Cupriavidus alkaliphilus sp. nov., a new species associated with agricultural plants that grow in alkaline soils

Systematic and Applied Microbiology 35 (2012) 310–314

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Cupriavidus alkaliphilus sp. nov., a new species associated with agricultural plants that grow in alkaline soils Paulina Estrada-de los Santos ∗ , Lourdes Martínez-Aguilar, Isabel M. López-Lara, Jesús Caballero-Mellado Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Ap. Postal 565-A, Cuernavaca, Morelos, Mexico

a r t i c l e

i n f o

Article history: Received 24 November 2011 Received in revised form 4 April 2012 Accepted 25 May 2012 Keywords: Cupriavidus Soil pH Rhizosphere Metal resistance Alkaline

a b s t r a c t A group of 20 bacterial strains was isolated from the rhizosphere of different agricultural plants growing in alkaline soils in the northeast of Mexico. The phylogenetic analysis of the 16S rRNA gene sequence from four strains showed that this novel group belonged to the Cupriavidus genus, with C. taiwanensis (∼98.9%) and C. necator (∼98.8%) as the closest species. However, DNA–DNA reassociation values were less than 20%. The novel group did not fix nitrogen and lacked nifH and nodA genes, unlike C. taiwanensis. Wholecell protein patterns were highly similar among the 20 strains but different from the closest Cupriavidus species. BOX-PCR patterns were distinct among the 20 strains but also differed from other Cupriavidus type species. The major cellular fatty acids from strains ASC-732T and SLV-2362 were C16:0 , C18:1 ␻7c/12t/9t and C16:1 ␻7c and/or C15:0 iso 2OH. The major polar lipids consisted of phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, 2-hydroxylated-phosphatidylethanolamine and an unknown aminolipid. The DNA G+C content of strain ASC-732T was 66.8 mol%. All 20 strains grew in the presence of 5–10 mg mL−1 arsenic, 1 mg mL−1 zinc, and 0.1 mg mL−1 copper. Consequently, the group of strains was considered to represent a novel species for which the name Cupriavidus alkaliphilus sp. nov. is proposed. The type strain is ASC-732T (=LMG 26294T = CIP 110330T ). © 2012 Elsevier GmbH. All rights reserved.

Introduction Ralstonia was proposed in 1995 to allocate Burkholderia pickettii, B. solanacearum and Alcaligenes eutrophus [43]. In 2004, Ralstonia species were divided in two sublineages, Ralstonia (sensu stricto) and the new genus Wautersia [40]. However, further analysis showed that W. eutropha was a synonym of Cupriavidus necator. Since C. necator was the oldest legitimate name, Wautersia species were transferred to the Cupriavidus genus [39] and, currently, the genus Cupriavidus is comprised of 13 species, with C. necator as the type species of the genus [39]. Recently, Wautersia numazuensis was also transferred to the genus Cupriavidus [25]. Cupriavidus species are inhabitants of diverse environments, such as soil, water and legume nodules, but they are also found in human medical samples [7,11,12,22,30]. C. taiwanensis is a plant-associated species that has been found forming nodules on legume plants [7]. Other

Abbreviations: BCCM/LMG, Belgian coordinated collections of microorganisms/LMG bacteria collection; CIP, Collection de l’Institut Pasteur. ∗ Corresponding author. Present address: Laboratorio de Microbiología Agrícola, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Plan de Ayala y Carpio, México, D.F., Mexico. E-mail address: [email protected] (P. Estrada-de los Santos). 0723-2020/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2012.05.005

Cupriavidus species-nodulating strains, phylogenetically related to C. taiwanensis, have been isolated from different Mimosa species growing in distinct regions of China, Costa Rica, Taiwan, Papua New Guinea and Texas [1,3,8,17,24]. Recently, C. pampae was isolated from an agricultural soil planted with alfalfa and other pasture species [13]. This Cupriavidus species was described as a herbicide degrading bacteria since it could use 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) as a sole carbon source. Like C. pampae, other Cupriavidus species are able to degrade toxic compounds or grow in the presence of heavy metals. For example, some Cupriavidus species are able to grow on media containing phenol or trichloroethylene (TCE) and can utilise different chlorophenols as sole carbon sources [9,33,47]. Furthermore, C. basilensis and C. campinensis were isolated from a zinc desert in Belgium [22]. C. metallidurans strain CH34T can grow in the presence of copper, chromium, mercury, nickel, silver, cadmium, cobalt, lead and zinc [27]. It also carries arsenite/arsenate-resistance genes [46] and, to date, this bacterium carries the largest number of genes encoding resistance to heavy metals. During a survey of Burkholderia species from agricultural soils in the northeast of Mexico, 47 Cupriavidus strains were isolated from the rhizosphere of maize, sugarcane, sorghum and agave plants growing in alkaline soils [19]. The strains were grouped by restriction analysis of the 16S rRNA gene (ARDRA). A partial taxonomic

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Table 1 Cupriavidus alkaliphilus strains isolated from distinct locations in Tamaulipas state in the northeast of Mexico. Strain

Plant

Source

Locality

Soil pH

ASC-634a ASC-743A1a , ASC-869c ASC-732Tc , ASC-738c , ASC-743c ASC-450a , ASC-15db , ASC-324db , ASC-327db CAG-122c MtRBr-3211b , MtRBr-3212b MLR2-44b MA1-22b SrRBr-232 SLV-2361c , SLV-2362c , SLV-2431c SNB1-3bc

Agave Agave Agave Agave Sugarcane Maize Maize Maize Sorghum Sorghum Sorghum

Rhizosphere Rhizosphere Rhizosphere Rhizosphere Rhizosphere Rhizosphere Rhizosphere Rhizosphere Rhizosphere Rhizosphere Rhizosphere

San Carlos San Carlos San Carlos San Carlos Gonzalez Rio Bravo La Rosita Abasolo Rio Bravo Los Vergeles Nicolas Bravo

9.0 8.9 6.5 ND 8.4 8.5 9.0 8.4 8.5 7.9 8.9

ND: not determined. a,b,c , isolation method (a: Az; b: BAz-Az; c: BAz-BAc). Locality coordinates: Rio Bravo, N 26◦ 00 26.7 , O 98◦ 10 01.28 ; Los Vergeles, N 24◦ 55 42.8 , O 97◦ 36 40.43 ; La Rosita, N 24◦ 36 59.22 , O 98◦ 22 57.42 ; San Carlos, N 24◦ 34 40.8, O 98◦ 56 38.36 ; Abasolo, N 24◦ 03 21.12 , O 98◦ 22 24.04 ; González, N 22◦ 49 42 , O 98◦ 25 46 ; Nicolás Bravo, N 22◦ 59 58.85 , O 98◦ 46 07.26 .

analysis of ARDRA profile 2, comprised of 20 strains, suggested the presence of a new Cupriavidus species. Further polyphasic taxonomy studies carried out in this work revealed that this group of strains actually belonged to a novel Cupriavidus species, for which the name Cupriavidus alkaliphilus sp. nov. is proposed.The group of 20 isolates is listed according to the source of isolation and soil pH in Table 1. The 16S rRNA gene sequence from four isolates (ASC-732T , CAG-122, SLV-2362 and SrBRr-232), randomly selected according to the plant isolation source and location, were amplified with the universal primers fD1/rD1 [41], as previously described [18]. The fragments were cloned into the pCR2.1 vector, according to the manufacturer’s instructions (Invitrogen). The sequencing of the fragments was performed at Macrogen (www.macrogen.com). A phylogenetic analysis, estimated with the maximum likelihood method, compared the novel isolates with type species of Cupriavidus and Ralstonia. The analysis unambiguously confirmed their allocation to the genus Cupriavidus (Fig. 1). The four strains shared more than 99.4% 16S rRNA gene sequence identity and the closest species, using the EzTaxon server 2.1 (http://www.eztaxon.org/), were C. taiwanensis LMG 19424T (98.9 ± 0.3%, accession number AF300324) and C. necator LMG 8453T (98.8 ± 0.1%, accession number CP002878), although there was high similarity with other Cupriavidus type species (Table 2). This high similarity seemed to be a general feature among Cupriavidus species (Table S1). The SDS-PAGE protein patterns from the 20 strains and a set of Cupriavidus type strains gathered from the BCCM/LMG, which

included C. taiwanensis LMG 19424T and the type species of the genus C. necator LMG 8453T , both being the closest species by 16S rRNA gene sequence analysis, and other Cupriavidus type species, selected randomly (according to the phylogenetic tree based on the 16S rRNA gene sequence analysis), were performed as previously described [18]. The protein patterns from novel strains were highly similar but clearly distinct from other Cupriavidus type species, including C. taiwanensis and C. necator (Fig. S1). It is known that bacteria with identical or very similar protein patterns have high levels of genome similarity but the similarity diminishes when the protein patterns are different [37]. Consequently, the protein patterns shown in this analysis confirmed that the group of novel strains represented a new species of Cupriavidus. Since the 20 strains showed homogeneous protein patterns, the intragenic diversity from the entire group of strains was tested by BOX-PCR and compared to the Cupriavidus type species set. The BOX element (BOXA1) was amplified according to the previous description [19]. The analysis showed different BOX-PCR profiles among the strains of this novel group, indicating a highly heterogeneous group (Fig. S2). As expected, the BOX-PCR patterns also differed from those of Cupriavidus type species. Even though it has been stated that DNA fingerprinting methods have a limited value for species descriptions [35], in this study it was important to reveal that the novel group of strains were not members of a single clone. Moreover, BOX profile heterogeneity has been documented in other Cupriavidus groups [24].

Table 2 16S rRNA gene sequence similarities and DNA–DNA reassociation values among Cupriavidus alkaliphilus and the type strains of species of Cupriavidus. Cupriavidus species

C. alkaliphilus ASC-732T C. alkaliphilus SLV-2362 C. alkaliphilus CAG-122 C. alkaliphilus SrBRr-232 C. necator LMG 8453T C. taiwanensis LMG 19424T C. numazuensis TE26T C. pinatubonensis LMG 23994T C. oxalaticus LMG 2235T C. gilardii LMG 5886T C. basilensis LMG 18990T C. pampae CPDB6T C. pauculus LMG 3413T C. respiraculi LMG 21510T C. campinensis LMG 19282T C. laharis LMG 23954T C. metallidurans CH34T

16S similarity with

DNA–DNA reassociation values with

C. alkaliphilus ASC-732T

ASC-732T

SLV-2362

100 99.3 99.3 99.3 98.7 98.6 98.2 97.8 97.7 97.9 97.8 97.6 97.5 97.6 97.4 97.4 97.4

100 71.2 93.4 72.3 19.7 14.5 26.3 27.5 12.2 37.8 34.9

92.3 100 85.3 88.3 14.8 32.3 18.0 21.5

The 16S similarity values were gathered from the EzTaxon database [10].

25.7 28.8

36.8

C. necator LMG 8453T 43.2 48.1

C. taiwanensis LMG 19424T 53.7 52.4

100 100

312

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C. alkaliphilus ASC-732T (HQ438078)

82

C. alkaliphilus SLV-2362 (HQ438080)

86 86

C. alkaliphilus SrBRr-232 (HQ438079) C. alkaliphilus CAG-122 (HQ438081)

77

T

C. taiwanensis LMG 19424 (AF300324) 89

C. oxalaticus LMG 2235T (AF155567)

90

C. necator LMG 8453T (AF191737) C. basilensis LMG 18990T (AF312022) C. numazuensis TE26T (AB104447)

91

99

C. laharis LMG 23954T (AB054961) 97

C. pinotubonensis LMG 23994T (AB121221)

C. respiraculi LMG 21510T (AF500583) C. gilardii LMG 5886T (AF076645)

92

C. pampae CPDB6 (FN430567) C. metallidurans CH34T (D87999) C. pauculus LMG 3413 (AF085226)

94

C. campinensis LMG 19282T (AF312020) 86

Characteristic

1

2

3

4

5

Nitrite reduction Presence ofb nifH gene nodA gene Activity of Arginine dihydrolase Urease Catalase Assimilation of N-acetyl glucosamine Trisodium citrate Erythritol Phenyl acetic acid Fructose

+

va

−a

−

va

− −

+ +

− −

nd nd

− −

− − +

+ va +

+a +a +

+ + +

+a +a +

− − − + +

− + − + −

+ + − + −

− + − + −

− + + + +

Strains: 1, C. alkaliphilus (ASC-732T , SLV-2362, CAG-122, SrBRr-232); 2, C. taiwanensis LMG 19424T ; 3, C. necator LMG 8453T ; 4, C. numazuensis TE26T ; 5, C. metallidurans CH34T .+, positive reaction (given when 3 or 4 strains were positive); −, negative reaction (given when 3 or 4 strains were negative); v, the strain showed variable results in independent analysis. a The result is different from other descriptions (Table S2). b All 20 strains were analysed. For nitrite reduction, strain SRBr-232 was negative. For arginine dihydrolase and urease activity strain ASC-732T gave a variable result in independent experiments.

T

73

Table 3 Differential phenotypic characteristics of Cupriavidus alkaliphilus sp. nov. and the closest Cupriavidus species.

R. pickettii LMG 5942T (X67042) R. insidiosa LMG 21421T (AF488779) R. mannitolilytica LMG 6866T (AJ270258)

100 88

T

R. solanacearum LMG 2299 (X67036) R. syzigii LMG 10661T (AB021403)

0.05

Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences of Cupriavidus and Ralstonia species. The multiple alignments of the sequences were performed with Muscle 3.57 [15]. The phylogenetic analysis was performed with maximum likelihood (ML) using the PhyML program [23]. Among-site rate variation was modelled by a gamma distribution with four-rate categories [44], with each category being represented by its mean. Tree searches were initiated from a BioNJ seed tree, retaining the best tree among those found with SPR (Subtree Pruning and Regrafting). The robustness of the ML topologies was evaluated using a Shimodaira-Hasegawa (SH)-like test [2]. The tree was visualised using the programme Mega version 5 [34]. The bar represents the number of expected substitutions per site under the GTR+G model.

The genomic DNA from strains ASC-732T , CAG-122, SLV-2362, SrBRr-232 and the closest Cupriavidus type species was extracted from liquid cultures grown in LB according to a previous description [18]. The DNA–DNA reassociation values performed in duplicate, according to Estrada-de los Santos et al. [18], between ASC-732T and other novel strains, were higher than 70% (Table 2), indicating a relationship at the species level [32,37]. The divergent DNA–DNA reassociation values among the novel strains (93.4, 72.3 and 71.1%; Table 2) have been reported previously in C. taiwanensis and C. pauculus strains [7,38]. In contrast, the value between strains ASC-732T and SLV-2362 with Cupriavidus type species was less than 40%, even with those very close to the novel group (Table 2). Additionally, the DNA–DNA reassociation values using C. taiwanensis and C. necator as probes against strains ASC-732T and SLV-2362 was low (Table 2), although they were higher than the reciprocal results with strains ASC-732T and SLV-2362 as probes, possibly as a result of the genomic content of the Cupriavidus species. These results confirmed that the group of strains could be delineated as a novel species of Cupriavidus. Subsequently, the phenotypic features of the novel strains were analysed. The 20 strains growing on LB agar plates produced colonies 2–3 mm in diameter after 48 h at 29 ◦ C. Colonies were whitish, creamy, glistening, with irregular margins and extended on the surface of the media after 4 days.

To determine the biochemical properties, strains ASC-732T , CAG-122, SLV-2362, SrBRr-232 and the closest Cupriavidus species, were precultured in liquid LB at 29 ◦ C for 16 h. The strains were analysed using different biochemical tests with the API 20NE system and by the assimilation of distinct carbon sources with the API 50CH system, according to the manufacturer’s instructions (bioMérieux). The novel strains produced catalase and oxidase but not indole. Urease and arginine dihydrolase activity was negative but the type strain gave variable results in independent experiments. Aesculin and gelatine hydrolysis was negative. d-Glucose was not fermented. The result for the assimilation of different carbon sources is presented in the species description. The 20 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. All strains grew on MacConkey agar plates at 29, 37 and 42 ◦ C. Additionally, the group of strains was able to grow at 15, 29, 37 and 42 ◦ C on LB + 1% NaCl agar plates and at 46 ◦ C in LB liquid media. One strain (ASC-743) grew at up to 50 ◦ C in liquid media. The pH range of growth on LB plates at 29 ◦ C was from 6 to 11, with an optimum of 7–9, although two strains grew only between pH 6 and 7. A total of 65% of the strains, excluding strain ASC-732T , were able to produce siderophores on CAA-CAS media, in assays performed as previously described [6]. All strains were unable to grow on SACC medium containing 0.2% phenol [6] or on LB or BSE agar plates [18] with different concentrations (from 0.1 to 10 mg mL−1 ) of cobalt (as CoCl2 –6H2 O) but the strains grew in the presence of 5–10 mg mL−1 arsenic (as Na2 HAsO4 –7H2 O), 1 mg mL−1 zinc (as ZnSO4 –7H2 O) and 0.1 mg mL−1 copper (as CuSO4 –5H2 O). The phenotypic features of the novel strains were compared to those of the closest Cupriavidus type species. The differential characteristics among these species are shown in Table 3. However, some discrepancies were found between the results obtained in this study and the results published in the literature for Cupriavidus type species (Table S2). The phenotypic analysis performed in these publications included the API 20NE system, which is a highly standardised method. Therefore, the phenotypic differences found might be a consequence of the experiment handling in each laboratory, according to the published study. The cellular fatty acid composition of strains ASC-732T and SLV2362 was performed according to the recommendations of the commercial identification system MIDI (Microbial Identification

P. Estrada-de los Santos et al. / Systematic and Applied Microbiology 35 (2012) 310–314

System, Sherlock version 3.10, Inc., Delaware, US) at BCCM/LMG, using an Agilent Technologies 6890N GC gas chromatograph (Santa Clara, CA, USA). The identification of the fatty acids was carried out with the TSBA identification library version 5.0 (peak naming table) after cells were grown for 24 h at 28 ◦ C on TSA medium. The major fatty acids in strains ASC-732T and SLV-2362 were C16:0 , C18:1 ␻7c/12t/9t and C16:1 ␻7c and/or C15:0 iso 2OH. A comparison of the fatty acid profiles from the novel species and the rest of the Cupriavidus species is shown in Table S3. The novel strains ASC-732T , SLV-2362, CAG-122 and SrBRr-232, and the closest Cupriavidus species were grown in LB liquid media for 16 h at 29 ◦ C, and the polar lipids were extracted following the Bligh and Dyer [4] technique. The chloroform phase was used for lipid analysis on TLC plates by two-dimensional separation [20]. Total polar lipids were observed by spraying with ANS reagent (8-anilino-1-naphtalenesulfonic acid) [45] and iodine vapour [31]. Polar lipids were identified on the basis of their migration in combination with specific staining. Lipids containing amino groups and glycolipids were identified with ninhydrin and ␣-naphthol, respectively, as described previously [14,42]. The polar lipids consisted of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), a 2-hydroxylated-phosphatidylethanolamine (2-OH-PE), cardiolipin (CL) and an unknown aminolipid (Fig. S3). The identification of 2OH-PE was based on the same mobility and staining properties as 2-OH-PE from Burkholderia cenocepacia [21]. The DNA G+C content of strain ASC-732T was determined using the HPLC technique of Mesbah et al. [26] at BCCM/LMG. The DNA G+C content was estimated to be 66.8 mol%. The novel strains were closely related to C. taiwanensis (∼99.0% 16S identity), a species that forms nodules and fixes nitrogen in legume root plants. However, even though most of the isolates were isolated in a semisolid medium lacking nitrogen, nitrogenase activity (N2 fixation measured indirectly by the acetylene-reduction method [5]) was not found in pure cultures growing in N-free semisolid media [18] or in semisolid media with added yeast extract (data not shown), which has been found useful to express nitrogen fixation in some Burkholderia species [16]. The presence of nifH and nodA genes was demonstrated with primers IGK [29]/NDR1 [36] and nodAB1/nodAB2 [28], respectively, in C. taiwanensis LMG 19424T and B. phymatum STM815T but was absent in the 20 novel strains (data not shown). nifH and nodA genes from C. taiwanensis LMG 19424T were used as probes for hybridisation experiments with strains ASC-732T , CAG-122, SrBRr-232 and SLV-2362, according to Estrada-de los Santos et al. [18]. The analysis confirmed the absence of both genes in the novel strains, thus showing an important difference compared with C. taiwanensis. Therefore, taking these results together, the group of 20 strains in this study represented a new species of the genus Cupriavidus, for which the name C. alkaliphilus sp. nov. is proposed, with the type strain being strain ASC-732T (=LMG 26294T = CIP 110330T ).

313

acid, adipic acid, malic acid and phenyl acetic acid. The following tests are negative: assimilation of glycerol, erythritol, d- and l-arabinose, d-ribose, d- and l-xylose, d-adonitol, d-galactose, d-glucose, d-mannose, l-sorbose, l-rhamnose, dulcitol, inositol, trisodium citrate, d-mannitol, d-sorbitol, N-acetylglucosamine, amygdaline, arbutin, aesculin, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-saccharose, d-threalose, inulin, dmelezitose, d-raffinose, starch, glycogen, xylitol, gentiobiose, d-turanose, d-lyxose, d- and l-fucose, d- and l-arabitol, potassium 2-ketogluconate, potassium 5-ketogluconate, methyl-␣dglucopyranoside, methyl-␣d-mannopyranoside, trisodium citrate and methyl-d-xylopyranoside. Nitrite reduction was positive except for strain SRBr-232, but nitrates were not reduced. The major fatty acids in strains ASC-732T and SLV-2362 were C16:0 , C18:1 ␻7c/12t/9t and C16:1 ␻7c and/or C15:0 iso 2OH. The polar lipids for strains ASC-732T , SLV-2362, CAG-122 and SrBRr-232 consisted of PG, PE, 2-OH-PE, CL and an unknown aminolipid. All strains were able to grow in the presence of between 5 and 10 mg mL−1 arsenic, 1 mg mL−1 zinc and 0.1 mg mL−1 copper. Strains ASC-732T and SLV-2362 were deposited at BCCM/LMG and CIP with the following identifiers: LMG 26294T = CIP 110330T and LMG 26295 = CIP 110331, respectively. The type strain is ASC-732T (=LMG 26294T = CIP 110330T ), and it was isolated from the rhizosphere of an agave plant (Agave montium-sancticaroli, http://www.ibiologia.unam.mx/) growing in alkaline soils in San Carlos, Tamulipas, Mexico. Arginine dihydrolase and urease activity was variable for this strain. Cells are coccoid to small rods (0.8 ␮m × 1.0–1.5 ␮m), single or in pairs and nonmotile. The DNA G+C content of strain ASC-732T is 66.8 mol%. Acknowledgements We are indebted to Dr. J.P. Euzéby for the etymological construction of the novel species name given in this paper. We are grateful to Cuauhtémoc Jacques and Angel Salazar-Bravo (Centro de Biotecnología Genómica, Instituto Politécnico Nacional) for collecting the agave and sugarcane plants. We are thankful to Marco Antonio Rogel-Hernández and Rafael Díaz-Méndez (Centro de Ciencias Genómicas, UNAM) for technical support. We thank Dr. Otto Geiger (Centro de Ciencias Genómicas, UNAM) for his help in designing the polar lipid experiments and discussion of the results. We thank Dr. Mike Dunn (Centro de Ciencias Genómicas, UNAM) for reading the manuscript and making constructive corrections to the English. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.syapm. 2012.05.005 References

Description of C. alkaliphilus sp. nov. C. alkaliphilus: al.ka.li’phi.lus. N.L. n. alkali, alkali; N.L. adj. philus -a -um (from Gr. adj. philos -ê -on), friend, loving; N.L. masc. adj. alkaliphilus, liking alkaline media. The cells are Gram-negative and aerobic. The pH range for growth is from 6 to 11. Growth occurs from 15 to 46 ◦ C on LB agar plates or in liquid media. The group is able to grow in the presence of 0.5, 1.0 and 1.5% NaCl but not with 3.0%. Catalase and oxidase activities are present. Indole production, d-glucose fermentation, aesculin and gelatine hydrolysis are negative. Arginine dihydrolase and urease activity are negative but the type strain gave variable results in independent experiments. There is assimilation of d-fructose, potassium gluconate, capric

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