were found in alkaline soils and proved to be representatives of two new species, Cupriavidus alkaliphilus and Cupriavidus plantarum (Estrada-de los Santos et al. 2012, 2014). The genome sequences of both species were obtained through the project “Genomic Encyclopedia of Bacteria and Archaea (GEBA) Phase III: The genomes of soil and plant-associated and newly described type strains” at the Joint Genome Institute (JGI) (Whitman et al. 2015), and the genome sequence of C. alkaliphilus ASC-732 T was reported (Rojas-Rojas et al. 2016). Later, through the project “GEBA Phase IV: Genome sequencing to study the core and pangenomes of soil and plant-associated prokaryotes”, the genomes of Reference strains from the species C. alkaliphilus and C. plantarum were also sequenced. In this study, we present the genome sequences and annotation of five C. plantarum strains as well as aspects of their phenotypes related to heavy metal resistance.

The C. plantarum strain ASC-64 T was isolated from agave (Agave sp.) rhizosphere in San Carlos county (N 24°34′40.8, O 98°56′38.36″), the strains MA1-4a, MA1-2za and MA2-19b were isolated from maize rhizosphere in the Abasolo county (N 24°03′21.12″, O 98°22′24.04″), and strain SLV-132 was isolated from sorghum rhizosphere in Los Vergeles county. All of these locations are in the northeast state of Tamaulipas, Mexico. All strains were isolated in 2010 (Estrada-de los Santos et al. 2011). Because of their resistance to metals, the five strains were selected for genome sequencing to explore the genotype associated with this phenotype. The strains were cultured in 5 mL of LB broth at 30 °C on a rotatory shaker at 200 rpm. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit (Promega). The concentration of DNA was determined in a Nanodrop 2000 (Thermo Scientific). The DNA was sent frozen to JGI, where the draft genomes were generated with Illumina technology (Bennett 2004). The genome sequencing and annotation was performed as described for C. alkaliphilus (Rojas-Rojas et al. 2016). The final draft assembly of C. plantarum ASC-64 T contained 15 scaffolds and 6,223,411 bp. The G + C content was 65.99 mol %, which is very close to the value of 65.7% determined by HPLC (Mesbah and Whitman 1989; Estrada-de los Santos et al. 2014). The genome was predicted to encode 5604 proteins and 67 stable RNAs (7 rRNAs, 51 tRNAs and 9 ncRNAs). The genomes of the reference strains included 9 to 27 scaffolds (Table 1). The number of genes associated with general COG functional categories was very similar among the five strains (Table 2).

Table 1.

Summary of general genomic features of five Cupriavidus plantarum species using the standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)

Attribute ASC-64T MA1-4a MA1-2za MA2-19b SLV-132
Genome size (bp) 6,223,411 6,296,586 6,278,407 6,433,748 6,390,750
DNA coding (bp) 5,658,554 5,730,421 5,711,369 5,823,124 5,787,240
DNA G + C (bp) [percentage] 4,106,759 [65.99%] 4,153,163 [65.96%] 4,141,105 [65.96%] 4,235,405 [65.83%] 4,210,199 [65.88%]
DNA scaffolds 15 19 15 9 27
Total genes 5672 5780 5753 5904 5786
Protein-coding genes 5605 5713 5685 5834 5717
RNA genes 67 67 68 59 69
Pseudo genes 56 46 46 nd nd
Genes in internal clusters 2172 2197 2181 2223 2207
Genes with function prediction 4743 4900 4882 4787 4914
Genes assigned to COGs 4376 4421 4406 4907 4415
Genes with Pfam domains 4972 5028 5018 5073 5039
Genes with signal peptides 654 659 646 nd 665
Genes with transmembrane helices 1391 1398 1399 nd 1405
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DOE-JGI Department of Energy, Joint Genome Institute. nd not detected

Table 2.

Number of genes and percentage associated with general Cluster of Orthologous Groups (COG) functional categories in the fives strains of Cupriavidus plantaruma, determined with the DOE-JGI pipeline

Code ASC-64T MA1-4a MA1-2za MA2-19b SLV-132 Description
J 231 (4.56) 231 (4.52) 230 (4.51) 243 (4.02) 229 (4.48) Translation, ribosomal structure and biogenesis
A 1 (0.02) 1 (0.02) 1 (0.02) 1 (0.02) 1 (0.02) RNA processing and modification
K 535 (10.55) 529 (10.34) 532 (10.43) 618 (10.22) 539 (10.55) Transcription
L 111 (2.19) 112 (2.19) 113 (2.22) 140 (2.31) 115 (2.25) Replication, recombination and repair
B 4 (0.08) 4 (0.08) 4 (0.08) 4 (0.07) 4 (0.08) Chromatin structure and dynamics
D 33 (0.65) 34 (0.66) 34 (0.67) 45 (0.74) 33 (0.65) Cell cycle control, Cell division, chromosome partitioning
V 97 (1.91) 97 (1.90) 98 (1.92) 123 (2.03) 100 (1.96) Defense mechanisms
T 266 (5.25) 272 (5.32) 266 (5.22) 352 (5.82) 265 (5.18) Signal transduction mechanisms
M 295 (5.82) 296 (5.79) 293 (5.75) 338 (5.59) 294 (5.75) Cell wall/membrane biogenesis
N 144 (2.84) 144 (2.82) 141 (2.76) 164 (2.71) 144 (2.82) Cell motility
U 81 (1.60) 96 (1.88) 91 (1.78) 121 (2.00) 92 (1.80) Intracellular trafficking and secretion
O 161 (3.18) 168 (3.29) 165 (3.24) 181 (2.99) 165 (3.23) Posttranslational modification, protein turnover, chaperones
C 491 (9.69) 487 (9.52) 486 (9.53) 512 (8.46) 489 (9.57) Energy production and conversion
G 257 (5.07) 253 (4.95) 258 (5.06) 307 (5.08) 258 (5.05) Carbohydrate transport and metabolism
E 473 (9.33) 468 (9.15) 479 (9.39) 572 (9.46) 476 (9.36) Amino acid transport and metabolism
F 88 (1.74) 88 (1.72) 89 (1.75) 88 (1.45) 87 (1.7) Nucleotide transport and metabolism
H 243 (4.79) 246 (4.81) 245 (4.80) 266 (4.4) 246 (4.81) Coenzyme transport and metabolism
I 317 (6.25) 316 (6.18) 320 (6.27) 338 (5.59) 314 (6.14) Lipid transport and metabolism
P 282 (5.56) 280 (5.48) 275 (5.39) 364 (6.02) 278 (5.44) Inorganic ion transport and metabolism
Q 187 (3.69) 200 (3.91) 200 (3.92) 238 (3.93) 200 (3.91) Secondary metabolites biosynthesis, transport and catabolism
R 506 (9.98) 507 (9.91) 503 (9.86) 691 (11.42) 512 (10.02) General function prediction only
S 193 (3.81) 197 (3.85) 193 (3.78) 239 (3.95) 195 (3.82) Function unknown
Z 1 (0.02) 1 (0.02) 1 (0.02) 3 (0.05) 1 (0.02) Cytoskeleton
W 63 (1.24) 63 (1.23) 61 (1.20) 75 (1.24) 63 (1.23) Extracellular structures
X 9 (0.18) 24 (0.47) 22 (0.43) 26 (0.43) 11 (0.22) Mobilome
– 1296 (22.85) 1359 (23.51) 1347 (23.41) 997 (16.89) 1371 (23.70) Not in COGs
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aDetermined with the DOE-JGI (U.S. Department of Energy, Joint Genome Institute) pipeline. The percentages are based on the total number of protein coding genes in each genome and enclosed in parenthesis

Given the ability of this species to grow in the presence of the metals zinc, copper and cobalt (Estrada-de los Santos et al. 2014), this analysis was extended. The minimum inhibitory concentrations (MIC) of various metals were determined using 96-well plates. Each well was filled with 100 μL of a sterile 1:100 dilution of LB broth and 100 μL of the inoculum supplemented with different amounts to metals obtain final concentrations of 0.25, 1, 2, 4, 10, 20, 30 and 50 mM. The metals tested were As5+ (NaH2AsO4), As3+ (NaAsO2), Co2+ [Co(NO2)3], Cr6+ (K2Cr2O7), Cu2+ (CuSO4), Ni2+ (NiCl2), and Zn2+ (ZnSO4). The use of minimal medium is important to avoid overestimating the metal resistance, which commonly occurs when rich medium is used (Rathnayake et al. 2013). However, Cupriavidus species were unable to grow in the minimal medium proposed by Rathnayake et al. (2013). Therefore, diluted LB broth was used instead. The bacterial inoculum was diluted to an OD600 = 0.9. The 96-well plates were incubated at 30 °C for 96 h, 120 rpm. The OD600 was measured with a Multiskan FC Microplate Photometer (Thermo Fischer Scientific). The MIC was the minimal concentration of metal that inhibited microbial growth (Lukasz et al. 2014). Although the resistance to metals was generally high, up to 50 mM for most of the species, they varied over a wide range depending upon the metal, strain and species (Table 3). For instance, the MICs of the strains of C. plantarum varied considerably, as did the MICs of other species. C. metallidurans CH34T, a bacterium highly resistant to heavy metals, possessed high MICs for most of the metals tested (Monchy et al. 2007) (Table 3).

Table 3.

Minimum inhibitory concentration of metal performed in vitro for Cupriavidus species and the five Cupriavidus plantarum strains

Cupriavidus species As 3+ (mM) As 5+ (mM) Co (mM) Cr (mM) Cu (mM) Ni (mM) Zn (mM)
C. plantarum
 ASC-64T 40 20 20 20 50 20 50
 MA1-4a 20 30 40 20 30 50 50
 MA1-2za 1 20 50 10 20 50 50
 MA2-19b 1 50 50 1 30 50 40
 SLV 132 1 30 50 1 40 50 50
C. metallidurans CH34T 50 50 40 20 50 10 50
C. numazuensis TE26T 30 20 5 10 50 1 10
C. respiraculi LMG 21510 T 10 40 30 10 50 10 50
C. taiwanensis LMG 19424T 20 40 10 50 50 30 50
C. oxalaticus Ox-1 T 50 50 50 10 50 50 10
C. alkaliphilus ASC-732 T 50 50 30 30 40 40 20
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The analysis was performed in triplicate and the replicates were all the same

Next, the presence of genes related to metal resistance was analyzed in the five genomes. Genes were considered homologous, when the amino acid sequence identity exceeded 50% when using the blastX tool for the comparison. The czc operon encodes a heavy metal efflux pump containing an inner membrane efflux pump (CzcA), a membrane fusion protein (CzcB) and an outer membrane factor (CzcC). It facilitates cell detoxification of the divalent ions cadmium, zinc and cobalt (Nies et al. 1989). This complex was first described in C. metallidurans (Nies et al. 1989) and has been shown to play a role on heavy metal resistance in the proteobacterial species Comamonas testosteroni (Xiong et al. 2011), Gluconobacter diazotrophicus (Intorne et al. 2012), Pseudomonas aeruginosa (Caille et al. 2007), Pseudomonas putida (Leedjärv et al. 2008) and the non-proteobacterial species Staphylococcus aureus (Kuroda et al. 1999), Helicobacter pylori (Stähler et al. 2006) and Marinobacter adhaerens (Stahl et al. 2015). The C. plantarum ASC-64 T genome carries three orthologs of the czc operon (locus tags Ga0116997_0029, Ga0116997_0030, Ga0116997_0031, Ga0116997_4230, Ga0116997_4231, Ga0116997_4232, Ga0116997_4808, Ga0116997_4809 and Ga0116997_4810) (Online Resource 1). Homologs of the czc operon were also found in the genomes of all four reference strains (Online Resource 1). While genetic manipulations are needed to confirm the role of the czc operon in heavy metal resistance of C. plantarum, the presence of three orthologs suggests that at least one of them could be linked to zinc and cobalt resistance phenotype observed in the five strains.

Genes that encode the cobalt transporter CbtAB are also present in the genome of the type strain of C. plantarum (Ga0116997_3073 and Ga0116997_3074) as well as those of the reference strains (Online Resource 1). This system plays an important role in vitamin B12 biosynthesis in prokaryotes (Rodionov et al. 2003; Zhang et al. 2009), although a role in cobalt resistance has been hypothesized for Brucella based on its part in regulation of cobalt transport (Roop et al. 2017). The presence of these genes could also play an important part in the cobalt resistance observed for C. plantarum strains.

Several putative chromate resistance proteins are encoded in the five C. plantarum genomes (Online Resource 1), including three with the pfam domain PF09828, which is related to the reduction of chromate accumulation in bacteria. This domain is present in the widely distributed chromate resistance protein ChrA (Ga0116997_1988, Ga0116997_1986 and Ga0116997_4558). The role of ChrA proteins in chromate resistance has been reported in other proteobacteria, including P. aeruginosa (Cervantes et al., 1990), C. metallidurans (Juhnke et al. 2002) and Paraburkholderia xenovorans (Acosta-Navarrete et al. 2014). Moreover, the ChrB protein plays a role in chrA expression (Nies et al. 1990), the gene encoding ChrB is also present in C. plantarum ASC-64 T (Ga0116997_4559) as well as the reference strains, which further supports the hypothesis that the at least one of the chrA homologs plays a role in chromate resistance reported here.

The MIC for copper for the C. plantarum strains was 20–50 mM. Therefore, we searched for genes related to the resistance of this metal. All the strains of C. plantarum encode parts of a putative Cop system, including CopA (Ga0116997_2928), CopB (Ga0116997_0947) and CopZ (Ga0116997_2927) (Online Resource 1). However, the cop operon in the C. plantarum genomes seems to be incomplete, because at least genes for CopC, CopD, CopR and CopS are missing. Therefore, it is unlikely that these genes are responsible for the divalent copper resistance phenotype. However, the C. plantarum strains also encode a putative cus operon (Ga0116997_4355, Ga0116997_4356, Ga0116997_4357 and Ga0116997_4358) (Online Resource 1). The cus gene cluster encodes a tetrapartite prokaryotic cooper resistance system comprising an inner membrane protein CusA (member of the resistance-nodulation-cell division superfamily, RND), a periplasmic membrane fusion protein CusB (belonging to the membrane fusion proteins family, MFP), an outer membrane protein CusC (member of the outer membrane factor family, OMF) and a periplasmic chaperone CusF (Bondarczuk and Piotrowska-Seget 2013). The participation of the Cus proteins in metal resistance has been demonstrated in other Gram-negative bacteria, such as Escherichia coli (Franke et al. 2003), Shewanella oneidensis (Toes et al. 2008) and Cupriavidus gilardii (Huang et al. 2019). Thus, it is a reasonable hypothesis that the Cus system is a major copper resistance mechanism in C. plantarum. All genomes also possessed an ortholog of the eukaryotic superoxide dismutase SOD1 (data not showed). This enzyme has been linked to copper buffering in Candida albicans (Li et al. 2015) and Saccharomyces cerevisiae (Culotta et al. 1995). However, this role is unclear in bacteria, and we do not consider that there is enough experimental evidence to propose this enzyme as a prokaryotic copper resistance mechanism.

Arsenite and arsenate resistance was observed in the five C. plantarum strains, with MIC’s of 1–40 mM and 20–50 mM, respectively. The genome of C. plantarum strain ASC-64 T encoded a putative transcriptional regulator ArsR (Ga0116997_2490), the arsenite efflux transporter Acr3 (Ga0116997_2491), the arsenate reductase ArsC (Ga0116997_2492), the trivalent organoarsenical oxidase ArsH (Ga0116997_2494) and the methylarsenite efflux permease ArsP (Ga0116997_2494), all encoded in a putative ars operon. Orthologs were also identified in all the C. plantarum reference genomes (Online Resource 1). Arsenic resistance conferred by the ars operon has been confirmed in the Gram-negative bacteria E. coli (Carlin et al. 1995), Pseudomonas putida (Fernandez et al., 2014), Pseudomonas fluorescens (Prithivirajsingh et al. 2001), P. xenovorans (Serrato-Gamiño et al. 2018) and C. metallidurans (Mergeay et al. 2003). The C. plantarum ars operon contains fewer genes than ars in C. metallidurans (Mergeay et al. 2003). Nevertheless, its genetic organization and composition is similar to the ars operon from P. xenovorans LB400T (data not showed), which also confers the arsenic resistance phenotype (Serrato-Gamiño et al. 2018). Nevertheless, heterologous expression or gene deletion experiments are needed to ensure a similar role of the ars operon in C. plantarum and to determine if the differences in operon composition compared to C. metallidurans is correlated with the differences in MIC.

Genes encoding for potential secondary metabolites, important as a source of antimicrobials and other bioactive compounds, were uncovered with the software antiSMASH 5.0 (Blin et al. 2019). The C. plantarum genome contains the genes for the biosynthesis of antimicrobials terpenes, phosphonates, bacteriocins, betalactones, nonribosomal peptides, phenazine and siderophores (Online Resource 2). However, the biosynthetic pathways for nonribosomal peptides and siderophores were incomplete, possibly because the genomes were in a draft status or the pathways differed from the canonical ones. The five C. plantarum strains also produced siderophores in CAA-CAS medium (Estrada-de los Santos et al. 2014), and their genomes encode cellulose biosynthesis (Online Resource 2). Cellulose biosynthesis bcs operons have been divided into three major types, namely those found in Komagataeibacter xylinus, E. coli and Agrobacterium tumefaciens (Romling et al. 2015). The C. plantarum bcs operon is similar that of K. xylinus, which is also found in P. xenovorans. Hydrolytic enzymes, such as chitinase and trehalase, are also encoded in the five C. plantarum genomes (Online Resource 2). C. plantarum has the gene cluster for the synthesis of tryptophan, but not the genes to produce indoleacetic acid, a plant growth-promoting hormone. Additionally, in regard with cellular appendages, C. plantarum genome encodes genes for flagellar biosynthesis as well as chemotaxis related genes. Among the pili encoded, it was noticeable that at least two type IV pilus gene clusters were present. The genetic information for the type I, II, III, VI and Sec secretion systems was also present.

Average Nucleotide Identity (ANI) calculations were obtained with JSpeciesWS (Richter et al. 2015), using different methods: ANIb (based on the BLAST algorithm), ANIm (based on the MUMmer algorithm) and TETRA (Tetranucleotides Signature Frequency Correlation Coefficient). The digital DNA-DNA hybridization (dDDH) was also calculated in silico by the Genome-to-Genome Distance Calculator (GGDC 2.1) using the BLAST method (Meier-Kolthoff et al. 2013). Results were based on recommended formula 2 (identities/HSP length), which is independent of genome length and appropriate for incomplete genomes. The genome of ASC-64 T was compared to C. plantarum reference strains and to other Cupriavidus species. The ANI results were consistent with the assignments of strain ASC-64 T and the four reference strains to the species C. plantarum (Table 4). For instance, the ANI was > 98% for all strains assigned to C. plantarum and < 86.0% for comparisons the other Cupriavidus species. Similarly, the dDDH values supported these assignments (Goris et al. 2007; Richter & Rosselló-Móra 2009). The genomes were also compared with Dot Plot, which employs MUMmer to generate dot plot diagrams between two genomes to analyze the synteny. The dot plot diagrams were built in the IMG/MER (Integrated Microbial Genomes & Microbiomes) platform at JGI. The results showed highly similarity, with parallel strands among each of two compared genomes, no inversions, and few breaks (Fig. 1). The synteny plot of C. plantarum genomes was performed with progressiveMauve (Darling et al. 2010), showing rearrangements, insertions and/or deletions through the species genomes (Fig. 1e). The highest synteny was observed between strains ASC-64 T, MA1-4a and MA2-19b. Plasticity of C. plantarum genomes was moderate compared to the C. metallidurans genomes (Mazhar et al. 2020).

Table 4.

Genome comparison of Cupriavidus plantarum ASC-64T with reference strains and other Cupriavidus species using average nucleotide identity (ANI) and genome-to genome distance calculator (GGDC)

Genome (ASC-64T vs) ANIb (%) ANIm (%) TETRA dDDH (%)
C. plantarum MA1-4a 99.03 99.09 0.99990 92.6
C. plantarum MA1-2za 98.97 99.08 0.99992 92.5
C. plantarum MA2-19b 98.89 99.08 0.99984 92.1
C. plantarum SLV-132 99.13 99.23 0.99984 93.8
C. metallidurans CH34T 78.28 85.59 0.90568 23.7
C. nantongensis X1T 78.04 85.76 0.87005 24.1
C. alkaliphilus ASC-732 T 77.99 85.80 0.87104 24.1
C. oxalaticus NBRC 13593T 77.96 85.82 0.87507 24.2
C. taiwanensis LMG 19424T 77.92 85.74 0.97759 24.1
C. necator N-1T 77.81 85.61 0.8674 28.8
C. gilardii CR3 77.23 85.48 0.8953 24.4
C. basilensis 4G11 77.13 85.22 0.87118 23.6
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ANIb average nucleotide identity, based on Blast, ANIm average nucleotide identity, based on Mummer, TETRA Tetranucleotides Signature Frequency Correlation Coefficient (above cutoff > 0.9999, in useful range > 0.989, below useful range < 0.989), dDDH digital DNA-DNA hybridization performed with the Genome-to-Genome Distance Calculator at the website ggdc.dsmz.de

Fig. 1.

Fig. 1

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Genome comparisons of Cupriavidus plantarum strains with Dot Plot and progressiveMauve. Strain ASC-64 T vs (a) MA1-2za, (b) MA1-4a, (c) MA2-19b and (d) SLV-132. Blue dots are parallel strands. e Synteny plot of C. plantarum genomes performed with progressiveMauve showing rearrangements, insertions and/or deletions through the species genomes

The position of C. plantarum within the genus Cupriavidus was examined with multilocus sequence analyses of genes atpD, gltB, gyrB, lepA, phaC, recA, trpB and 16S rRNA following the methodology implemented previously for Burkholderia senu lato (Estrada-de los Santos et al. 2016). In addition, a phylogenomic analysis by virtual genome fingerprint using the VAMPhyRE software (Muñoz-Ramírez et al. 2017), was also performed. The phylogenomic tree was constructed with the Neighbor-Joining method with the program MEGA X (Kumar et al. 2018). In both cases, the phylogenetic and phylogenomic analysis positioned C. plantarum in the genus Cupriavidus (Fig. 2). The increasing availability of genome sequence data has yielded more reliable phylogenetic reconstructions than single gene or protein analyses. Consequently, the taxonomy of many prokaryotic groups has been revised, and several new genera and higher taxonomic groups have been described (Campbell et al. 2015; Naushad et al. 2015; Estrada-de los Santos et al. 2018).

Fig. 2.

Fig. 2

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Phylogenetic trees-based on the analyses of Cupriavidus species. a Multilocus sequence analyses-based on the concatenated atpD, gltB, gyrB, lepA, phaC, recA, trpB and 16S rRNA sequences with maximum likelihood. The bar represents the expected sustitutions per site under the model GTR + G + I. b Phylogenomic analysis by virtual genome fingerprinting using the software Vamphyre, the tree was was inferred with Neighbor Joining method of available Cupriavidus species genome sequences. The bar represents the amount of changes in the nucleotides. In bold the species C. plantarum

In summary, the analyses of the genome sequences of C. plantarum strains in this study showed that the original description of the species was accurate. Moreover, a number of genes likely to play a role in metal resistance were identified, and these strains may be useful for bioremediation of contaminated environments.

Nucleotide sequence accession numbers

The genome sequences of C. plantarum strains have been deposited at the Genbank with the following BioProject accession numbers: strain ASC-64 T (PRJNA329822), strain MA1-2za (PRJNA439959), strain SLV-132 (PRJNA439960), strain MA1-4a (PRJNA439958) and strain MA2-19b (PRJNA547271). The genome sequences are also freely available at IMG/MER in the JGI-DOE, USA.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 28 kb) (28.6KB, docx)
Supplementary file2 (DOCX 25 kb) (25.3KB, docx)

Acknowledgements

I.A.H., K.D.L.C. and F.U.R.R. received a fellowship from Conacyt. The genome sequencing was conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, and was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. We thank Marcel Huntemann, Alicia Clum, Manoj Pillay, Krishnaveni Palaniappan, Neha Varghese, Natalia Mikhailova, Dimitrios Stamatis, T.B.K. Reddy, Natalia Ivanova, Nikos Kyrpides, Tanja Woyke, and Nicole Shapiro for the genome sequencing and annotation at JGI-DOE, USA.

Author contributions

I-AH: methodology, validation, formal analysis, investigation, data curation, writing—original draft, visualization. FU-RR: methodology, validation, formal analysis, investigation, data curation, writing—original draft, visualization. KD-LC: methodology, validation, formal analysis, writing-reviewing & editing. V-LS: formal analysis, review & editing. MSV-M: funding acquisition, writing—review & editing. WBW: funding acquisition, writing—review & editing. JA-I: investigation, writing—review & editing. P-ES: conceptualization, validation, formal analysis, investigation, resources, data curation, writing—original draft, writing—reviewing & editing, visualization, supervision, project administration, funding acquisition.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Ivan Arroyo-Herrera and Fernando Uriel Rojas-Rojas both authors contributed equally to the work.

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