Cupriavidus plantarum sp. nov., a plantassociated species
Paulina Estrada-de los Santos, Roosivelt Solano-Rodríguez, Lucía Tomiko Matsumura-Paz, María Soledad Vásquez-Murrieta, et al. Archives of Microbiology ISSN 0302-8933 Volume 196 Number 11 Arch Microbiol (2014) 196:811-817 DOI 10.1007/s00203-014-1018-7
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Author's personal copy Arch Microbiol (2014) 196:811–817 DOI 10.1007/s00203-014-1018-7
ORIGINAL PAPER
Cupriavidus plantarum sp. nov., a plant‑associated species Paulina Estrada‑de los Santos · Roosivelt Solano‑Rodríguez · Lucía Tomiko Matsumura‑Paz · María Soledad Vásquez‑Murrieta · Lourdes Martínez‑Aguilar
Received: 19 March 2014 / Revised: 13 July 2014 / Accepted: 15 July 2014 / Published online: 7 August 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract During a survey of plant-associated bacteria in northeast Mexico, a group of 13 bacteria was isolated from agave, maize and sorghum plants rhizosphere. This group of strains was related to Cupriavidus respiraculi (99.4 %), but a polyphasic investigation based on DNA–DNA hybridization analysis, other genotypic studies and phenotypic features showed that this group of strains actually belongs to a new Cupriavidus species. Consequently, taking all the results together, the description of Cupriavidus plantarum sp. nov. is proposed. Keywords Cupriavidus · Rhizosphere · Plant-associated bacteria
Introduction The genus Cupriavidus was described in 2004 to allocate a sublineage of the genus Ralstonia sensu lato (Vaneechoutte et al. 2004; Vandamme and Coenye 2004). Currently,
Communicated by Erko Stackebrandt. Electronic supplementary material The online version of this article (doi:10.1007/s00203-014-1018-7) contains supplementary material, which is available to authorized users. P. Estrada‑de los Santos (*) · R. Solano‑Rodríguez · L. T. Matsumura‑Paz · M. S. Vásquez‑Murrieta Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Prolongacion de Carpio y Plan de Ayala s/n, Col. Santo Tomas, Delegacion Miguel Hidalgo, 11340 Mexico, DF, Mexico e-mail:
[email protected] L. Martínez‑Aguilar Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, 565‑A, Cuernavaca, Morelos, Mexico
the genus comprises 14 species, which are inhabitants of diverse ecological environments, such as soil, water, legume plant nodules and human clinical samples (Vandamme et al. 2004; Sato et al. 2006; Cuadrado et al. 2010; Estradade los Santos et al. 2012; Martínez-Aguilar et al. 2012). Particularly, some Cupriavidus species have been found in association with plants. This is the case of Cupriavidus taiwanensis, which is able to form nodules in legumes and strains related to this species have been isolated from different Mimosa species growing around the world (Chen et al. 2001, 2003; Barret and Parker 2006; Andam et al. 2007; Elliot et al. 2008; Liu et al. 2011). Also, C. necator nodulates leguminous plants (da Silva et al. 2012). C. pampae, though not directly isolated from a plant, was collected from an agricultural field where alfalfa and other pasture species were cultivated (Cuadrado et al. 2010). The latest species described in the genus, C. alkaliphilus, was found in the rhizosphere of agave, sugarcane, maize and sorghum plants growing at northeast of Mexico (Estrada-de los Santos et al. 2011, 2012). Additionally, some strains related to C. campinensis, a species originally isolated from soil, were found to be able to colonize torrefied (thermally treated) grass fibers, which represent a colonizable niche potentially useful to establish a beneficial interaction that improves plant growth (Trifonova et al. 2008). Besides the ability to fix nitrogen and nodulate legumes, Cupriavidus has other plant growth promotion activities. For example, Cupriavidus sp. can synthetize the indol acetic acid phytohormone (Pongsilp et al. 2012), C. necator can solubilize phosphate (Yu et al. 2011), and C. alkaliphilus can synthetize siderophores on culture media (Estrada-de los Santos et al. 2012). Moreover, the ability to grow in the presence of heavy metals has been shown on several Cupriavidus species. For instance, C. metallidurans CH34T successfully thrives in the presence of copper, chromium, mercury, nickel, silver, cadmium, cobalt,
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lead, zinc and arsenic (Monchy et al. 2007; Zhang et al. 2009). C. basilensis and C. campinensis were isolated from a zinc desert in Belgium (Goris et al. 2001). Furthermore, C. alkaliphilus was able to grow effectively in the presence of arsenic, copper and zinc (Estrada-de los Santos et al. 2012). Inoculation of Helianthus annuus (sunflower) with Ralstonia eutropha (now Cupriavidus necator) has been proposed as a phytoremediation strategy to reduce the toxicity of heavy metals to plants in polluted environments (Marques et al. 2013). Given the diverse features contained in different Cupriavidus species, it is interesting to study these characteristics on novel species. In 2008, we started a project to study the distribution of Burkholderia species associated to agricultural plants in northeast of Mexico. Interestingly, in addition to Burkholderia, other diverse isolates were found. The isolated bacteria belongs to the genus Cupriavidus, and among the strains Cupriavidus respiraculi was identified (Estradade los Santos et al. 2011). The analysis of these bacteria using Amplified rDNA Restriction Analysis, (ARDRA) showed different Cupriavidus groups, and C. alkaliphilus was recently described as a new species, referring to the soil alkalinity existing in the surveyed area (Estrada-de los Santos et al. 2012). Following this study, a second ARDRA group of Cupriavidus was further analyzed. The taxonomical analysis showed that this new group of strains belongs to a novel species, and consequently Cupriavidus plantarum sp.nov. is proposed.
Materials and methods Microorganisms and isolation source A group of 13 Cupriavidus strains was isolated from different plants at northeast of Mexico (Estrada-de los Santos et al. Table 1 Cupriavidus plantarum sp. nov. strains isolated from distinct location of Tamaulipas state in the northeast of Mexico Strain
Plant
Source
Locality
Soil pH
ASC-64T MA1-1, MA1-2a, MA1-4a, MA11za, MA1-2za, MA1-2zb, MA1-4z MA2-18b, MA218c, MA2-19b SLV-132
Agave Maize
Rhizosphere Rhizosphere
San Carlos Abasolo
9.0 8.4
SLV-2261
Maize
Rhizosphere
Abasolo
8.7
Sorghum
Rhizosphere
Los Vergeles
7.7
Sorghum
Rhizosphere
Los Vergeles
7.9
Locality coordinates: San Carlos N 24°34′40.8″, O 98°56′38.36″; Abasolo N 24°03′21.12″, O98°22′24.04″; Los Vergeles N 24°55´42.8″, O 97°36´40.43″
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2011). The isolation source is indicated in Table 1. Reference strains used in this study (C. respiraculi LMG 21510T, C. necator LMG 8453T, C. taiwanensis LMG 19424T, C. oxalaticus LMG 2235T and C. gilardii LMG 5886T) were gathered from the culture collection LMG/BCCM in Belgium. Phenotypic and biochemical analysis To determine the biochemical properties, the 13 isolates were precultured in liquid LB at 29 °C for 16 h. Then, all bacteria were analyzed using the API 20NE system and by the assimilation of distinct carbon sources with the API 50CH system, according to the manufacturer’s instructions (bioMérieux). Additionally, the strains were grown 72 h on (1) LB at 29 °C with 0.5, 1.0,1.5 and 3.0 % NaCl, (2) MacConkey agar plates at 29, 37 and 42 °C, (3) LB at 15, 29, 37 and 42 °C, (4) LB at pH from 4.5 to 12.5 at 29 °C, (5) the colony morphology was revised on LB and BSE agar plates (Estrada-de los Santos et al. 2001), grown 48 h at 29 °C, (6) SACC medium containing 0.2 % phenol (Caballero-Mellado et al. 2007), (7) BSE agar plates containing different concentrations (from 0.1 to 10 mg/mL) of cobalt (as CoCl2– 6H2O), arsenic (as Na2HAsO4–7H2O), zinc (as ZnSO4– 7H2O) and copper (as CuSO4–5H2O), (8) CAS-CAA medium for siderophore production, (9) NBRIP medium for phosphate solubilization (Caballero-Mellado et al. 2007), (10) Castañeda’s medium added with starch for amylolytic activity (Hernández-Montañez et al. 2012) and (11) SkimMilk (DIFCO) for proteolitic activity, (12) semisolid BSE and JMV (Reis et al. 2004) medium to test strains ability to fix nitrogen through acetylene reduction. All analyses were performed in duplicate. Motility assays were performed on semisolid LB, trypticase soy agar (TSA) and potato dextrose agar media with 0.25 % agar, as previously described (Ibarra et al. 2010). The 13 strains were overnight cultured on TSB media, and 5 μL was spotted in the middle of the semisolid media. Strain ASC-64T was overnight cultured on JP liquid media (Jain and Patriquin 1985), and transmission electron microscopy was performed as described previously (Ventura-Suarez et al. 2011). Chemotaxonomical analysis The SDS-PAGE protein patterns from the strains were performed growing the strains in LB medium with reciprocal shaking (200 rpm) for 15 h at 29 °C. After the incubation time, 1.0 mL sample was harvested by centrifugation at 12,300×g for 10 min. The pellets were 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 performed as described by Laemmli (1970). The fatty acid profiles were carried out at BCCM/LMG. In short, the cells were grown for 24 h at
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28 °C on TSA. Inoculation and harvesting of the cells and extraction and analysis of the fatty acids were performed according to the recommendations of the commercial identification system MIDI (Microbial Identification System, Inc., DE, USA). The whole-cell fatty acid composition was determined by gas chromatography. Genotypic analysis The 16S rRNA gene sequences (c.a. 1,500 bp) were amplified with the universal primers fD1/rD1 (Weisburg et al. 1991). The amplified 16S rRNA genes were directly sequenced at Macrogen (www.macrogen.com) using the primers fD1/rD1. A multiple alignment was performed with Muscle 3.57 (Edgar 2004), and a phylogenetic analysis was carried out with maximum likelihood (ML) using PhyML program (Guindon and Gascuel 2003). Among-site rate variation was modeled by a gamma distribution with four rate categories (Yang 1996), with each category being represented by its mean, under the GTR+G model. Tree searches were initiated from a Bio NJ seed tree, retaining the best tree among those found with Nearest Neighbor Interchange. The robustness of the ML topologies was evaluated using a Shimodaira–Hasegawa (SH)-like test (Anisimova and Gascuel 2006). ML trees were visualized with program MEGA version 5 (Tamura et al. 2011). Bacterial genomic DNA was extracted from liquid cultures grown in LB for the DNA–DNA hybridization (DDH) experiments. These experiments were performed twice as described previously (Estrada-de los Santos et al. 2001) using a DNA probe (from strain ASC-64T) labeled radioactively with P32. The DNA G+C content determination was carried out at BCCM/LMG. The strain was cultivated on TSA. Genomic DNA was extracted according to a modification of the procedure of Wilson (Wilson 1987). The DNA G+C content was determined using the HPLC technique (Mesbah et al.1989). The given value is the mean of two independent analyses. The BOX element (BOXA1) was amplified using the BOXA1R primers (Versalovic et al. 1994) according to a previous description (Estrada-de los Santos et al. 2011). The presence of nifH and nodA genes were analyzed with primers IGK (Poly et al. 2001)/NDR-1 (Valdes et al. 2005) and nodAB1/nodAB2 (Moulin et al. 2001), respectively. Burkholderia phymatum STM815T and C. taiwanensis LMG 19424T were used as positive control.
Results and discussion The 13 isolates were previously grouped by ARDRA (Estrada-de los Santos et al. 2011). Therefore, the 16S rRNA gene sequence from four isolates (ASC-64T, MA14a, MA1-1za and SLV-2261 with the accession numbers:
813
HQ438086, HQ438088, HQ438087 and HQ438089, respectively) was obtained and compared to all type species of Cupriavidus using a phylogenetic analysis estimated with the ML method. The results showed unambiguously the allocation of the novel species within the genus Cupriavidus (Fig. 1). The novel-analyzed strains formed a robust group closely related to C. respiraculi LMG 21510T. The intraspecies similarity of the novel species was 99.8 %, and the average similarity of the novel species with C. respiraculi LMG 21510T (AF500583), C. metallidurans CH34T (D87999), C. necator LMG 8453T (AF191737), C. taiwanensis LMG 19424T (AF300324) and C. alkaliphilus ASC-732T (HQ438078) was 99.4, 98.8, 98.2, 98.6 and 97.7 %, respectively. We previously found that there is a high 16S rRNA gene sequence similarity among Cupriavidus species (Estrada-de los Santos et al. 2012), and the novel species also shares this feature. To show the similarity of the 13 isolates, a SDS-PAGE was carried out. The protein patterns from novel strains were compared to a set of type strains of Cupriavidus species, including C. necator LMG 8453T, C. respiraculi LMG 21510T, C. taiwanensis LMG 19424T, C. oxalaticus LMG 2235T and C. gilardii LMG 5886T. The analysis revealed highly similar protein patterns, which were completely different from the closest species C. respiraculi and other type strains of Cupriavidus species (Suppl. Fig. S1). It is known that bacteria with identical or very similar protein patterns have high levels of genome similarity, but this similarity diminishes when the protein patterns are different (Vandamme et al. 1996). Consequently, the results suggested that the group of 13 novel strains represents a new Cupriavidus species. The genetic diversity of the isolates was further examined by BOX-PCR. The amplification products yielded different patterns consisting of fragments in size from 200 to 2,000 bp indicating a highly heterogeneous group (Suppl. Fig. S2). As it was anticipated, the BOX-PCR patterns were completely different from Cupriavidus type species. Previously, it was stated that DNA-fingerprinting methods have a limited value for species description (Tindall et al. 2010); however, in this study, it was important to show that the novel group of strains was not a single clone. Besides, the genomic heterogeneity of strains by the BOX approach has been documented in other Cupriavidus groups (Liu et al. 2011; Estrada-de los Santos et al. 2012). The analysis of the total genome by DDH experiments to delineate the new species from related species showed that strains ASC-64T shares 94.2 % similarity to MA1-4a, MA1-1za and SLV2261 (88.3 % with MA1-4a, 96.0 % with MA1-1za and 98.3 % with SLV-2261), showing a relationship at the species level. However, the DDH values between strain ASC64T and type strains of Cupriavidus species was less than 70 % which is the cutoff value to define a new species (Tindall et al. 2010): 25.9 % with C. respiraculi, 34.6 % with C.
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Arch Microbiol (2014) 196:811–817 C. plantarum MA1-4a (HQ438088)
66 95
C. plantarum MA1-1za (HQ438087) C. plantarum SLV-2261 (HQ438089)
67
C. plantarum ASC-64T (HQ438086) C. respiraculi LMG 21510T (AF500583)
76
C. metallidurans CH34T (D87999) 84
81
C. pauculus LMG 3413 (AF085226) C. pampae CPDB6T (FN430567) C gilardii LMG 5886T (AF076645)
79
C. taiwanensis LMG 19424T (AF300324) C. necator LMG 8453T (AF191737)
99 94
C. necator LMG 1199 (M32021) C. oxalaticus LMG 2235T (AF155567) C. alkaliphilus ASC-732T (HQ438078)
84
C. basilensis LMG 18990T (AF312022) C. laharis LMG 23954T (AB054961)
53
C. pinotubonensis LMG 23994T (AB121221)
99
C. campinensis LMG 19282T (AF312020) 99
R. insidiosa LMG 21421T (AF488779) R. pickettii LMG 5942T (X67042) T
R. mannitolilytica LMG 6866 (AJ270258)
99
R. solanacearum LMG 2299T (X67036)
97 92
R. syzigii LMG 10661T (AB021403)
0.005
Fig. 1 Phylogenetic tree based on the alignment of 16S rRNA gene sequences of Cupriavidus and Ralstonia species. The bar represents the number of expected substitutions per site under the GTR+G model
taiwanensis, 20.6 % with C. metallidurans, 20.4 % with C. necator and 27.5 % with C. alkaliphilus. This result confirmed that the group of strains could be defined as a novel species of Cupriavidus. Accordingly, the phenotypic properties from the 13 strains were tested. All had uniform colony morphology; on BSE agar plates, colonies were round, 1–2 mm diameter, whitish in the center and transparent in margins with entire borders. On LB agar plates, colonies were round, convex, 1–2 mm in diameter and creamy color with entire margins. The strains were able to grow on LB at 29 °C with 0.5, 1.0 and 1.5 % NaCl, but not with 3.0 % or higher concentrations. They grew on MacConkey agar plates at 29, 37 and 42 °C and on LB at 15, 29, 37 and 42 °C. All strains grew from pH 4.5 to 11.0, and only two (MA14z and MA1-4a) were able to grow on phenol; all strains lack the ability to fix nitrogen in culture media, and nifH and nodA genes were not found, a feature present in both control species B. phymatum and C. taiwanensis (data not shown). To determine the biochemical properties, the 13 strains were tested with the API 20NE and API 50CH systems. There was assimilation of potassium gluconate, capric acid, adipic acid, malic acid and weakly of trisodium citrate, but the strains were unable to grow on other
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carbon sources listed in Suppl. Table S1. Fermentation of d-glucose was negative. The 13 strains were unable to solubilize phosphate, synthesize very low amounts of IAA at 48 and 72 h (around 1 μg/mL) and produce siderophores halos from 8 to 13 mm diameter. The strains were unable to degrade casein and starch. The ability to grow with increasing metal concentrations was dependent upon the type of media used for bacterial growth. The resistance to zinc and arsenic was more evident on BSE medium and the resistance to copper and cobalt was higher on LB medium. This effect has been previously observed in C. metallidurans CH34T (Mergeay et al. 1985). The medium composition has strong effects on the ability of this strain to resist metals. The differential features between the novel strain and other Cupriavidus species is shown in Table 2. The major cellular fatty acids for strains ASC-64T and MA1-4a were C16:0 (23.6 %), C17:0 cyclo (19.9 %), C18:1 w7c (19.5 %); summed feature 3 corresponds to C16:1 w7c and/or C15:0 ISO 2-OH (13.5 %), and summed feature 2 corresponds to C14:0 3-OH and/or 16:1 ISO I, an unidentified fatty acid with equivalent chain length value of 10.928, 12:0 ALDE or any five combination of these fatty acids (9.6 %) (Suppl. Table S1).
Author's personal copy Arch Microbiol (2014) 196:811–817 Table 2 Differential phenotypic characteristics of C. plantarum sp. nov. with other Cupriavidus species
Strains: 1, C. plantarum sp. nov. (n = 13); 2, C. taiwanensis LMG 19424T; 3, C. oxalaticus LMG 2235T; 4, C. respiraculi LMG 21510T; 5, C. metallidurans CH34T; 6, C. necator LMG 8453T; 7, C. alkaliphilus ASC-732T + Positive reaction
− Negative reaction w weak reaction
a Bacterial growth in the presence of metal in mg/ mL, arsenic and zinc on BSE medium, cobalt and copper on LB medium, results from Estrada-de los Santos et al. (2011) b
Diameter of the halo given in mm by subtracting colony diameter from the total diameter, for the novel species the number is the average from all strains, each tested twice c
Two strains gave a positive reaction (MA2-19b and SLV132)
815 Characteristic
1
2
3
4
5
6
7
+ + +
+ + +
w + −
+ + +
+ − −
+ + +
+ + +
+ +
+ +
+ +
− −
+ +
+ +
10 1.0 0.1 −
1.0 1.0 0.5 −
1.0 − 0.5 −
10 1.0 0.5 −
10 10 0.1 1.0
1.0 − 0.5 −
5.0 1.0 0.1 −
− + +
− − +
+ + −
+ + +
+ + +
+ + +
− − +
N-acetyl glucosamine Trisodium citrate Erythritol Phenyl acetic acid Fructose Nitrite reduction Nitrate reduction Presence of:
−
w − − − − –c
−
+ − + − − +
−
+ − + − − −
−
− − − − − −
−
− + + + + +
+
+ − + − − −
−
− − + + + +
nifH gene
−
+
−
−
−
−
−
10.2
9.0
nd
5.0
3.0
8.0
6.0
Growth on MacConkey at 29 °C 37 °C 42 °C
Growth on LB + 1 % NaCl at: 37 °C + 42 °C +
Growth with metalsa Arsenic Zinc Copper Cobalt
Enzyme activity Arginine dihydrolase Urease Catalase Assimilation of
nodA gene Siderophore synthesisb
−
Type strain ASC-64 showed flagella, but mostly the appendages were lost (Suppl. Figure S3). Moreover, all strains were motile on semisolid agar plates (Suppl. Fig. S4). The DNA G+C content of strain ASC-64T was 65.7 mol %, and the given value is the mean of two independent analysis. According to the taxonomical properties presented in this study, the group of 13 strains represents a new Cupriavidus species; therefore, C. plantarum sp. nov., is proposed.
+
−
−
−
−
−
at 15–42 °C, up to 1.5 % NaCl and at pH 4.5–11.0. The strains lack the ability to fix nitrogen, degrade casein and starch and solubilize phosphate. Catalase and oxidase is produced, but no indole. Cells are negative for gelatin and aesculine hydrolysis. The most abundant fatty acids were C16:0, C17:0 cyclo and C18:1 w7c. The G+C content of the type strain is 65.7 mol %. ASC-64 type strain was isolated from agave rhizosphere at San Carlos, Tamulipas, Mexico, and it was deposited in BCCM/LMG as LMG 26296T and CIP as CIP 110328T.
Description of Cupriavidus plantarum sp. nov Cupriavidus plantarum : plan’ta.rum. L. fem. n. planta, any vegetable production that serves to propagate the species, a plant; L. gen. pl. n. plantarum, of plants. Cells are strictly aerobic, Gram-negative, non-sporeforming, coccoid to small rods (0.8 μm × 1.1–1.5 μm), single, pairs and motile. The colony morphology was uniform 1–2 mm diameter, creamy with entire margins in LB medium. Grows on potassium gluconate, capric acid, adipic acid, malic acid and weakly on trisodium citrate. Grows
Acknowledgments We are indebted to Dr. J.P. Euzeby for the ethymology of the novel species name given in the paper. We are grateful to Cuauhtémoc Jacques and Angel Salazar Bravo (Centro de Biotecnología Genómica, IPN) for collecting the agave plants. We are thankful to Marco Antonio Rogel-Hernández and Rafael Diaz-Méndez for technical support (Centro de Ciencias Genómicas, UNAM). We thank Dr. E.O. Lopez-Villegas and M.R. Espinoza-Mellado (Escuela Nacional de Ciencias Biolgicas, IPN) for transmission electron microscopy analysis. We are always grateful to Jesús CaballeroMellado for the teaching and invaluable time discussing about bacterial taxonomy and plant-associated bacteria. RSR was supported by a MSc fellowship from Consejo Nacional de Ciencia y Tencología
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Author's personal copy 816 (CONACyT). This research was partially funded by grant SIP 20130940 and SIP 2014-0353.
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