International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2338–2345
DOI 10.1099/ijs.0.063560-0
Pseudomonas helmanticensis sp. nov., isolated from forest soil Martha-Helena Ramı´rez-Bahena,1,2 Maria Jose´ Cuesta,1 Jose´ David Flores-Fe´lix,3 Rebeca Mulas,4 Rau´l Rivas,2,3 Joao Castro-Pinto,5 Javier Bran˜as,5 Daniel Mulas,5 Fernando Gonza´lez-Andre´s,4 Encarna Vela´zquez2,3 and A´lvaro Peix1,2 Correspondence A´lvaro Peix [email protected]
1
Instituto de Recursos Naturales y Agrobiologı´a, IRNASA-CSIC, Salamanca, Spain
2
Unidad Asociada Grupo de Interaccio´n Planta-Microorganismo, Universidad de Salamanca-IRNASA (CSIC), Salamanca, Spain
3
Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca, Salamanca, Spain
4
Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de Leo´n, Leo´n, Spain
5
Fertiberia S. A., Madrid, Spain
A bacterial strain, OHA11T, was isolated during the course of a study of phosphate-solubilizing bacteria occurring in a forest soil from Salamanca, Spain. The 16S rRNA gene sequence of strain OHA11T shared 99.1 % similarity with respect to Pseudomonas baetica a390T, and 98.9 % similarity with the type strains of Pseudomonas jessenii, Pseudomonas moorei, Pseudomonas umsongensis, Pseudomonas mohnii and Pseudomonas koreensis. The analysis of housekeeping genes rpoB, rpoD and gyrB confirmed its phylogenetic affiliation to the genus Pseudomonas and showed similarities lower than 95 % in almost all cases with respect to the above species. Cells possessed two polar flagella. The respiratory quinone was Q9. The major fatty acids were C16 : 0, C18 : 1v7c and summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH). The strain was oxidase-, catalase- and urease-positive, positive for arginine dihydrolase but negative for nitrate reduction, b-galactosidase production and aesculin hydrolysis. It was able to grow at 31 6C and at pH 11. The DNA G+C content was 58.1 mol%. DNA–DNA hybridization results showed values lower than 49 % relatedness with respect to the type strains of the seven closest related species. Therefore, the combined genotypic, phenotypic and chemotaxonomic data support the classification of strain OHA11T to a novel species of the genus Pseudomonas, for which the name Pseudomonas helmanticensis sp. nov. is proposed. The type strain is OHA11T (5LMG 28168T5CECT 8548T).
Many species of bacteria are able to solubilize inorganic phosphates in vitro, and some of them can mobilize phosphorus to plants (Antoun et al., 1998; Peix et al., 2001; Kaur & Reddy, 2013). Within soil microbiota, the groups of pseudomonads, bacilli and rhizobia are considered the most effective phosphate solubilizers (Rodrı´guez & Fraga, 1999). The genus Pseudomonas includes several species reported as phosphate-solubilizing bacteria, some of which were isolated from soil, such as Pseudomonas rhizosphaerae (Peix et al., 2003), Pseudomonas lutea (Peix et al., 2004) or The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, rpoD, rpoB and gyrB gene sequences of strain OHA11T are HG940537, HG940517, HG940518 and HG940516, respectively. One supplementary table and one supplementary figure are available with the online version of this paper.
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the recently described Pseudomonas guariconensis (Toro et al., 2013). During a study of phosphate-solubilizing rhizospheric bacteria in soils from northern Spain, we isolated a strain (OHA11T) in a forest soil located in Salamanca province that is able to produce a large transparent ‘halo’ surrounding its colonies in media containing insoluble bicalcium phosphate as the phosphorus source, and subjected this strain to a polyphasic taxonomic study. The isolation procedure was the same as described by Peix et al. (2003). Briefly, 10 g of rhizospheric soil was suspended in 90 ml sterile water and stirred for 30 min. From this suspension, 100 ml was spread on YED-P medium and incubated at 28 uC for 7 days. The isolate formed a transparent ‘halo’ around the colonies, indicating in vitro phosphate solubilization activity. This strain was classified to the genus Pseudomonas within the Gammaproteobacteria based on 16S rRNA and 063560 G 2014 IUMS Printed in Great Britain
Pseudomonas helmanticensis sp. nov.
housekeeping gene sequence analyses, and the phylogenetic, chemotaxonomic and phenotypic data obtained showed that it represents a novel species. Cells were stained according to the Gram procedure described by Doetsch (1981). Motility was checked by phase-contrast microscopy after growing cells in nutrient agar medium at 22 uC for 48 h. The flagellation type was determined by electron microscopy after 48 h of incubation on tripticase soy agar (TSA; Becton Dickinson, BBL) at 22 uC as previously described (Rivas et al., 2007). Strain OHA11T was Gram-stain-negative, rod-shaped (0.6–0.86 1.6–2.0 mm) and motile by means of two polar flagella (Fig. S1, available in the online Supplementary Material). Cells grew as round translucent beige to yellowish colonies on nutrient agar. For 16S rRNA gene sequencing and comparison analysis, DNA extraction, amplification and sequencing were performed as reported by Rivas et al. (2007). Amplification and partial sequencing of the gyrB, rpoB and rpoD housekeeping genes were performed as described by Mulet et al. (2010), using the primers PsEG30F (59-ATYGAAATCGCCAARCG-39) and PsEG790R (59-CGGTTGATKTCCTTGA-39) for the rpoD gene (Mulet et al., 2009), LAPS5F (59-TGGCCGAGAACCAGTTCCGCGT-39) and LAPS27R (59-CGGCTTCGTCCAGCTTGTTCAG-39) for the rpoB gene (Tayeb et al., 2005), and GyrBPUN1F (59-AAGGAGCTGGTGYTGACC-39) and GyrBPUN1R (59-GCGTCGATCATCTTGCCG-39) for the gyrB gene (Ramos et al., 2013). The sequences obtained (1472 nt for 16S, 721 nt for rpoD, 1125 nt for rpoB and 713 nt for gyrB) were compared with those from GenBank using the BLASTN (Altschul et al., 1990) and EzTaxon (Kim et al., 2012) programs and levels of similarity were calculated after pairwise comparison. For phylogenetic analysis, sequences were aligned using the CLUSTAL X software (Thompson et al., 1997). Distances were calculated according to Kimura’s two-parameter model (Kimura, 1980). Phylogenetic trees based on 16S rRNA gene sequences were reconstructed using the neighbourjoining (Saitou & Nei, 1987) and maximum-likelihood (Rogers & Swofford, 1998) methods. The MEGA5 software (Tamura et al., 2011) was used for all analyses. Comparison of the 16S rRNA gene sequence of strain OHA11T against the type strains of bacterial species recorded in the EzTaxon-e database showed the affiliation of the novel strain to the genus Pseudomonas. The closest relative was Pseudomonas baetica a390T (Lo´pez et al., 2012) with 99.1 % pairwise similarity (13 nt difference), and the next closest relatives with 98.9 % similarity (15–17 nt differences) were the type strains of Pseudomonas jessenii (Verhille et al., 1999), Pseudomonas moorei, Pseudomonas mohnii (Ca´mara et al., 2007), Pseudomonas umsongensis and Pseudomonas koreensis (Kwon et al., 2003). The type strain of Pseudomonas reinekei (Ca´mara et al., 2007) shared 98.5 % similarity (21 nt difference) and the type strains of other species of the genus Pseudomonas shared less than 98.5 % similarity with strain OHA11T. Phylogenetic analysis of 16S rRNA gene sequences was carried out http://ijs.sgmjournals.org
including all the closest related species to the novel strain as well as the type species of the genus, Pseudomonas aeruginosa LMG 1242T. According to the maximum-likelihood phylogenetic tree (Fig. 1), strain OHA11T clustered in a separate branch related to P. baetica a390T. The results were congruent with the tree topology obtained after neighbour-joining phylogenetic analysis (data not shown). Additionally to the 16S rRNA gene, three housekeeping genes widely used in the phylogenetic analysis of species of the genus Pseudomonas were analysed here (Tayeb et al., 2005; Mulet et al., 2009, 2010, 2012; Ramos et al., 2013; Toro et al., 2013). The phylogenies obtained for the three genes were congruent with those based on the 16S rRNA gene sequence analysis, supporting the affiliation of strain OHA11T to the genus Pseudomonas as a separate species related to the P. baetica cluster. The concatenated rpoD, rpoB and gyrB gene phylogenetic tree showed that strain OHA11T clusters in a separate branch that is related to a group formed by P. baetica, P. koreensis and Pseudomonas moraviensis (Fig. 2). Levels of rpoD, rpoB and gyrB gene sequence similarity were about 87–92, 94–95 and 93–97 %, respectively, between strain OHA11T and the type strains of P. baetica, P. jessenii, P. moorei, P. mohnii, P. umsongensis, P. koreensis and P. reinekei. These values are similar to or lower than those found among several species of the genus Pseudomonas. For example, in the case of the rpoD gene, the type strain of P. jessenii showed about 92 % similarity with respect to those of Pseudomonas vancouverensis, P. moorei and P. mohnii; the type strain of P. reinekei showed 94 % similarity with respect to those of P. moorei and P. mohnii; the type strains of P. moorei and P. mohnii showed 96 % similarity between themselves and 93.7 % similarity with those of P. koreensis and P. moraviensis; and the type strain of Pseudomonas punonensis showed 91.6 % similarity with respect to those of Pseudomonas argentinensis and Pseudomonas straminea. In the case of the rpoB gene, the type strains of P. vancouverensis and P. mohnii shared 95.6 % similarity; the type strains of P. moorei and P. mohnii, P. jessenii and P. reinekei, P. koreensis and P. moraviensis, and P. vancouverensis, P. jessenii and P. reinekei shared about 97 % identity; the type strain of P. punonensis had 95.8 % similarity with respect to that of P. argentinensis and 90.5–90.7 % with respect to those of P. straminea and Pseudomonas flavescens; and the type strain of P. guariconensis had 86 % similarity with respect to that of Pseudomonas entomophila and 84–85 % with respect to those of Pseudomonas plecoglossicida, Pseudomonas monteilii and Pseudomonas taiwanensis. All these species showed gyrB gene sequence similarities ranging from 86 to 97 %. Therefore, the results of the rpoD, rpoB and gyrB gene sequence analyses also indicated that strain OHA11T represents an undescribed species of the genus Pseudomonas. DNA–DNA hybridization was carried according to the method of Ezaki et al. (1989), following the recommendations of Willems et al. (2001). Strain OHA11T was hybridized with the type strains of the seven species of the genus Pseudomonas showing more than 98.5 % 16S 2339
M.-H. Ramı´rez-Bahena and others T 81 Pseudomonas chlororaphis subsp. piscium JF3835 (FJ168539) 16 Pseudomonas chlororaphis subsp. aurantiaca NCIB 10068T (DQ682655) T 24 Pseudomonas frederiksbergensis JAJ28 (AJ249382) Pseudomonas thivervalensis CFBP 11261T (AF100323)
0.02
54
20
Pseudomonas brassicacearum subsp. brassicacearum DBK11T (AF100321) Pseudomonas brassicacearum subsp. neoaurantiaca CIP 109457T (EU391388)
Pseudomonas kilonensis 520-20T (AJ292426)
66 Pseudomonas corrugata ATCC 29736T (D84012) Pseudomonas lundensis ATCC 49968T (AB021395)
15
Pseudomonas fragi ATCC 4973T (AF094733) 98 96 Pseudomonas deceptionensis M1T (GU936597) Pseudomonas syringae ICMP 3023T (AJ308316)
13
Pseudomonas lini CFBP 5737T (AY035996)
35 Pseudomonas arsenicoxydans VC-1T (FN645213) Pseudomonas mandelii CIP 105273T (AF058286)
67
Pseudomonas migulae CIP 105470T (AF074383) T 85 Pseudomonas brenneri CFML 97-391 (AF268968)
Pseudomonas panacis CG20106T (AY787208)
17
63
Pseudomonas veronii CIP 104663T (AF064460)
85 Pseudomonas extremaustralis CT14-3T (AJ583501) Pseudomonas mohnii IpA-2T (AM293567) 55 Pseudomonas umsongensis Ps 3-10T (AF468450) 29 Pseudomonas jessenii CIP 105274T (AF068259) 57
Pseudomonas reinekei MT-1T (AM293565) Pseudomonas baetica a390T (FM201274)
27 35 68
Pseudomonas helmanticensis OHA11T (HG940537) Pseudomonas koreensis Ps 9-14T (AF468452) Pseudomonas moraviensis CCM 7280T (AY970952)
60 52
Pseudomonas moorei RW10T (AM293566) Pseudomonas vancouverensis ATCC 700688T (AJ011507)
Pseudomonas graminis DSM 11363T (Y11150) 95
Pseudomonas lutea OK2T (AY364537)
Pseudomonas agarici LMG 2112T (Z76652) Pseudomonas fuscovaginae MAFF 301177T (AB021381) Pseudomonas asplenii LMG 2137T (Z76655)
57
Pseudomonas putida DSM 291T (Z76667) T 88 Pseudomonas parafulva AJ 2129 (AB060132) Pseudomonas cremoricolorata NRIC 0181T (AB060136) Pseudomonas guariconensis PCAVU11T (HF674459)
64 60
47
T 85 Pseudomonas mosselii CIP 105259 (AF072688) Pseudomonas entomophila L48T (AY907566) 39 Pseudomonas taiwanensis BCRC 17751T (EU103629)
Pseudomonas plecoglossicida FPC951T (AB009457) 45 Pseudomonas monteilii CIP 104883T (AF064458) Pseudomonas oleovorans subsp. lubricantis RS1T (DQ842018) Pseudomonas aeruginosa LMG 1242T (Z76651) Acinetobacter baumannii DSM 30007T (X81660)
Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences (1242 nt) of strain OHA11T and the type strains of closely related species of the genus Pseudomonas. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. Bar, 2 nt substitutions per 100 nt. 2340
International Journal of Systematic and Evolutionary Microbiology 64
Pseudomonas helmanticensis sp. nov. Pseudomonas gessardii (FN554468, AJ717438, FN554186)
100 0.02
Pseudomonas brenneri (FN554457, AJ717482, FN554176)
99
73
Pseudomonas libanensis (FN554477, AJ717454, FN554195) Pseudomonas orientalis (FN554493, AJ717434, FN554209)
95
Pseudomonas grimontii (FN554470, AJ717439, FN554188) 67
47
Pseudomonas veronii (FN554518, AJ717445, FN554233) Pseudomonas migulae (FN554486, AJ717446, FN554204)
54
Pseudomonas lini (FN554478, AJ717466, FN554196)
95
Pseudomonas arsenicoxydans (HE800488, HE800503, HE800469) Pseudomonas frederiksbergensis (AM084335, AJ717465, AM084676)
99 84
72
Pseudomonas mandelii (FN554482, AJ717435, FN554200) Pseudomonas reinekei (FN554508, FN554754, AM293559)
69
100
80
Pseudomonas mohnii (FN554487, FN554741, AM293561) Pseudomonas moorei (FN678363, FN554742, AM293560) Pseudomonas umsongensis (FN554516, FN554763, FN554231)
58
Pseudomonas vancouverensis (FN554517, AJ717473, AM293558)
59 87
95
Pseudomonas jessenii (FN678364, AJ717447, FN554191) Pseudomonas helmanticensis OHA11T (HG940517, HG940518, HG940516) Pseudomonas baetica (FN678357, HE800504, HE800470)
98
Pseudomonas moraviensis (FN554490, FN554743, FN554206)
94 93
Pseudomonas koreensis (FN554476, FN554737, FN554194)
Pseudomonas kilonensis (AM084336, AJ717472, AM084677) 100
Pseudomonas brassicacearum (AM084334, AJ717436, AM084675)
Pseudomonas chlororaphis subsp. aureofaciens (FN554453, AJ717426, FN554172) Pseudomonas aeruginosa (AJ633568, AJ717442, AJ633104)
Fig. 2. Maximum-likelihood phylogenetic tree based on concatenated partial rpoD, rpoB and gyrB gene sequences (682, 855 and 717 nt, respectively) of strain OHA11T and the type strains of closely related species of the genus Pseudomonas. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. Bar, 2 nt substitutions per 100 nt.
rRNA gene sequence similarity: P. baetica a390T, P. jessenii DSM 17150T, P. moorei DSM 12647T, P. mohnii DSM 18327T, P. umsongensis DSM 16611T, P. koreensis DSM 16610T and P. reinekei DSM 18361T (Table S1). Mean hybridization values were less than 49 % in all cases. Therefore, strain OHA11T represents a novel species of the genus Pseudomonas when the recommendation of a threshold value of 70 % DNA–DNA relatedness for the definition of a bacterial species is considered (Wayne et al., 1987). For base composition analysis, DNA was prepared according to Chun & Goodfellow (1995). The G+C content of the DNA was determined using the thermal denaturation method (Mandel & Marmur, 1968). The DNA G+C content of strain OHA11T was 58.1 mol%. This value is within the range reported for species of the genus Pseudomonas (Palleroni, 2005). The cellular fatty acids were analysed by using the Microbial Identification System (MIDI; Microbial ID) Sherlock 6.1 and the library RTSBA6 according to the technical instructions provided for this system (Sasser, 1990). Strain OHA11T was grown on TSA plates for 24 h at 28 uC and harvested in lateexponential growth phase. The major fatty acids of strain OHA11T were C16 : 0 (31.9 %), C18 : 1v7c (15.6 %) and summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH, 32.9 %). As expected, all the relatives clustering in the same phylogenetic http://ijs.sgmjournals.org
group as strain OHA11T shared similar fatty acid profiles (Table 1). Strain OHA11T has the three fatty acids typically present in the genus Pseudomonas according to Palleroni (2005), namely C10 : 0 3-OH, C12 : 0 and C12 : 0 3-OH. Strain OHA11T was cultivated for 24 h on TSA plates at 28 uC to obtain the cell mass required for quinone analysis, which was carried out by the Identification Service of the DSMZ (Braunschweig, Germany) from freeze-dried cells using the methods described by Tindall (1990a, b). Strain OHA11T contained Q9 as the respiratory quinone (100 %). The presence of Q9 as a major ubiquinone is in agreement with the results obtained for species of the genus Pseudomonas (Palleroni, 2005). For fluorescent pigment analysis, cells were grown on King B agar and tested for pigment production (King et al., 1954). Strain OHA11T produced a fluorescent pigment on this medium, similarly to P. baetica, P. jessenii, P. umsongensis and P. koreensis. Physiological and biochemical tests were performed as previously described (Peix et al., 2005) including the same species of the genus Pseudomonas used for DNA–DNA hybridization experiments. Additionally, the API 20NE, API 32 GN and API 50CH systems with API 50 CHB/E medium (bioMe´rieux) as well as Biolog GN2 Microplates 2341
M.-H. Ramı´rez-Bahena and others
Table 1. Cellular fatty acid contents (%) of strain OHA11T, and the type strains of its closest related species and the type species of the genus Pseudomonas, P. aeruginosa Strains: 1, OHA11T; 2, P. baetica a390T; 3, P. jessenii DSM 17150T; 4, P. moorei DSM 12647T; 5, P. mohnii DSM 18327T; 6, P. umsongensis DSM 16611T; 7, P. koreensis DSM 16610T; 8, P. reinekei DSM 18361T; 9, P. aeruginosa ATCC 10145T. Data for taxa 2–8 are from Lo´pez et al. (2012); data for taxon 9 are from Xiao et al. (2009) using the same conditions. ND, Not detected; TR, trace. Fatty acid C10 : 0 3-OH C12 : 0 2-OH C12 : 0 3-OH C10 : 0 C12 : 0 C14 : 0 C16 : 0 C17 : 0 cyclo C17 : 0 C16 : 1v5c C18 : 1v7c C18 : 0 Summed feature 3*
1
2
3
4
5
6
7
8
9
2.4 4.9 2.9 0.1 2.0 0.5 31.9 5.1 0.2 0.1 15.6 0.8 32.9
3.4 5.5 3.2 0.1 1.7 0.5 29.4 3.2 0.1 0.1 12.2 0.3 39.5
2.8 2.3 3.4 0.1 4.7 0.3 29.0 0.9 0.1 0.1 17.2 0.7 38.1
2.6 3.7 3.3 0.2 2.9 0.3 27.5 2.8 0.1 0.1 16.6 0.4 39.1
3.9 3.7 4.1
3.5 3.4 3.5
1.8 4.2 4.0
3.7 4.0 3.9 0.1 2.7 0.4 31.5
3.6 3.7 4.5 4.8 1.3 20.5
ND
ND
ND
ND
ND
3.5 0.7 33.2 11.7
2.9 0.6 33.3 1.9
ND
ND
ND
ND
12.5 0.7 23.9
13.5 0.7 35.6
3.4 0.4 25.3 2.3 0.2 0.1 21.0 0.7 35.8
TR
3.8
TR
ND
ND
13.5 0.7 35.5
38.9 TR
20.0
*Summed feature 3: C16 : 1v7c/iso-C15 : 0 2-OH.
were used following the manufacturers’ instructions. The API 20NE and API 50CH results were recorded after 48 h of incubation at 28 uC. Phenotypic characteristics of the novel strain are reported below in the species description and differences with respect to the closest relatives in the genus Pseudomonas and the type species of the genus, P. aeruginosa, are given in Table 2. The phenotypic characteristics of strain OHA11T support its classification within the genus Pseudomonas as it is a motile, Gramnegative rod that is strictly aerobic, catalase- and oxidasepositive and produces a fluorescent pigment typical of the genus (Hildebrand et al., 1994). Nevertheless, as stated by Palleroni (2005), these characteristics do not allow definitive differentiation of members of the genus Pseudomonas from other rRNA groups of aerobic ‘pseudomonads’. Analysis of 16S rRNA genes and of chemotaxonomic characteristics such as fatty acids and ubiquinone composition are necessary for this purpose (Palleroni, 2005). Strain OHA11T can be differentiated from recognized species of the genus Pseudomonas based on 16S rRNA and housekeeping gene sequences, DNA–DNA hybridization values, as well as overall phenotypic and chemotaxonomic characteristics.
Pseudomonas helmanticensis (hel.man.tic.en9sis. N.L. fem. adj. helmanticensis pertaining to Helmantica, the name of Salamanca in Roman times).
Gram-stain-negative, strictly aerobic, non-spore-forming rod-shaped cells of 1.6–2.0 mm length and 0.6–0.8 mm diameter, motile by means of two polar flagella. Colonies grown on nutrient agar are circular, convex, beige to yellowish, translucent and usually 1.5–2.0 mm in diameter within 2 days of growth at 28 uC. Growth temperature range is 5–31 uC and pH range for growth is pH 5–11. Able to grow with 0–5 % NaCl in nutrient broth. A diffusible fluorescent pigment is produced on King B medium. Strictly aerobic with oxidative metabolism and no fermentation of sugars in peptone media. The respiratory ubiquinone is Q9. Major fatty acids are C16 : 0, C18 : 1v7c and summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH). Oxidase- and catalase-positive. In the API 20 NE system, positive for arginine dihydrolase and urease. Negative for indole and b-galactosidase production. Negative for nitrate reduction and aesculin hydrolysis. Positive for assimilation of glucose, gluconate, caprate, L-arabinose, mannose, mannitol, malate, N-acetylglucosamine and citrate. Negative for assimilation of maltose, adipate and phenylacetate. In the API 32GN system, positive for assimilation of N-acetylglucosamine, L-ribose, acetate, L-alanine, L-serine, mannitol, glucose, L-sorbose, L-arabinose, propionate, L-histidine, L-proline, 2-ketogluconate, 3-hydroxybutyrate and 3-hydroxybenzoate. Negative for assimilation of L-rhamnose, inositol, sucrose, maltose, itaconate, suberate, lactate, 5-ketogluconate, glycogen, 4-hydroxybenzoate, salicin, melibiose, L-fucose, caprate, valerate, citrate and malonate. In the API 50CH system, positive for acid production from D-fucose and gluconate and assimilation of 2-ketogluconate. Negative for glycerol, erythritol, D-arabinose, L-xylose, adonitol, methyl b-Dxyloside, sorbose, L-rhamnose, dulcitol, inositol, mannitol,
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Given the phylogenetic, chemotaxonomic and phenotypic data presented, strain OHA11T should be assigned to a novel species of the genus Pseudomonas, for which the name Pseudomonas helmanticensis sp. nov. is proposed. Description of Pseudomonas helmanticensis sp. nov.
Pseudomonas helmanticensis sp. nov.
Table 2. Differential phenotypic characteristics between strain OHA11T, and the type strains of its phylogenetically closest related species and the type species of the genus, P. aeruginosa Strains: 1, OHA11T; 2, P. baetica a390T; 3, P. jessenii DSM 17150T; 4, P. moorei DSM 12647T; 5, P. mohnii DSM 18327T; 6, P. umsongensis DSM 16611T; 7, P. koreensis DSM 16610T; 8, P. reinekei DSM 18361T ; 9, P. aeruginosa ATCC 10145T. All data were obtained in this study, except for taxon 9, which were from Palleroni (2005), Clark et al. (2006) and Xiao et al. (2009). +, Positive; 2, negative; w, weak. Characteristic
1
2
3
4
5
6
7
8
9
Fluorescent pigments on King B agar Nitrate reduction Gelatin hydrolysis Growth at 41 uC Assimilation of (API20NE): D-Mannitol N-Acetylglucosamine Phenylacetate Acid production from (API 50CH): Glycerol Maltose Lactose Sucrose D-Fucose 2-Ketogluconate (assimilation) Assimilation of (Biolog GN2): Tween 40 Tween 80 D-Arabitol D-Fructose myo-Inositol D-Mannitol D-Mannose Acetate Formate D-Galacturonate D-Glucuronate a-Hydroxybutyrate a-Ketobutyrate a-Ketoglutarate a-Ketovalerate Malonate Propionate Glucuronamide D-Alanine Glycyl L-glutamate L-Histidine Hydroxy-L-Proline L-Leucine L-Ornithine D-Serine L-Threonine c-Aminobutyrate Inosine Uridine Phenylethylamine Putrescine
+ 2 2 2
+ 2 + 2
+ + 2 2
2 2 2 2
2 2 2 2
+ + 2 2
+ 2 2 2
2 2 2 2
+ + + +
+ + 2
+ + 2
+ + +
+ 2 +
+ 2 +
2 2 +
+ + 2
+ 2 +
+ + 2
2 2 2 2 + +
2 + + + + 2
2 2 2 2 2 2
+ 2 2 2 2 +
2 2 2 2 + +
2 2 + 2 w 2
+ 2 2 2 w w
2 2 2 2 + 2
+ 2 2 2 2 +
+ + + + + + + 2 2 2 2 2 2 + 2 w w 2 + 2 + + + + 2 2 + + w 2 +
+ + + + 2 + + + 2 2 2 2 2 + 2 + + 2 + 2 2 2 + 2 2 2 + + + 2 2
+ + + + 2 + + + + 2 2 2 + + + + + 2 + 2 + + + + + + + 2 2 + +
2 2 2 2 2 + 2 2 2 + + 2 2 + 2 2 2 2 w 2 + + 2 2 2 2 + 2 2 2 +
2 2 2 2 2 + + 2 w + + 2 2 + 2 + + 2 + 2 2 + 2 2 2 2 + 2 2 + +
+ + 2 + 2 2 + + + + 2 + + + + + + 2 + + + + + + 2 + + 2 2 + +
+ + + + 2 + + + 2 + + + + 2 + + + + + + + + + + 2 + + + + 2 +
2 2 + 2 2 2 2 + + + 2 w 2 + 2 w + 2 2 2 2 2 2 2 2 2 2 2 2 + 2
+ + 2 + 2 + 2 + 2 + + 2 + + + + + 2 + 2 2 + + 2 2 2 + + 2 2 +
sorbitol, methyl a-D-mannoside, methyl a-D-glucoside, Nacetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, http://ijs.sgmjournals.org
inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, lyxose, tagatose, L-fucose, D-arabitol and L-arabitol. Assimilation of 5-ketogluconate is negative 2343
M.-H. Ramı´rez-Bahena and others
and that of L-arabinose, D-ribose, D-xylose, galactose, glucose, fructose and mannose is weak. In Biolog GN2 plates, positive for assimilation of Tween 40, Tween 80, Nacetyl-D-glucosamine, L-arabinose, D-arabitol, D-fructose, D-galactose, a-D-glucose, myo-inositol, D-mannitol, Dmannose, methyl pyruvate, cis-aconitate, citrate, D-galactonic acid lactone, D-gluconate, D-glucosaminic acid, b-hydroxybutyrate, a-ketoglutarate, DL-lactic acid, quinic acid, D-saccharic acid, succinate, bromosuccinic acid, Dalanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartate, L-glutamate, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-proline, L-pyroglutamate, L-serine, DL-carnitine, c-aminobutyrate, urocanate, inosine, putrescine, 2aminoethanol and glycerol. Negative for a-cyclodextrin, dextrin, glycogen, N-acetyl-D-galactosamine, adonitol, cellobiose, i-erythritol, L-fucose, gentiobiose, a-lactose, lactulose, maltose, melibiose, methyl b-D-glucoside, Dpsicose, raffinose, L-rhamnose, D-sorbitol, sucrose, trehalose, turanose, xylitol, monomethyl succinate, acetate, formate, D-galacturonate, D-glucuronate, a-hydroxybutyrate, c-hydroxybutyrate, p-hydroxyphenylacetate, itaconate, a-ketobutyrate, a-ketovalerate, sebacate, succinamic acid, glucuronamide, L-alaninamide, glycyl L-aspartate, glycyl L-glutamate, L-phenylalanine, D-serine, L-threonine, thymidine, phenylethylamine, 2,3-butanediol, DL-a-glycerol phosphate, glucose 1-phosphate and glucose 6phosphate. Assimilation of malonate, propionate and uridine is weak. The type strain is OHA11T (5LMG 28168T5CECT 8548T), isolated from a forest soil in Salamanca province, Spain. The DNA G+C content of the type strain is 58.1 mol%.
Doetsch, R. N. (1981). Determinative methods of light microscopy. In
Manual of Methods for General Bacteriology, pp. 21–33. Edited by P. Gerdhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
deoxyribonucleic acid-deoxyribonucleic acid acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229. Hildebrand, D. C., Palleroni, N. J., Hendson, M., Toth, J. & Johnson, J. L. (1994). Pseudomonas flavescens sp. nov., isolated from walnut
blight cankers. Int J Syst Bacteriol 44, 410–415. Kaur, G. & Reddy, M. S. (2013). Phosphate solubilizing rhizobacteria
from an organic farm and their influence on the growth and yield of maize (Zea mays L.). J Gen Appl Microbiol 59, 295–303. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. Kimura, M. (1980). A simple method for estimating evolutionary rates
of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120. King, E. O., Ward, M. K. & Raney, D. E. (1954). Two simple media for
the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44, 301–307. Kwon, S. W., Kim, J. S., Park, I. C., Yoon, S. H., Park, D. H., Lim, C. K. & Go, S. J. (2003). Pseudomonas koreensis sp. nov., Pseudomonas
umsongensis sp. nov. and Pseudomonas jinjuensis sp. nov., novel species from farm soils in Korea. Int J Syst Evol Microbiol 53, 21–27. Lo´pez, J. R., Die´guez, A. L., Doce, A., De la Roca, E., De la Herran, R., Navas, J. I., Toranzo, A. E. & Romalde, J. L. (2012). Pseudomonas
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Acknowledgements This research was funded by MINECO (Spanish Central Government) Grant INNPACTO IPT-2011-1283-060000. M.-H. R.-B. is recipient of a JAE-Doc researcher contract from CSIC co-financed by ERDF. M. J .C., J. D. F.-F. and R. M. are recipients of contracts supported by this project.
temperature profile for determining the guanine plus cytosine content of DNA. Methods Enzymol 12B, 195–206. Mulet, M., Bennasar, A., Lalucat, J. & Garcı´a-Valde´s, E. (2009). An
rpoD-based PCR procedure for the identification of Pseudomonas species and for their detection in environmental samples. Mol Cell Probes 23, 140–147. Mulet, M., Lalucat, J. & Garcı´a-Valde´s, E. (2010). DNA sequence-
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DOI 10.1099/ijs.0.063560-0
Pseudomonas helmanticensis sp. nov., isolated from forest soil Martha-Helena Ramı´rez-Bahena,1,2 Maria Jose´ Cuesta,1 Jose´ David Flores-Fe´lix,3 Rebeca Mulas,4 Rau´l Rivas,2,3 Joao Castro-Pinto,5 Javier Bran˜as,5 Daniel Mulas,5 Fernando Gonza´lez-Andre´s,4 Encarna Vela´zquez2,3 and A´lvaro Peix1,2 Correspondence A´lvaro Peix [email protected]
1
Instituto de Recursos Naturales y Agrobiologı´a, IRNASA-CSIC, Salamanca, Spain
2
Unidad Asociada Grupo de Interaccio´n Planta-Microorganismo, Universidad de Salamanca-IRNASA (CSIC), Salamanca, Spain
3
Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca, Salamanca, Spain
4
Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de Leo´n, Leo´n, Spain
5
Fertiberia S. A., Madrid, Spain
A bacterial strain, OHA11T, was isolated during the course of a study of phosphate-solubilizing bacteria occurring in a forest soil from Salamanca, Spain. The 16S rRNA gene sequence of strain OHA11T shared 99.1 % similarity with respect to Pseudomonas baetica a390T, and 98.9 % similarity with the type strains of Pseudomonas jessenii, Pseudomonas moorei, Pseudomonas umsongensis, Pseudomonas mohnii and Pseudomonas koreensis. The analysis of housekeeping genes rpoB, rpoD and gyrB confirmed its phylogenetic affiliation to the genus Pseudomonas and showed similarities lower than 95 % in almost all cases with respect to the above species. Cells possessed two polar flagella. The respiratory quinone was Q9. The major fatty acids were C16 : 0, C18 : 1v7c and summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH). The strain was oxidase-, catalase- and urease-positive, positive for arginine dihydrolase but negative for nitrate reduction, b-galactosidase production and aesculin hydrolysis. It was able to grow at 31 6C and at pH 11. The DNA G+C content was 58.1 mol%. DNA–DNA hybridization results showed values lower than 49 % relatedness with respect to the type strains of the seven closest related species. Therefore, the combined genotypic, phenotypic and chemotaxonomic data support the classification of strain OHA11T to a novel species of the genus Pseudomonas, for which the name Pseudomonas helmanticensis sp. nov. is proposed. The type strain is OHA11T (5LMG 28168T5CECT 8548T).
Many species of bacteria are able to solubilize inorganic phosphates in vitro, and some of them can mobilize phosphorus to plants (Antoun et al., 1998; Peix et al., 2001; Kaur & Reddy, 2013). Within soil microbiota, the groups of pseudomonads, bacilli and rhizobia are considered the most effective phosphate solubilizers (Rodrı´guez & Fraga, 1999). The genus Pseudomonas includes several species reported as phosphate-solubilizing bacteria, some of which were isolated from soil, such as Pseudomonas rhizosphaerae (Peix et al., 2003), Pseudomonas lutea (Peix et al., 2004) or The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, rpoD, rpoB and gyrB gene sequences of strain OHA11T are HG940537, HG940517, HG940518 and HG940516, respectively. One supplementary table and one supplementary figure are available with the online version of this paper.
2338
the recently described Pseudomonas guariconensis (Toro et al., 2013). During a study of phosphate-solubilizing rhizospheric bacteria in soils from northern Spain, we isolated a strain (OHA11T) in a forest soil located in Salamanca province that is able to produce a large transparent ‘halo’ surrounding its colonies in media containing insoluble bicalcium phosphate as the phosphorus source, and subjected this strain to a polyphasic taxonomic study. The isolation procedure was the same as described by Peix et al. (2003). Briefly, 10 g of rhizospheric soil was suspended in 90 ml sterile water and stirred for 30 min. From this suspension, 100 ml was spread on YED-P medium and incubated at 28 uC for 7 days. The isolate formed a transparent ‘halo’ around the colonies, indicating in vitro phosphate solubilization activity. This strain was classified to the genus Pseudomonas within the Gammaproteobacteria based on 16S rRNA and 063560 G 2014 IUMS Printed in Great Britain
Pseudomonas helmanticensis sp. nov.
housekeeping gene sequence analyses, and the phylogenetic, chemotaxonomic and phenotypic data obtained showed that it represents a novel species. Cells were stained according to the Gram procedure described by Doetsch (1981). Motility was checked by phase-contrast microscopy after growing cells in nutrient agar medium at 22 uC for 48 h. The flagellation type was determined by electron microscopy after 48 h of incubation on tripticase soy agar (TSA; Becton Dickinson, BBL) at 22 uC as previously described (Rivas et al., 2007). Strain OHA11T was Gram-stain-negative, rod-shaped (0.6–0.86 1.6–2.0 mm) and motile by means of two polar flagella (Fig. S1, available in the online Supplementary Material). Cells grew as round translucent beige to yellowish colonies on nutrient agar. For 16S rRNA gene sequencing and comparison analysis, DNA extraction, amplification and sequencing were performed as reported by Rivas et al. (2007). Amplification and partial sequencing of the gyrB, rpoB and rpoD housekeeping genes were performed as described by Mulet et al. (2010), using the primers PsEG30F (59-ATYGAAATCGCCAARCG-39) and PsEG790R (59-CGGTTGATKTCCTTGA-39) for the rpoD gene (Mulet et al., 2009), LAPS5F (59-TGGCCGAGAACCAGTTCCGCGT-39) and LAPS27R (59-CGGCTTCGTCCAGCTTGTTCAG-39) for the rpoB gene (Tayeb et al., 2005), and GyrBPUN1F (59-AAGGAGCTGGTGYTGACC-39) and GyrBPUN1R (59-GCGTCGATCATCTTGCCG-39) for the gyrB gene (Ramos et al., 2013). The sequences obtained (1472 nt for 16S, 721 nt for rpoD, 1125 nt for rpoB and 713 nt for gyrB) were compared with those from GenBank using the BLASTN (Altschul et al., 1990) and EzTaxon (Kim et al., 2012) programs and levels of similarity were calculated after pairwise comparison. For phylogenetic analysis, sequences were aligned using the CLUSTAL X software (Thompson et al., 1997). Distances were calculated according to Kimura’s two-parameter model (Kimura, 1980). Phylogenetic trees based on 16S rRNA gene sequences were reconstructed using the neighbourjoining (Saitou & Nei, 1987) and maximum-likelihood (Rogers & Swofford, 1998) methods. The MEGA5 software (Tamura et al., 2011) was used for all analyses. Comparison of the 16S rRNA gene sequence of strain OHA11T against the type strains of bacterial species recorded in the EzTaxon-e database showed the affiliation of the novel strain to the genus Pseudomonas. The closest relative was Pseudomonas baetica a390T (Lo´pez et al., 2012) with 99.1 % pairwise similarity (13 nt difference), and the next closest relatives with 98.9 % similarity (15–17 nt differences) were the type strains of Pseudomonas jessenii (Verhille et al., 1999), Pseudomonas moorei, Pseudomonas mohnii (Ca´mara et al., 2007), Pseudomonas umsongensis and Pseudomonas koreensis (Kwon et al., 2003). The type strain of Pseudomonas reinekei (Ca´mara et al., 2007) shared 98.5 % similarity (21 nt difference) and the type strains of other species of the genus Pseudomonas shared less than 98.5 % similarity with strain OHA11T. Phylogenetic analysis of 16S rRNA gene sequences was carried out http://ijs.sgmjournals.org
including all the closest related species to the novel strain as well as the type species of the genus, Pseudomonas aeruginosa LMG 1242T. According to the maximum-likelihood phylogenetic tree (Fig. 1), strain OHA11T clustered in a separate branch related to P. baetica a390T. The results were congruent with the tree topology obtained after neighbour-joining phylogenetic analysis (data not shown). Additionally to the 16S rRNA gene, three housekeeping genes widely used in the phylogenetic analysis of species of the genus Pseudomonas were analysed here (Tayeb et al., 2005; Mulet et al., 2009, 2010, 2012; Ramos et al., 2013; Toro et al., 2013). The phylogenies obtained for the three genes were congruent with those based on the 16S rRNA gene sequence analysis, supporting the affiliation of strain OHA11T to the genus Pseudomonas as a separate species related to the P. baetica cluster. The concatenated rpoD, rpoB and gyrB gene phylogenetic tree showed that strain OHA11T clusters in a separate branch that is related to a group formed by P. baetica, P. koreensis and Pseudomonas moraviensis (Fig. 2). Levels of rpoD, rpoB and gyrB gene sequence similarity were about 87–92, 94–95 and 93–97 %, respectively, between strain OHA11T and the type strains of P. baetica, P. jessenii, P. moorei, P. mohnii, P. umsongensis, P. koreensis and P. reinekei. These values are similar to or lower than those found among several species of the genus Pseudomonas. For example, in the case of the rpoD gene, the type strain of P. jessenii showed about 92 % similarity with respect to those of Pseudomonas vancouverensis, P. moorei and P. mohnii; the type strain of P. reinekei showed 94 % similarity with respect to those of P. moorei and P. mohnii; the type strains of P. moorei and P. mohnii showed 96 % similarity between themselves and 93.7 % similarity with those of P. koreensis and P. moraviensis; and the type strain of Pseudomonas punonensis showed 91.6 % similarity with respect to those of Pseudomonas argentinensis and Pseudomonas straminea. In the case of the rpoB gene, the type strains of P. vancouverensis and P. mohnii shared 95.6 % similarity; the type strains of P. moorei and P. mohnii, P. jessenii and P. reinekei, P. koreensis and P. moraviensis, and P. vancouverensis, P. jessenii and P. reinekei shared about 97 % identity; the type strain of P. punonensis had 95.8 % similarity with respect to that of P. argentinensis and 90.5–90.7 % with respect to those of P. straminea and Pseudomonas flavescens; and the type strain of P. guariconensis had 86 % similarity with respect to that of Pseudomonas entomophila and 84–85 % with respect to those of Pseudomonas plecoglossicida, Pseudomonas monteilii and Pseudomonas taiwanensis. All these species showed gyrB gene sequence similarities ranging from 86 to 97 %. Therefore, the results of the rpoD, rpoB and gyrB gene sequence analyses also indicated that strain OHA11T represents an undescribed species of the genus Pseudomonas. DNA–DNA hybridization was carried according to the method of Ezaki et al. (1989), following the recommendations of Willems et al. (2001). Strain OHA11T was hybridized with the type strains of the seven species of the genus Pseudomonas showing more than 98.5 % 16S 2339
M.-H. Ramı´rez-Bahena and others T 81 Pseudomonas chlororaphis subsp. piscium JF3835 (FJ168539) 16 Pseudomonas chlororaphis subsp. aurantiaca NCIB 10068T (DQ682655) T 24 Pseudomonas frederiksbergensis JAJ28 (AJ249382) Pseudomonas thivervalensis CFBP 11261T (AF100323)
0.02
54
20
Pseudomonas brassicacearum subsp. brassicacearum DBK11T (AF100321) Pseudomonas brassicacearum subsp. neoaurantiaca CIP 109457T (EU391388)
Pseudomonas kilonensis 520-20T (AJ292426)
66 Pseudomonas corrugata ATCC 29736T (D84012) Pseudomonas lundensis ATCC 49968T (AB021395)
15
Pseudomonas fragi ATCC 4973T (AF094733) 98 96 Pseudomonas deceptionensis M1T (GU936597) Pseudomonas syringae ICMP 3023T (AJ308316)
13
Pseudomonas lini CFBP 5737T (AY035996)
35 Pseudomonas arsenicoxydans VC-1T (FN645213) Pseudomonas mandelii CIP 105273T (AF058286)
67
Pseudomonas migulae CIP 105470T (AF074383) T 85 Pseudomonas brenneri CFML 97-391 (AF268968)
Pseudomonas panacis CG20106T (AY787208)
17
63
Pseudomonas veronii CIP 104663T (AF064460)
85 Pseudomonas extremaustralis CT14-3T (AJ583501) Pseudomonas mohnii IpA-2T (AM293567) 55 Pseudomonas umsongensis Ps 3-10T (AF468450) 29 Pseudomonas jessenii CIP 105274T (AF068259) 57
Pseudomonas reinekei MT-1T (AM293565) Pseudomonas baetica a390T (FM201274)
27 35 68
Pseudomonas helmanticensis OHA11T (HG940537) Pseudomonas koreensis Ps 9-14T (AF468452) Pseudomonas moraviensis CCM 7280T (AY970952)
60 52
Pseudomonas moorei RW10T (AM293566) Pseudomonas vancouverensis ATCC 700688T (AJ011507)
Pseudomonas graminis DSM 11363T (Y11150) 95
Pseudomonas lutea OK2T (AY364537)
Pseudomonas agarici LMG 2112T (Z76652) Pseudomonas fuscovaginae MAFF 301177T (AB021381) Pseudomonas asplenii LMG 2137T (Z76655)
57
Pseudomonas putida DSM 291T (Z76667) T 88 Pseudomonas parafulva AJ 2129 (AB060132) Pseudomonas cremoricolorata NRIC 0181T (AB060136) Pseudomonas guariconensis PCAVU11T (HF674459)
64 60
47
T 85 Pseudomonas mosselii CIP 105259 (AF072688) Pseudomonas entomophila L48T (AY907566) 39 Pseudomonas taiwanensis BCRC 17751T (EU103629)
Pseudomonas plecoglossicida FPC951T (AB009457) 45 Pseudomonas monteilii CIP 104883T (AF064458) Pseudomonas oleovorans subsp. lubricantis RS1T (DQ842018) Pseudomonas aeruginosa LMG 1242T (Z76651) Acinetobacter baumannii DSM 30007T (X81660)
Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences (1242 nt) of strain OHA11T and the type strains of closely related species of the genus Pseudomonas. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. Bar, 2 nt substitutions per 100 nt. 2340
International Journal of Systematic and Evolutionary Microbiology 64
Pseudomonas helmanticensis sp. nov. Pseudomonas gessardii (FN554468, AJ717438, FN554186)
100 0.02
Pseudomonas brenneri (FN554457, AJ717482, FN554176)
99
73
Pseudomonas libanensis (FN554477, AJ717454, FN554195) Pseudomonas orientalis (FN554493, AJ717434, FN554209)
95
Pseudomonas grimontii (FN554470, AJ717439, FN554188) 67
47
Pseudomonas veronii (FN554518, AJ717445, FN554233) Pseudomonas migulae (FN554486, AJ717446, FN554204)
54
Pseudomonas lini (FN554478, AJ717466, FN554196)
95
Pseudomonas arsenicoxydans (HE800488, HE800503, HE800469) Pseudomonas frederiksbergensis (AM084335, AJ717465, AM084676)
99 84
72
Pseudomonas mandelii (FN554482, AJ717435, FN554200) Pseudomonas reinekei (FN554508, FN554754, AM293559)
69
100
80
Pseudomonas mohnii (FN554487, FN554741, AM293561) Pseudomonas moorei (FN678363, FN554742, AM293560) Pseudomonas umsongensis (FN554516, FN554763, FN554231)
58
Pseudomonas vancouverensis (FN554517, AJ717473, AM293558)
59 87
95
Pseudomonas jessenii (FN678364, AJ717447, FN554191) Pseudomonas helmanticensis OHA11T (HG940517, HG940518, HG940516) Pseudomonas baetica (FN678357, HE800504, HE800470)
98
Pseudomonas moraviensis (FN554490, FN554743, FN554206)
94 93
Pseudomonas koreensis (FN554476, FN554737, FN554194)
Pseudomonas kilonensis (AM084336, AJ717472, AM084677) 100
Pseudomonas brassicacearum (AM084334, AJ717436, AM084675)
Pseudomonas chlororaphis subsp. aureofaciens (FN554453, AJ717426, FN554172) Pseudomonas aeruginosa (AJ633568, AJ717442, AJ633104)
Fig. 2. Maximum-likelihood phylogenetic tree based on concatenated partial rpoD, rpoB and gyrB gene sequences (682, 855 and 717 nt, respectively) of strain OHA11T and the type strains of closely related species of the genus Pseudomonas. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. Bar, 2 nt substitutions per 100 nt.
rRNA gene sequence similarity: P. baetica a390T, P. jessenii DSM 17150T, P. moorei DSM 12647T, P. mohnii DSM 18327T, P. umsongensis DSM 16611T, P. koreensis DSM 16610T and P. reinekei DSM 18361T (Table S1). Mean hybridization values were less than 49 % in all cases. Therefore, strain OHA11T represents a novel species of the genus Pseudomonas when the recommendation of a threshold value of 70 % DNA–DNA relatedness for the definition of a bacterial species is considered (Wayne et al., 1987). For base composition analysis, DNA was prepared according to Chun & Goodfellow (1995). The G+C content of the DNA was determined using the thermal denaturation method (Mandel & Marmur, 1968). The DNA G+C content of strain OHA11T was 58.1 mol%. This value is within the range reported for species of the genus Pseudomonas (Palleroni, 2005). The cellular fatty acids were analysed by using the Microbial Identification System (MIDI; Microbial ID) Sherlock 6.1 and the library RTSBA6 according to the technical instructions provided for this system (Sasser, 1990). Strain OHA11T was grown on TSA plates for 24 h at 28 uC and harvested in lateexponential growth phase. The major fatty acids of strain OHA11T were C16 : 0 (31.9 %), C18 : 1v7c (15.6 %) and summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH, 32.9 %). As expected, all the relatives clustering in the same phylogenetic http://ijs.sgmjournals.org
group as strain OHA11T shared similar fatty acid profiles (Table 1). Strain OHA11T has the three fatty acids typically present in the genus Pseudomonas according to Palleroni (2005), namely C10 : 0 3-OH, C12 : 0 and C12 : 0 3-OH. Strain OHA11T was cultivated for 24 h on TSA plates at 28 uC to obtain the cell mass required for quinone analysis, which was carried out by the Identification Service of the DSMZ (Braunschweig, Germany) from freeze-dried cells using the methods described by Tindall (1990a, b). Strain OHA11T contained Q9 as the respiratory quinone (100 %). The presence of Q9 as a major ubiquinone is in agreement with the results obtained for species of the genus Pseudomonas (Palleroni, 2005). For fluorescent pigment analysis, cells were grown on King B agar and tested for pigment production (King et al., 1954). Strain OHA11T produced a fluorescent pigment on this medium, similarly to P. baetica, P. jessenii, P. umsongensis and P. koreensis. Physiological and biochemical tests were performed as previously described (Peix et al., 2005) including the same species of the genus Pseudomonas used for DNA–DNA hybridization experiments. Additionally, the API 20NE, API 32 GN and API 50CH systems with API 50 CHB/E medium (bioMe´rieux) as well as Biolog GN2 Microplates 2341
M.-H. Ramı´rez-Bahena and others
Table 1. Cellular fatty acid contents (%) of strain OHA11T, and the type strains of its closest related species and the type species of the genus Pseudomonas, P. aeruginosa Strains: 1, OHA11T; 2, P. baetica a390T; 3, P. jessenii DSM 17150T; 4, P. moorei DSM 12647T; 5, P. mohnii DSM 18327T; 6, P. umsongensis DSM 16611T; 7, P. koreensis DSM 16610T; 8, P. reinekei DSM 18361T; 9, P. aeruginosa ATCC 10145T. Data for taxa 2–8 are from Lo´pez et al. (2012); data for taxon 9 are from Xiao et al. (2009) using the same conditions. ND, Not detected; TR, trace. Fatty acid C10 : 0 3-OH C12 : 0 2-OH C12 : 0 3-OH C10 : 0 C12 : 0 C14 : 0 C16 : 0 C17 : 0 cyclo C17 : 0 C16 : 1v5c C18 : 1v7c C18 : 0 Summed feature 3*
1
2
3
4
5
6
7
8
9
2.4 4.9 2.9 0.1 2.0 0.5 31.9 5.1 0.2 0.1 15.6 0.8 32.9
3.4 5.5 3.2 0.1 1.7 0.5 29.4 3.2 0.1 0.1 12.2 0.3 39.5
2.8 2.3 3.4 0.1 4.7 0.3 29.0 0.9 0.1 0.1 17.2 0.7 38.1
2.6 3.7 3.3 0.2 2.9 0.3 27.5 2.8 0.1 0.1 16.6 0.4 39.1
3.9 3.7 4.1
3.5 3.4 3.5
1.8 4.2 4.0
3.7 4.0 3.9 0.1 2.7 0.4 31.5
3.6 3.7 4.5 4.8 1.3 20.5
ND
ND
ND
ND
ND
3.5 0.7 33.2 11.7
2.9 0.6 33.3 1.9
ND
ND
ND
ND
12.5 0.7 23.9
13.5 0.7 35.6
3.4 0.4 25.3 2.3 0.2 0.1 21.0 0.7 35.8
TR
3.8
TR
ND
ND
13.5 0.7 35.5
38.9 TR
20.0
*Summed feature 3: C16 : 1v7c/iso-C15 : 0 2-OH.
were used following the manufacturers’ instructions. The API 20NE and API 50CH results were recorded after 48 h of incubation at 28 uC. Phenotypic characteristics of the novel strain are reported below in the species description and differences with respect to the closest relatives in the genus Pseudomonas and the type species of the genus, P. aeruginosa, are given in Table 2. The phenotypic characteristics of strain OHA11T support its classification within the genus Pseudomonas as it is a motile, Gramnegative rod that is strictly aerobic, catalase- and oxidasepositive and produces a fluorescent pigment typical of the genus (Hildebrand et al., 1994). Nevertheless, as stated by Palleroni (2005), these characteristics do not allow definitive differentiation of members of the genus Pseudomonas from other rRNA groups of aerobic ‘pseudomonads’. Analysis of 16S rRNA genes and of chemotaxonomic characteristics such as fatty acids and ubiquinone composition are necessary for this purpose (Palleroni, 2005). Strain OHA11T can be differentiated from recognized species of the genus Pseudomonas based on 16S rRNA and housekeeping gene sequences, DNA–DNA hybridization values, as well as overall phenotypic and chemotaxonomic characteristics.
Pseudomonas helmanticensis (hel.man.tic.en9sis. N.L. fem. adj. helmanticensis pertaining to Helmantica, the name of Salamanca in Roman times).
Gram-stain-negative, strictly aerobic, non-spore-forming rod-shaped cells of 1.6–2.0 mm length and 0.6–0.8 mm diameter, motile by means of two polar flagella. Colonies grown on nutrient agar are circular, convex, beige to yellowish, translucent and usually 1.5–2.0 mm in diameter within 2 days of growth at 28 uC. Growth temperature range is 5–31 uC and pH range for growth is pH 5–11. Able to grow with 0–5 % NaCl in nutrient broth. A diffusible fluorescent pigment is produced on King B medium. Strictly aerobic with oxidative metabolism and no fermentation of sugars in peptone media. The respiratory ubiquinone is Q9. Major fatty acids are C16 : 0, C18 : 1v7c and summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH). Oxidase- and catalase-positive. In the API 20 NE system, positive for arginine dihydrolase and urease. Negative for indole and b-galactosidase production. Negative for nitrate reduction and aesculin hydrolysis. Positive for assimilation of glucose, gluconate, caprate, L-arabinose, mannose, mannitol, malate, N-acetylglucosamine and citrate. Negative for assimilation of maltose, adipate and phenylacetate. In the API 32GN system, positive for assimilation of N-acetylglucosamine, L-ribose, acetate, L-alanine, L-serine, mannitol, glucose, L-sorbose, L-arabinose, propionate, L-histidine, L-proline, 2-ketogluconate, 3-hydroxybutyrate and 3-hydroxybenzoate. Negative for assimilation of L-rhamnose, inositol, sucrose, maltose, itaconate, suberate, lactate, 5-ketogluconate, glycogen, 4-hydroxybenzoate, salicin, melibiose, L-fucose, caprate, valerate, citrate and malonate. In the API 50CH system, positive for acid production from D-fucose and gluconate and assimilation of 2-ketogluconate. Negative for glycerol, erythritol, D-arabinose, L-xylose, adonitol, methyl b-Dxyloside, sorbose, L-rhamnose, dulcitol, inositol, mannitol,
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International Journal of Systematic and Evolutionary Microbiology 64
Given the phylogenetic, chemotaxonomic and phenotypic data presented, strain OHA11T should be assigned to a novel species of the genus Pseudomonas, for which the name Pseudomonas helmanticensis sp. nov. is proposed. Description of Pseudomonas helmanticensis sp. nov.
Pseudomonas helmanticensis sp. nov.
Table 2. Differential phenotypic characteristics between strain OHA11T, and the type strains of its phylogenetically closest related species and the type species of the genus, P. aeruginosa Strains: 1, OHA11T; 2, P. baetica a390T; 3, P. jessenii DSM 17150T; 4, P. moorei DSM 12647T; 5, P. mohnii DSM 18327T; 6, P. umsongensis DSM 16611T; 7, P. koreensis DSM 16610T; 8, P. reinekei DSM 18361T ; 9, P. aeruginosa ATCC 10145T. All data were obtained in this study, except for taxon 9, which were from Palleroni (2005), Clark et al. (2006) and Xiao et al. (2009). +, Positive; 2, negative; w, weak. Characteristic
1
2
3
4
5
6
7
8
9
Fluorescent pigments on King B agar Nitrate reduction Gelatin hydrolysis Growth at 41 uC Assimilation of (API20NE): D-Mannitol N-Acetylglucosamine Phenylacetate Acid production from (API 50CH): Glycerol Maltose Lactose Sucrose D-Fucose 2-Ketogluconate (assimilation) Assimilation of (Biolog GN2): Tween 40 Tween 80 D-Arabitol D-Fructose myo-Inositol D-Mannitol D-Mannose Acetate Formate D-Galacturonate D-Glucuronate a-Hydroxybutyrate a-Ketobutyrate a-Ketoglutarate a-Ketovalerate Malonate Propionate Glucuronamide D-Alanine Glycyl L-glutamate L-Histidine Hydroxy-L-Proline L-Leucine L-Ornithine D-Serine L-Threonine c-Aminobutyrate Inosine Uridine Phenylethylamine Putrescine
+ 2 2 2
+ 2 + 2
+ + 2 2
2 2 2 2
2 2 2 2
+ + 2 2
+ 2 2 2
2 2 2 2
+ + + +
+ + 2
+ + 2
+ + +
+ 2 +
+ 2 +
2 2 +
+ + 2
+ 2 +
+ + 2
2 2 2 2 + +
2 + + + + 2
2 2 2 2 2 2
+ 2 2 2 2 +
2 2 2 2 + +
2 2 + 2 w 2
+ 2 2 2 w w
2 2 2 2 + 2
+ 2 2 2 2 +
+ + + + + + + 2 2 2 2 2 2 + 2 w w 2 + 2 + + + + 2 2 + + w 2 +
+ + + + 2 + + + 2 2 2 2 2 + 2 + + 2 + 2 2 2 + 2 2 2 + + + 2 2
+ + + + 2 + + + + 2 2 2 + + + + + 2 + 2 + + + + + + + 2 2 + +
2 2 2 2 2 + 2 2 2 + + 2 2 + 2 2 2 2 w 2 + + 2 2 2 2 + 2 2 2 +
2 2 2 2 2 + + 2 w + + 2 2 + 2 + + 2 + 2 2 + 2 2 2 2 + 2 2 + +
+ + 2 + 2 2 + + + + 2 + + + + + + 2 + + + + + + 2 + + 2 2 + +
+ + + + 2 + + + 2 + + + + 2 + + + + + + + + + + 2 + + + + 2 +
2 2 + 2 2 2 2 + + + 2 w 2 + 2 w + 2 2 2 2 2 2 2 2 2 2 2 2 + 2
+ + 2 + 2 + 2 + 2 + + 2 + + + + + 2 + 2 2 + + 2 2 2 + + 2 2 +
sorbitol, methyl a-D-mannoside, methyl a-D-glucoside, Nacetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, http://ijs.sgmjournals.org
inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, lyxose, tagatose, L-fucose, D-arabitol and L-arabitol. Assimilation of 5-ketogluconate is negative 2343
M.-H. Ramı´rez-Bahena and others
and that of L-arabinose, D-ribose, D-xylose, galactose, glucose, fructose and mannose is weak. In Biolog GN2 plates, positive for assimilation of Tween 40, Tween 80, Nacetyl-D-glucosamine, L-arabinose, D-arabitol, D-fructose, D-galactose, a-D-glucose, myo-inositol, D-mannitol, Dmannose, methyl pyruvate, cis-aconitate, citrate, D-galactonic acid lactone, D-gluconate, D-glucosaminic acid, b-hydroxybutyrate, a-ketoglutarate, DL-lactic acid, quinic acid, D-saccharic acid, succinate, bromosuccinic acid, Dalanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartate, L-glutamate, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-proline, L-pyroglutamate, L-serine, DL-carnitine, c-aminobutyrate, urocanate, inosine, putrescine, 2aminoethanol and glycerol. Negative for a-cyclodextrin, dextrin, glycogen, N-acetyl-D-galactosamine, adonitol, cellobiose, i-erythritol, L-fucose, gentiobiose, a-lactose, lactulose, maltose, melibiose, methyl b-D-glucoside, Dpsicose, raffinose, L-rhamnose, D-sorbitol, sucrose, trehalose, turanose, xylitol, monomethyl succinate, acetate, formate, D-galacturonate, D-glucuronate, a-hydroxybutyrate, c-hydroxybutyrate, p-hydroxyphenylacetate, itaconate, a-ketobutyrate, a-ketovalerate, sebacate, succinamic acid, glucuronamide, L-alaninamide, glycyl L-aspartate, glycyl L-glutamate, L-phenylalanine, D-serine, L-threonine, thymidine, phenylethylamine, 2,3-butanediol, DL-a-glycerol phosphate, glucose 1-phosphate and glucose 6phosphate. Assimilation of malonate, propionate and uridine is weak. The type strain is OHA11T (5LMG 28168T5CECT 8548T), isolated from a forest soil in Salamanca province, Spain. The DNA G+C content of the type strain is 58.1 mol%.
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Acknowledgements This research was funded by MINECO (Spanish Central Government) Grant INNPACTO IPT-2011-1283-060000. M.-H. R.-B. is recipient of a JAE-Doc researcher contract from CSIC co-financed by ERDF. M. J .C., J. D. F.-F. and R. M. are recipients of contracts supported by this project.
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