[Postprint]
International Journal of Systematic and Evolutionary Microbiology 64(7): 2338-2345 (2014)
Pseudomonas helmanticensis sp. nov., isolated from a forest soil
Martha-Helena Ramírez-Bahena1, 2, Maria José Cuesta1, José David Flores-Félix3,
Rebeca Mulas4, Raúl Rivas2,3, Joao Castro-Pinto5, Javier Brañas5, Daniel Mulas5,
Fernando González-Andrés4, Encarna Velázquez2,3, Álvaro Peix1,2*
1
Instituto de Recursos Naturales y Agrobiología. IRNASA-CSIC, Salamanca.
Spain
2
Unidad Asociada Grupo de Interacción Planta-Microorganismo Universidad de
Salamanca-IRNASA (CSIC)
3
Departamento de Microbiología y Genética. Universidad de Salamanca. Spain.
4
Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad. Universidad
de León. Spain
5
Fertiberia S. A. Spain
*Corresponding author: Alvaro Peix. Instituto de Recursos Naturales y Agrobiología,
IRNASA-CSIC, c/Cordel de Merinas 40-52, 37008 Salamanca, Spain. E-mail:
alvaro.peix@csic.es
Running title: Pseudomonas helmanticensis sp. nov.
Keywords: Pseudomonas/ taxonomy/ Salamanca /forest soil/ phosphate solubilizing
bacteria
Contents List Category: New taxa Gram negative bacteria (Proteobacteria)
Accession numbers for strain OHA11T gene sequences: HG940537 for 16S rRNA
gene, HG940517 for rpoD, HG940518 for rpoB and HG940516 for gyrB
1
Summary
A bacterial strain named OHA11T was isolated in the course of a study of phosphate
solubilizing bacteria occurring in a forest soil from Salamanca, Spain. The 16S rRNA
gene sequence had 99.1% identity with respect to the closest relative Pseudomonas
baetica a390T, and the following closest related species with 98.9 % similarity were
P. jessenii, P. moorei, P. umsongensis, P. mohnii and P. koreensis, for which
OHA11T was classified within genus Pseudomonas. The analysis of housekeeping
genes rpoB, rpoD and gyrB confirmed its phylogenetic affiliation and showed
identities lower than 95% in almost all cases with respect to the mentioned closest
relatives. The strain has two polar flagella. The respiratory quinone is Q9. The major
fatty acids are 16:0, 18:1 7c and 16:1 7c/ 15:0 iso 2OH in summed feature 3. The
strain is oxidase, catalase and urease positive, the arginine dihydrolase system is
present but nitrate reduction, –galactosidase production and esculine hydrolysis are
negative. It can grow at 31ºC 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 results of genotypic, phenotypic and chemotaxonomic data support the
classification of strain OHA11 into a novel species of Pseudomonas, for which the
name P. helmanticensis sp. nov. is proposed. The type strain is OHA11T (=LMG
28168T, CECT 8548T).
2
Many species of bacteria are able to solubilize inorganic phosphates in vitro, and
some of them can mobilize P to plants (Antoun et al., 1998; Peix et al., 2001; Kaur
and Reddy, 2013). Within soil microbiota the groups of pseudomonads, bacilli and
rhizobia are considered the most effective phosphate solubilizers (Rodriguez and
Fraga, 1999). Genus Pseudomonas includes several species reported as phosphate
solubilising bacteria (PSB), and some of them were isolated from soil, such as P.
rhizosphaerae (Peix et al., 2003), P. lutea (Peix et al., 2004) or the recently described
P. guariconensis (Toro et al., 2013). During a study of phosphate-solubilizing
rhizospheric bacteria in soils from Northern Spain, we isolated a strain in a forest soil
located in Salamanca province (Spain), able to produce a great transparent ‘halo’
surrounding its colonies in media containing insoluble bicalcium phosphate as
phosphorus source, which was coded as OHA11T, and subjected to a polyphasic
taxonomy study. The isolation procedure was the same described in 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 µl was spread on YED-P medium and incubated at
28ºC for 7 days. The isolate formed a transparent ‘halo’ around the colonies
indicating in vitro phosphate solubilization activity.
This strain was classified into genus Pseudomonas within Gammaproteobacteria
according to 16S rRNA and housekeeping gene analyses and the phylogenetic,
chemotaxonomic and phenotypic data obtained showed that it represents a novel
species for which we propose the name Pseudomonas helmanticensis sp. nov.
The cells were stained according to the Gram procedure described by Doetsch (1981).
Motility was checked by phase-contrast microscopy after growing them in nutrient
agar medium at 22°C for 48 h. The flagellation type was determined by electron
microscopy after 48h incubation in TSA at 22°C as was previously described (Rivas
et al., 2007). Strain OHA11T is Gram negative, rod-shaped (0.6-0.8 x 1.6-2.0m) and
motile by two polar flagella (Fig. S1, available in IJSEM Online). Cells grew as round
translucent beige to yellowish coloured 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). The amplification and partial
sequencing of gyrB, rpoB and rpoD housekeeping genes was performed as described
by Mulet et al. (2010), using the primers PsEG30F/PsEG790R for rpoD gene (Mulet
et al., 2009), LAPS5F/LAPS27R for rpoB gene (Tayeb et al., 2005) and GyrBPUN1F
3
(5’-AAGGAGCTGGTGYTGACC-3’)
and
GyrBPUN1R
(5’-
GCGTCGATCATCTTGCCG-3’) for 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 identities were calculated
after pairwise comparison. For phylogenetic analysis sequences were aligned using
the Clustal_X software (Thompson et al., 1997). The distances were calculated
according to Kimura´s two-parameter model (Kimura, 1980). Phylogenetic trees of
16S rRNA gene were inferred using the neighbour-joining analysis (NJ, Saitou & Nei,
1987), and maximum likelihood (ML; Rogers & Swofford, 1998). MEGA5 software
(Tamura et al., 2011) was used for all analyses.
The 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 new strain to genus Pseudomonas. The closest related species is
Pseudomonas baetica a390T (López et al., 2012) with 99.1% pairwise similarity (13
nucleotides difference), and the following closest related species with 98.9 %
similarity (15-17 different nucleotides) were P. jessenii (Verhille et al., 1999), P.
moorei, P. mohnii (Cámara et al., 2007), P. umsongensis and P. koreensis (Kwon et
al., 2003). The species P. reinekei (Cámara et al., 2007) showed 98.5 % similarity (21
nucleotides difference) and the remaining Pseudomonas species showed identities
below 98.5%. The phylogenetic analysis of 16S rRNA gene was carried out including
all the closest related species to the new species as well as the type species of the
genus, P. aeruginosa LMG 1242T. According to the ML phylogenetic tree (Figure 1)
OHA11T clustered in a separate branch related with P. baetica a390T. The results
were congruent with the tree topology obtained after NJ phylogenetic analysis (data
not shown).
Additionally to the 16S rRNA gene, three housekeeping genes widely used in the
phylogenetic analysis of Pseudomonas species were analysed in this work (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 analysis, supporting the affiliation of OHA11T to genus Pseudomonas as a
separated species related to the P. baetica cluster. The concatenated rpoD, rpoB and
gyrB genes phylogenetic tree showed that OHA11T clusters in a separate branch
which is related to a group formed by P. baetica, P. koreensis and P. moraviensis
4
(Figure 2). The identities of rpoD gene, rpoB gene and gyrB gene were about 87-92%,
94-95% and 93-97% respectively with respect to P. baetica, P. jessenii, P. moorei, P.
mohnii, P. umsongensis, P. koreensis and P. reinekei. These values are similar or
lower than those found among several species of Pseudomonas. For example, in the
case of rpoD gene, P. jessenii showed about 92% identity with respect to P.
vancouverensis, P.moorei and P. mohnii; P. reinekii showed 94% with respect to
P.moorei and P. mohnii; P.moorei and P. mohnii showed 96% identity between them
and P. koreensis and P. moraviensis 93.7% identity; P. punonensis showed 91.6%
with respect to P. argentinensis and P. straminea. In the case of rpoB gene P.
vancouverensis and P. mohnii have 95.6% identity; P.moorei and P. mohnii, P.
jessenii and P. reinekii, P. koreensis and P. moraviensis and P. vancouverensis, P.
jessenii and P. reinekii showed about 97% identity; P. punonensis has 95.8% identity
with respect to P. argentinensis and 90.5-90.7% with respect to P. straminea and P.
flavescens. P. guariconensis has 86% identity with respect to P. entomophila and 8485% with respect to P. plecoglossicida, P. monteilii and P. taiwanensis. All these
species showed values ranging from 86% to 97% in the gyrB gene among them.
Therefore the results of the rpoD, rpoB and gyrB gene analysis also indicated that
OHA11T represents an undescribed species of Pseudomonas.
DNA-DNA hybridization was carried out by the method of Ezaki et al. (1989),
following the recommendations of Willems et al. (2001). OHA11T was hybridized
with the type strains of the seven Pseudomonas species showing more than 98.5%
identity in 16S rRNA gene: Pseudomonas baetica a390T, P. jessenii DSM17150T, P.
moorei DSM12647T, P. mohnii DSM18327T, P. umsongensis DSM16611T, P.
koreensis DSM16610T and P. reinekei DSM18361T (Table S1, available in IJSEM
Online). The hybridization average values were less than 49% in all cases. Therefore
the strain OHA11T represents a different species of Pseudomonas when the
recommendation of a threshold value of 70% DNA-DNA similarity for definition of a
bacterial species is considered (Wayne et al., 1987).
For base composition analysis, DNA was prepared according to Chun & Goodfellow
(1995). The mol % G+C content of DNA was determined using the thermal
denaturation method (Mandel & Marmur, 1968). The G+C content of strain OHA11T
was 58.1 mol %. This value is within the range obtained for Pseudomonas species
(Palleroni, 2005).
5
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 by this system (Sasser, 1990). OHA11T was grown on TSA
plates (Becton Dikinson, BBL) for 24h at 28°C and harvested in late log growth
phase. The major fatty acids of strain OHA11T are 16:0 (31.9%), 18:1 7c (15.6%),
and 16:1 7c/ 15:0 iso 2OH in summed feature 3 (32.9%). As expected, all the
relatives clustering in the same phylogenetic group that OHA11T shared similar fatty
acid profiles (Table 1). OHA11T has the three fatty acids typically present in genus
Pseudomonas according to Palleroni (2005) which are C10:0 3OH, C12:0 and C12:0 3OH.
The strain OHA11T was cultivated for 24h in TSA plates (Becton Dikinson, BBL) at
28°C to obtain the cell mass required for quinone analysis that was carried out by the
Identification Service and Dr. Brian Tindall at DSMZ (Braunschweig, Germany) from
freeze dried cells using the methods described by Tindall (1990a; 1990b). The novel
isolate OHA11T contained Q9 as respiratory quinone (100%). The presence of Q9 as
major ubiquinone is in agreement with the results obtained in the species of genus
Pseudomonas (Palleroni, 2005).
For fluorescent pigment analysis, cells were grown in King B agar and testing for
pigment production (King et al., 1954). Strain OHA11T produced a fluorescent
pigment in this medium, similarly to the close relatives P. baetica, P. jessenii, P.
umsongensis and P. koreensis.
The physiological and biochemical tests were performed as previously described
(Peix et al., 2005) including the same Pseudomonas species chosen for DNA-DNA
hybridization experiments. Additionally API 20NE, API 32 GN and API 50CH with
API 50 CHB/E medium (BioMérieux, France) as well as Biolog GN2 Microplates
(Biolog, USA) were used following the manufacturer’s instructions. The results of
API 20NE and API 50CH were recorded after 48h incubation at 28ºC. Phenotypic
characteristics of the new species are reported below in the species description and the
differences with respect to the closest Pseudomonas species and the type species of
the genus, P. aeruginosa are recorded in Table 2. The phenotypic characteristics of
strain OHA11T support its classification within genus Pseudomonas since it is a
motile Gram negative rod strictly aerobic, catalase and oxidase positive and produces
a fluorescent pigment typical of this genus (Hildebrand et al., 1994). Nevertheless as
was stated by Palleroni (2005) these characteristics do not allow an unquestionable
6
differentiation of genus Pseudomonas to other ribosomal RNA groups of aerobic
‘pseudomonads’. The analysis of the 16S rRNA genes and that of chemotaxonomic
characteristics such as fatty acids and ubiquinone composition are necessary for this
purpose (Palleroni, 2005). The strain OHA11T can be differentiated from other species
of the genus Pseudomonas in the 16S rRNA and housekeeping gene sequences, DNADNA hybridization values, as well as in the overall set of phenotypic and
chemotaxonomic characteristics.
Therefore, taking into account all the phylogenetic, chemotaxonomic and phenotypic
data, OHA11T should be assigned to a novel species within genus Pseudomonas, for
which the name Pseudomonas helmanticensis sp. nov. is proposed.
Description of Pseudomonas helmanticensis sp. nov.
Pseudomonas helmanticensis (hel.man.tic.en'sis. N.L. fem. adj. helmanticensis
pertaining to Helmantica, the name of Salamanca in Roman times).
Gram negative, strictly aerobic, non-spore forming rod-shaped cells of 1.6-2.0m in
length and 0.6-0.8 m in diameter, motile by two polar flagella. Colonies morphology
on nutrient agar is circular convex, beige to yellowish, translucid and usually 1.5 to
2.0 mm in diameter within 2 days growth at 28°C. Growth temperature range is 5ºC31ºC and pH range for growth is 5 to 11. It can grow at 0-5% NaCl concentration 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 16:0, 18:1 7c and
16:1 7c/ 15:0 iso 2OH in summed feature 3. Oxidase and catalase positive. In API
20 NE system arginine dihydrolase and urease are positive. Indole and –
galactosidase production is negative. Nitrate reduction and esculine hydrolysis are
negative. Assimilation of glucose, gluconate, caprate, L-arabinose, mannose,
mannitol, malate, N-acetyl-glucosamine and citrate is positive. Assimilation of Dmaltose, adipate and phenylacetate was negative. In API 32GN assimilation of Nacetyl-glucosamine, L-ribose, acetate, L-alanine, L-serine, mannitol, glucose, Lsorbose, L-arabinose, propionate, L-histidine, L-proline, 2-ketogluconate, 3hydroxybutyrate and 3-hydroxybenzoate is positive. Assimilation of L-rhamnose,
inositol, sucrose, maltose, itaconate, suberate, lactate, 5-ketogluconate, glycogen, 4hydroxybenzoate, salicine, melibiose, L-fucose, caprate, valerate, citrate and
7
malonate, was negative. In API 50CH system the acid production from D-fucose,
gluconate and assimilation of 2-ketogluconate is positive. That of glycerol, erythritol,
D-arabinose, L-xylose, adonitol, methyl--D-xyloside, sorbose, L-rhamnose, dulcitol,
inositol, mannitol, sorbitol, methyl-α-D-mannoside, methyl-α-D-glucoside, N-acetylglucosamine, amygdaline, arbutine, esculine, salicine, cellobiose, D-maltose, lactose,
melibiose, D-sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen,
xylitol, gentiobiose, turanose, lyxose, tagatose, L-fucose, and D and L-arabitol is
negative. Assimilation of 5-ketogluconate is negative and that of L-arabinose, Dribose, D-xylose, galactose, glucose, fructose and mannose is weak. In Biolog GN2
plates the assimilation of tween 40, tween80, N-acetyl-D-glucosamine, L-arabinose,
D-arabitol, D-fructose, D-galactose, α-D-glucose, m-inositol, D-mannitol, Dmannose, methyl-piruvate, cis-aconitate, citrate, D-galactonate lactone, D-gluconate,
D-glucosaminate,-hydroxy-butyrate, α-ketoglutarate, D, L-lactate, quinate, Dsaccharate, succinate, bromo-succinate, D-alanine, L-alanine, L-alanyl-glycine, Lasparagine, L-aspartate, L-glutamate, L-histidine, hydroxyl-L-proline, L-leucine, Lornithine, L-proline, L-pyroglutamate, L-serine, D,L carnitine, γ-aminobutyrate,
urocanate, inosine, putrescine, 2-aminoethanol and glycerol is positive. Negative
results
were
obtained
for
α-cyclodextrin,
dextrin,
glycogen,
N-acetyl-D-
galactosamine, adonitol, D-cellobiose, i-erythritol, L-fucose, gentibiose, α-D-lactose,
lactulose, maltose, D-melibiose, -methyl-D-glucoside, D-psicose, D-raffinose, Lrhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, mono-methyl-succinate,
acetate,
formate,
D-galacturonate,
D-glucuronate,
α-hydroxybutirate,
γ-
hydroxybutyrate, p-hydroxyphenylacetate, itaconate, α-ketobutyrate, α-ketovalerate,
sebacate, succinamic, glucuronamide, L-alaninamide, glycyl-L- aspartate, glycyl-Lglutamate, L-phenylalanine, D-serine, L-threonine, thymidine, phenylethylamine, 2,
3-butanediol,
D,L-
α-glycerolphosphate,
glucose-1-phosphate
and
glucose-6-
phosphate. Finally, Assimilation of malonate, propionate, uridine was weak. G+C
base composition was 58.1 mol%. The type strain is OHA11T (LMG 28168T, CECT
8548T), isolated from a forest soil in Salamanca province, Spain.
Acknowledgements
This research was funded by MINECO (Spanish Central Government) Grant
INNPACTO IPT-2011-1283-060000. MHRB is recipient of a JAE-Doc researcher
8
contract from CSIC cofinanced by ERDF. MJC, JDFF and RM are recipients of
contracts supported by this project.
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13
14
Figure legends:
Figure 1. Maximum Likelihood phylogenetic tree based on 16S rRNA gene sequences
(1242 nt) of Pseudomonas helmanticensis OHA11T and closely related Pseudomonas
species. Bootstrap values (expressed as percentages of 1000 replications) are shown at
the branching points. Bar, 2 nt substitutions per 100 nt.
Figure 2. Maximum Likelihood phylogenetic tree based on concatenated partial rpoD,
rpoB and gyrB gene sequences (682, 855 and 717 nt, respectively) of Pseudomonas
helmanticensis OHA11T and the type strains of the closely related Pseudomonas
species. Bootstrap values (expressed as percentages of 1000 replications) are shown at
the branching points. Bar, 5 nt substitutions per 100 nt.
15
Table 1. Cellular fatty acid composition (%) of P. helmanticensis OHA11T, its closest related species and the type species of the genus Pseudomonas.
P. aeruginosa.
Taxa: 1, P. helmanticensis OHA11T; 2, P. baetica a390T; 3, P. jessenii DSM17150T; 4, P. moorei DSM12647T; 5, P. mohnii DSM18327T; 6, P.
umsongensis DSM16611T; 7, P. koreensis DSM16610T ; 8, P. reinekei DSM18361T ; 9, P. aeruginosa ATCC 10145T. Data data were obtained in this
study and from §López et al. (2012) and ∫Xiao et al. (2009) using the same conditions. nd: no detected, tr: traces.
Fatty acids
10:0 3OH
12:0 2OH
12:0 3OH
10:0
12:0
14:0
16:0
17:0 cyclo
17:0
C16:1 5c
C18:1 7c
18:0
Summed feature 3*
1
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
2§
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
3§
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
4§
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
5§
3.9
3.7
4.1
nd
3.5
0.7
33.2
11.7
nd
nd
12.5
0.7
23.9
6§
3.5
3.4
3.5
nd
2.9
0.6
33.3
1.9
nd
nd
13.5
0.7
35.6
*Summed feature 3: C16:1 7c/15 iso 2OH.
7§
1.8
4.2
4.0
nd
3.4
0.4
25.3
2.3
0.2
0.1
21.0
0.7
35.8
16
8§
3.7
4.0
3.9
0.1
2.7
0.4
31.5
nd
3.8
nd
13.5
0.7
35.5
∫
9
3.6
3.7
4.5
tr
4.8
1.3
20.5
nd
tr
nd
38.9
tr
20.0
Table 2. Differential phenotypic characteristics of P. helmanticensis OHA11T, its phylogenetically closest related species and the type species of
this genus, P. aeruginosa.
Taxa: 1, P. helmanticensis OHA11T; 2, P. baetica a390T; 3, P. jessenii DSM17150T; 4, P. moorei DSM12647T; 5, P. mohnii DSM18327T; 6, P.
umsongensis DSM16611T; 7, P. koreensis DSM16610T ; 8, P. reinekei DSM18361T ; 9, P. aeruginosa ATCC 10145T. All data were obtained in
this study unless specifically indicated. +, positive; -, negative; nd, no data.
Fluorescent pigments King B Agar
Nitrate reduction
Gelatin hydrolysis
Growth at:
41°C
Assimilation of (API20NE):
D-Mannitol
N-acetyl-glucosamine
Phenylacetate
Acid prodution of (API 50CH):
Glycerol
Maltose
Lactose
Sucrose
D-Fucose
2-ketogluconate (assimilation)
Assimilation of (Biolog GN2):
Tween 40
Tween 80
D-Arabitol
D-Fructose
m-Inositol
D-Mannitol
D-Mannose
Acetate
Formate
D-Galacturonate
D-Glucuronate
α-Hydroxy-Butyrate
α-Keto-Butyrate
α-Keto-Glutarate
1
+
-
2
+
+
3
+
+
-
4
-
5
-
6
+
+
-
7
+
-
8
-
9¥
+
+
+
-
-
-
-
-
-
-
-
+
+
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
-
+
+
+
+
+
+
-
-
+
+
+
+
+
w
-
+
w
w
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
17
α-Keto-Valerate
Malonate
Propionate
Glucuronamide
D-Alanine
Glycyl-L-Glutamate
L-Histidine
Hydroxy-L-Proline
L-Leucine
L-Ornithine
D-Serine
L-Threonine
-Aminobutyrate
Inosine
Uridine
Phenylethylamine
Putrescine
¥
w
w
+
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
+
Data for P. aeruginosa ATCC 10145T are from Palleroni (2005), Clark et al. (2006) and Xiao et al. (2009)
18
+
+
+
+
+
+
+
+
+
+
+
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