IJSEM Papers in Press. Published November 10, 2014 as doi:10.1099/ijs.0.065201-0
Pseudomonas yamanorum sp. nov., a new psychrotolerant bacterium isolated from Sub-Antarctic environment (Tierra del Fuego, Ushuaia) Víctor Gonzalo Arnau†, Leandro Arturo Sánchez†, Osvaldo Daniel Delgado* †
PROIMI - CONICET, Av. Belgrano y Pje. Caseros, 4000. Tucumán, Argentina.
*
Present address: Centro de Investigación y Transferencia Catamarca (CITCa), Universidad Nacional de
Catamarca, CONICET. Av. Belgrano 300. (K4700AAP). San Fernando del Valle de Catamarca, Catamarca, Argentina.
Running title: Pseudomonas yamanorum sp. nov. Subject category: New taxa Gram negative bacteria (Proteobacteria) Keywords: Pseudomonas / taxonomy / Tierra del Fuego / Ushuaia / Sub-Antarctic
†
These authors contributed equally to this work
*
Corresponding author:
Dr. Osvaldo Daniel Delgado Av. Belgrano y Pje. Caseros S. M. de Tucumán 4000, Argentina. E-mail: [email protected] Tel: +54 381 4344888 Fax: +54 381 4344887
GenBank accession numbers for 16S rRNA, gyrB, rpoB and rpoD gene sequences of strain 8H1T are EU557337, EU557346, JX987893 and JX987895, respectively. GenBank accession numbers for other sequences used in this publication are shown in phylogenetic trees based on single genes (available as supplementary data).
One supplementary table; nine supplementary figures and 16S rRNA, gyrB, rpoD, rpoB nucleotide sequence alignments and MLSA are available with the online version of this paper.
1
Summary
2
A psychrotolerant strain, 8H1T was isolated from soil samples collected in Isla de los Estados, Ushuaia,
3
Argentina. Cells were Gram negative, aerobic, straight rods, occurring singly or in pairs, non-spore-forming
4
and motile by mean of two polar flagella. The isolate was able to grow in the 4-35 °C range, with an optimum
5
rate at 28 °C. The predominant cellular fatty acids were summed feature 3 (C 16:1 ω6c and/or C16:1 ω7c; 38.0
6
%), C16:0 (35.4 %) and summed feature 8 (C18:1 ω7c; 9.5 %). The polar lipid pattern of strain 8H1T comprised
7
phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phospholipid. Ubiquinone 9 (Q-9)
8
was the predominant lipoquinone. DNA G+C content was 59.8 mol%. 16S rRNA gene sequence-based
9
phylogeny suggested the affiliation of strain 8H1T to the Pseudomonas fluorescens group; displaying ≥98.5 %
10
sequence similarities among 29 type strains. MLSA study by concatenating 16S rRNA, gyrB, rpoD and rpoB
11
genes showed that isolate 8H1T could be discriminated from closely related Pseudomonas species and
12
placed in the Pseudomonas gessardii subgroup, including the species with the highest MLSA sequence
13
similarities: P. brenneri (96.2 %), P. gessardii (96.1 %), P. proteolytica (96.0 %), P. meridiana (96.0 %), P.
14
mucidolens (95.4 %). DNA-DNA hybridization analysis between 8H1T and these closely related type strains
15
revealed relatedness values of 27.0, 8.8, 41.2, 39.7 and 46.1 %, respectively. These results together with
16
differences in several phenotypic features support the classification of a novel species for which the name
17
Pseudomonas yamanorum sp. nov. is proposed. The type strain is 8H1T (=DSM 26522T =CCUG 63249T
18
=LMG 27247T).
2
19
The genus Pseudomonas Migula 1894 was originally described to include Gram negative, strictly aerobic rods
20
and motile by polar flagella on the Approved List of Bacterial Names (Skerman et al., 1980). They are
21
characterized by a high level of metabolic diversity and it is one of the most diverse and ubiquitous bacterial
22
genera whose species have been isolated worldwide in several kinds of environments, from the Antarctica to
23
the Tropics; found in sediments, water, soil, sea, deserts, plant rhizosphere, fungi, diseased animal
24
specimens and human clinical samples (Peix et al., 2009). The genus Pseudomonas (sensu lato) has been
25
reclassified on the basis of phenotypic features (Lysenko, 1961; Stanier et al., 1966; Sneath et al., 1981),
26
rRNA-DNA hybridization (Palleroni et al., 1973), 16S rRNA gene sequence similarity (Anzai et al., 2000) and
27
chemotaxonomic data (Oyaizu & Komagata, 1983; Vancanneyt et al., 1996). Currently, the species belonging
28
to the genus Pseudomonas sensu stricto are now restricted to the rRNA similarity group I of Palleroni (1984),
29
which belongs to the γ subclass of Proteobacteria, and they include close to 140 species with standing in
30
nomenclature according to the LPSN (http://www.bacterio.cict.fr/p/pseudomonas.html; Euzéby, 1997). Since,
31
the ad hoc committee (Stackebrandt et al., 2002) recommended the evaluation and application of protein-
32
coding gene sequence analysis to ‘genomically circumscribe the taxon species and in order to differentiate it
33
from neighbouring species’, phylogenetic analysis of sequences of several housekeeping genes defined as
34
multilocus sequence analysis (MLSA) became a common method of genotypic characterization. Based on this
35
approach, Mulet et al. (2010) have provided an updated study for Pseudomonas genus phylogeny, splitting it
36
into two main lineages or intrageneric groups (IG), named IG P. aeruginosa and IG P. fluorescens. In this
37
way, MLSA has been applied for recently description of new Pseudomonas species (Campos et al., 2010;
38
Hirota et al., 2011; López et al., 2012; Pascual et al., 2012; Ramos et al., 2013). However, identification of
39
new species requires not only of genetic data, but also it must be supported with phenotypic and
40
chemotaxonomic data (polyphasic approach) (Kämpfer & Glaeser, 2012).
41
Strain 8H1T was isolated from a soil sample collected from Isla de los Estados Reservation, Ushuaia,
42
Argentina at Observatory Island location (54º 39´ S, 64º 08´ W) and selected for antimicrobial production
43
(Sánchez et al., 2009). The Island raises 800 m.a.s.l. and its mountainous geography could be considered as
44
the south-eastern end of the Fuegian Andes. The climate is humid due to abundant rains (>2,000 mm/year)
45
and the horizontal contribution of marine winds, recording an average temperature of 11 °C during summer;
46
whereas, average temperature is around 1 °C during winter.
47
For antimicrobial producers isolation, 1 g of defrosted soil samples was inoculated into 50 ml of either Luria-
48
Bertani (LB) or M9 media and incubated at 8 and 20 °C with shaking at 200 r.p.m. for 24 h. Cultures were
3
49
serially diluted and 0.1 ml aliquots were plated onto the same solid media and incubated at the same
50
temperatures for 2 days or longer when necessary. Colonies were counted and their morphological
51
characteristics recorded. Duplicate plates were made by randomly picking over 8,000 colony morphotypes.
52
The antimicrobial producers were identified by the deferred antagonism procedure of Gratia (1946) and
53
Fredericq (1948) and also by a modified agar-well diffusion assay as described by Portrait et al. (1999).
54
In order to assess isolate 8H1T taxonomic position, a polyphasic approach including: phenotypic
55
characterization, chemotaxonomic analysis (FAME, polar lipids and respiratory quinones), DNA G+C content
56
determination, phylogenetic analysis based on 16S rRNA, gyrB, rpoD and rpoB genes individually and
57
concatenated and DNA-DNA hybridization experiments were carried out along this work. The obtained data
58
showed that isolate 8H1T represents a novel species of the genus Pseudomonas, for which the name
59
Pseudomonas yamanorum sp. nov. is herein proposed.
60
Strain 8H1T was routinely cultured aerobically on LB broth or agar as basal medium (pH~7) at 25 °C. Cell
61
suspensions were prepared in 20 % (w/v) glycerol in LB medium for long-term storage at -80 °C.
62
The colony characteristics of strain 8H1T were studied after growth on LB agar at 25 °C after 48 h incubation.
63
Cell morphology and flagellation were examined by scanning electron microscopy (SEM) at the Centro
64
Integral de Microscopía Electrónica (LAMENOA), Tucumán, Argentina. Scanning electron observation was
65
conducted using a Zeiss Supra 55 VP (Carl Zeiss, Oberkochen, Germany) scanning electron microscope at ×
66
10,000-50,000 magnification (7.0 kV). For this, cells were harvested after 24 h growth on LB agar at 25 °C,
67
fixed overnight at 4 °C with Karnovsky (1965) fixative, washed three times with 0.1 M phosphate buffer (pH
68
7.0) and increasing concentrations of ethanol (from 50-100 % v/v) followed by a 100 % acetone washing,
69
mounted on 12 mm cover slips, dried to a critical point in CO2, and finally gold-palladium (80/20) coated.
70
Gram staining was performed using a Difco Gram stain set. Catalase and oxidase activities were assayed as
71
previously described (Smibert & Krieg, 1994). Acid production from carbohydrates, its assimilation as carbon
72
source; and enzymatic activities, were determined by using API 50 CH, API 20 NE, API 20 E and API ZYM
73
systems (bioMérieux, France) according to the manufacturer’s instructions with adaptation for strictly aerobic
74
metabolism of Pseudomonas. Adaptation consisted to avoid mineral oil sealing in carbohydrate acidification
75
tests. Susceptibility to antibiotics was determined by the disk diffusion assay using Gram negative 1, 2, 3 and
76
Staphylococcus A and B series disks (Britania Laboratories; Buenos Aires, Argentina). All determinations
77
were performed at 25 °C by duplicate.
4
78
Other physiological and phenotypic features of strain 8H1T were studied in parallel with its closely related
79
species by using commercial and conventional tests, all performed at 25 °C by duplicate. API 20 Strep system
80
was used for assessing acid production from carbohydrates and some enzymatic activities with adaptation
81
described above. The ability of each strain to utilize a carbon compound as sole carbon source was
82
determined as described by Shivaji et al. (1989). Hydrolysis of starch, gelatin (gelatinase) and tributyrin
83
(tributyrin esterase), tolerance to 1 % (w/v) 2, 3, 5-triphenyltetrazolium chloride, and production of fluorescent
84
pigments on King’s A and King’s B media were assessed as described by Gavini et al. (1989). The optimum
85
temperature for growth was determined by incubation at 4, 10, 15, 20, 22, 25, 28, 30, 35, 37 and 41 °C in
86
both, Nutrient broth (NB, Merck) and Nutrient agar (NA, Merck) media, in a pH range of 3-13 (adjusted with
87
HCl or NaOH). Optimal growth temperature was estimated by assessing changes in OD 600 along incubation
88
period. Halo-tolerance was tested in NB medium containing 0-10 % (w/v) NaCl. All tests were read within 2 to
89
8 days of incubation.
90
Cells of 8H1T were Gram negative, aerobic, straight rods (0.4-0.45 x 1.3-1.8 µm), occurring singly and in pairs,
91
non-spore-forming and motile by means of two polar flagella (Fig. 1; Fig. S1, available in IJSEM Online).
92
Colonies of the novel isolate grown on LB agar at 25 °C for 48 h were 1.5-2.5 mm in diameter, greenish,
93
mucoid with irregular edges and production of fluorescent pigments on King’s A and B media was observed
94
under UV light (365 nm).
95
Strain 8H1T was able to grow at temperatures ranging from 4 to 35 °C, with an optimum at 28 °C and pH
96
values between 4.0 and 9.5 in NB and NA media. Tolerance to NaCl was observed at concentrations up to 6
97
% (w/v) in NB medium. 8H1T isolate was negative for the Voges-Proskauer test, indole and hydrogen sulfide
98
production, hydrolysis of hippuric acid, nitrate reduction and denitrification. The novel strain 8H1T displayed
99
features consistent with those reported for members of the genus Pseudomonas, and as shown in Table 1,
100
our isolate can be readily differentiated from other phylogenetically related species by several phenotypic
101
properties. Optimum temperature for growth, tolerance up to 6 % (w/v) NaCl, production of fluorescent
102
pigment on King’s A medium and assimilation of D-sorbitol distinguish strain 8H1T from its closely related
103
species.
104
To further characterize strain 8H1T, analysis of cellular fatty acid methyl esters (FAME), polar lipids,
105
respiratory quinones and DNA G+C content were carried out by the Identification Service of DSMZ,
106
Braunschweig, Germany. Fatty acids, polar lipids, respiratory quinones (freeze-dried biomass) and DNA G+C
5
107
content analyses were carried out from cell biomass growing on Trypticase Soy Broth (TSB) agar (30 g TSB l -
108
1
109
Fatty acids were extracted and prepared according to standard protocols described for the MIDI Microbial
110
Identification System (Sasser, 1990). Cellular fatty acid content was analyzed by GC with an Agilent 6890N
111
gas chromatograph, with the MIDI Microbial Identification System using the TSBA6 method (MIDI, 2008) and
112
Microbial Identification Sherlock software package version 6.1. The fatty acid profile of strain 8H1 T contained
113
summed feature 3 (C16:1 ω6c and/or C16:1 ω7c; 38.0 %), C16:0 (35.4 %) and summed feature 8 (C18:1 ω7c; 9.5
114
%), as the major fatty acids and small proportions of C10:0 3-OH (3.0 %), C12:0 (2.4 %), C12:0 2-OH (3.7 %),
115
C12:0 3-OH (3.7 %) and C17:0 cyclo (2.2 %) and other fatty acids traces (
Pseudomonas yamanorum sp. nov., a new psychrotolerant bacterium isolated from Sub-Antarctic environment (Tierra del Fuego, Ushuaia) Víctor Gonzalo Arnau†, Leandro Arturo Sánchez†, Osvaldo Daniel Delgado* †
PROIMI - CONICET, Av. Belgrano y Pje. Caseros, 4000. Tucumán, Argentina.
*
Present address: Centro de Investigación y Transferencia Catamarca (CITCa), Universidad Nacional de
Catamarca, CONICET. Av. Belgrano 300. (K4700AAP). San Fernando del Valle de Catamarca, Catamarca, Argentina.
Running title: Pseudomonas yamanorum sp. nov. Subject category: New taxa Gram negative bacteria (Proteobacteria) Keywords: Pseudomonas / taxonomy / Tierra del Fuego / Ushuaia / Sub-Antarctic
†
These authors contributed equally to this work
*
Corresponding author:
Dr. Osvaldo Daniel Delgado Av. Belgrano y Pje. Caseros S. M. de Tucumán 4000, Argentina. E-mail: [email protected] Tel: +54 381 4344888 Fax: +54 381 4344887
GenBank accession numbers for 16S rRNA, gyrB, rpoB and rpoD gene sequences of strain 8H1T are EU557337, EU557346, JX987893 and JX987895, respectively. GenBank accession numbers for other sequences used in this publication are shown in phylogenetic trees based on single genes (available as supplementary data).
One supplementary table; nine supplementary figures and 16S rRNA, gyrB, rpoD, rpoB nucleotide sequence alignments and MLSA are available with the online version of this paper.
1
Summary
2
A psychrotolerant strain, 8H1T was isolated from soil samples collected in Isla de los Estados, Ushuaia,
3
Argentina. Cells were Gram negative, aerobic, straight rods, occurring singly or in pairs, non-spore-forming
4
and motile by mean of two polar flagella. The isolate was able to grow in the 4-35 °C range, with an optimum
5
rate at 28 °C. The predominant cellular fatty acids were summed feature 3 (C 16:1 ω6c and/or C16:1 ω7c; 38.0
6
%), C16:0 (35.4 %) and summed feature 8 (C18:1 ω7c; 9.5 %). The polar lipid pattern of strain 8H1T comprised
7
phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phospholipid. Ubiquinone 9 (Q-9)
8
was the predominant lipoquinone. DNA G+C content was 59.8 mol%. 16S rRNA gene sequence-based
9
phylogeny suggested the affiliation of strain 8H1T to the Pseudomonas fluorescens group; displaying ≥98.5 %
10
sequence similarities among 29 type strains. MLSA study by concatenating 16S rRNA, gyrB, rpoD and rpoB
11
genes showed that isolate 8H1T could be discriminated from closely related Pseudomonas species and
12
placed in the Pseudomonas gessardii subgroup, including the species with the highest MLSA sequence
13
similarities: P. brenneri (96.2 %), P. gessardii (96.1 %), P. proteolytica (96.0 %), P. meridiana (96.0 %), P.
14
mucidolens (95.4 %). DNA-DNA hybridization analysis between 8H1T and these closely related type strains
15
revealed relatedness values of 27.0, 8.8, 41.2, 39.7 and 46.1 %, respectively. These results together with
16
differences in several phenotypic features support the classification of a novel species for which the name
17
Pseudomonas yamanorum sp. nov. is proposed. The type strain is 8H1T (=DSM 26522T =CCUG 63249T
18
=LMG 27247T).
2
19
The genus Pseudomonas Migula 1894 was originally described to include Gram negative, strictly aerobic rods
20
and motile by polar flagella on the Approved List of Bacterial Names (Skerman et al., 1980). They are
21
characterized by a high level of metabolic diversity and it is one of the most diverse and ubiquitous bacterial
22
genera whose species have been isolated worldwide in several kinds of environments, from the Antarctica to
23
the Tropics; found in sediments, water, soil, sea, deserts, plant rhizosphere, fungi, diseased animal
24
specimens and human clinical samples (Peix et al., 2009). The genus Pseudomonas (sensu lato) has been
25
reclassified on the basis of phenotypic features (Lysenko, 1961; Stanier et al., 1966; Sneath et al., 1981),
26
rRNA-DNA hybridization (Palleroni et al., 1973), 16S rRNA gene sequence similarity (Anzai et al., 2000) and
27
chemotaxonomic data (Oyaizu & Komagata, 1983; Vancanneyt et al., 1996). Currently, the species belonging
28
to the genus Pseudomonas sensu stricto are now restricted to the rRNA similarity group I of Palleroni (1984),
29
which belongs to the γ subclass of Proteobacteria, and they include close to 140 species with standing in
30
nomenclature according to the LPSN (http://www.bacterio.cict.fr/p/pseudomonas.html; Euzéby, 1997). Since,
31
the ad hoc committee (Stackebrandt et al., 2002) recommended the evaluation and application of protein-
32
coding gene sequence analysis to ‘genomically circumscribe the taxon species and in order to differentiate it
33
from neighbouring species’, phylogenetic analysis of sequences of several housekeeping genes defined as
34
multilocus sequence analysis (MLSA) became a common method of genotypic characterization. Based on this
35
approach, Mulet et al. (2010) have provided an updated study for Pseudomonas genus phylogeny, splitting it
36
into two main lineages or intrageneric groups (IG), named IG P. aeruginosa and IG P. fluorescens. In this
37
way, MLSA has been applied for recently description of new Pseudomonas species (Campos et al., 2010;
38
Hirota et al., 2011; López et al., 2012; Pascual et al., 2012; Ramos et al., 2013). However, identification of
39
new species requires not only of genetic data, but also it must be supported with phenotypic and
40
chemotaxonomic data (polyphasic approach) (Kämpfer & Glaeser, 2012).
41
Strain 8H1T was isolated from a soil sample collected from Isla de los Estados Reservation, Ushuaia,
42
Argentina at Observatory Island location (54º 39´ S, 64º 08´ W) and selected for antimicrobial production
43
(Sánchez et al., 2009). The Island raises 800 m.a.s.l. and its mountainous geography could be considered as
44
the south-eastern end of the Fuegian Andes. The climate is humid due to abundant rains (>2,000 mm/year)
45
and the horizontal contribution of marine winds, recording an average temperature of 11 °C during summer;
46
whereas, average temperature is around 1 °C during winter.
47
For antimicrobial producers isolation, 1 g of defrosted soil samples was inoculated into 50 ml of either Luria-
48
Bertani (LB) or M9 media and incubated at 8 and 20 °C with shaking at 200 r.p.m. for 24 h. Cultures were
3
49
serially diluted and 0.1 ml aliquots were plated onto the same solid media and incubated at the same
50
temperatures for 2 days or longer when necessary. Colonies were counted and their morphological
51
characteristics recorded. Duplicate plates were made by randomly picking over 8,000 colony morphotypes.
52
The antimicrobial producers were identified by the deferred antagonism procedure of Gratia (1946) and
53
Fredericq (1948) and also by a modified agar-well diffusion assay as described by Portrait et al. (1999).
54
In order to assess isolate 8H1T taxonomic position, a polyphasic approach including: phenotypic
55
characterization, chemotaxonomic analysis (FAME, polar lipids and respiratory quinones), DNA G+C content
56
determination, phylogenetic analysis based on 16S rRNA, gyrB, rpoD and rpoB genes individually and
57
concatenated and DNA-DNA hybridization experiments were carried out along this work. The obtained data
58
showed that isolate 8H1T represents a novel species of the genus Pseudomonas, for which the name
59
Pseudomonas yamanorum sp. nov. is herein proposed.
60
Strain 8H1T was routinely cultured aerobically on LB broth or agar as basal medium (pH~7) at 25 °C. Cell
61
suspensions were prepared in 20 % (w/v) glycerol in LB medium for long-term storage at -80 °C.
62
The colony characteristics of strain 8H1T were studied after growth on LB agar at 25 °C after 48 h incubation.
63
Cell morphology and flagellation were examined by scanning electron microscopy (SEM) at the Centro
64
Integral de Microscopía Electrónica (LAMENOA), Tucumán, Argentina. Scanning electron observation was
65
conducted using a Zeiss Supra 55 VP (Carl Zeiss, Oberkochen, Germany) scanning electron microscope at ×
66
10,000-50,000 magnification (7.0 kV). For this, cells were harvested after 24 h growth on LB agar at 25 °C,
67
fixed overnight at 4 °C with Karnovsky (1965) fixative, washed three times with 0.1 M phosphate buffer (pH
68
7.0) and increasing concentrations of ethanol (from 50-100 % v/v) followed by a 100 % acetone washing,
69
mounted on 12 mm cover slips, dried to a critical point in CO2, and finally gold-palladium (80/20) coated.
70
Gram staining was performed using a Difco Gram stain set. Catalase and oxidase activities were assayed as
71
previously described (Smibert & Krieg, 1994). Acid production from carbohydrates, its assimilation as carbon
72
source; and enzymatic activities, were determined by using API 50 CH, API 20 NE, API 20 E and API ZYM
73
systems (bioMérieux, France) according to the manufacturer’s instructions with adaptation for strictly aerobic
74
metabolism of Pseudomonas. Adaptation consisted to avoid mineral oil sealing in carbohydrate acidification
75
tests. Susceptibility to antibiotics was determined by the disk diffusion assay using Gram negative 1, 2, 3 and
76
Staphylococcus A and B series disks (Britania Laboratories; Buenos Aires, Argentina). All determinations
77
were performed at 25 °C by duplicate.
4
78
Other physiological and phenotypic features of strain 8H1T were studied in parallel with its closely related
79
species by using commercial and conventional tests, all performed at 25 °C by duplicate. API 20 Strep system
80
was used for assessing acid production from carbohydrates and some enzymatic activities with adaptation
81
described above. The ability of each strain to utilize a carbon compound as sole carbon source was
82
determined as described by Shivaji et al. (1989). Hydrolysis of starch, gelatin (gelatinase) and tributyrin
83
(tributyrin esterase), tolerance to 1 % (w/v) 2, 3, 5-triphenyltetrazolium chloride, and production of fluorescent
84
pigments on King’s A and King’s B media were assessed as described by Gavini et al. (1989). The optimum
85
temperature for growth was determined by incubation at 4, 10, 15, 20, 22, 25, 28, 30, 35, 37 and 41 °C in
86
both, Nutrient broth (NB, Merck) and Nutrient agar (NA, Merck) media, in a pH range of 3-13 (adjusted with
87
HCl or NaOH). Optimal growth temperature was estimated by assessing changes in OD 600 along incubation
88
period. Halo-tolerance was tested in NB medium containing 0-10 % (w/v) NaCl. All tests were read within 2 to
89
8 days of incubation.
90
Cells of 8H1T were Gram negative, aerobic, straight rods (0.4-0.45 x 1.3-1.8 µm), occurring singly and in pairs,
91
non-spore-forming and motile by means of two polar flagella (Fig. 1; Fig. S1, available in IJSEM Online).
92
Colonies of the novel isolate grown on LB agar at 25 °C for 48 h were 1.5-2.5 mm in diameter, greenish,
93
mucoid with irregular edges and production of fluorescent pigments on King’s A and B media was observed
94
under UV light (365 nm).
95
Strain 8H1T was able to grow at temperatures ranging from 4 to 35 °C, with an optimum at 28 °C and pH
96
values between 4.0 and 9.5 in NB and NA media. Tolerance to NaCl was observed at concentrations up to 6
97
% (w/v) in NB medium. 8H1T isolate was negative for the Voges-Proskauer test, indole and hydrogen sulfide
98
production, hydrolysis of hippuric acid, nitrate reduction and denitrification. The novel strain 8H1T displayed
99
features consistent with those reported for members of the genus Pseudomonas, and as shown in Table 1,
100
our isolate can be readily differentiated from other phylogenetically related species by several phenotypic
101
properties. Optimum temperature for growth, tolerance up to 6 % (w/v) NaCl, production of fluorescent
102
pigment on King’s A medium and assimilation of D-sorbitol distinguish strain 8H1T from its closely related
103
species.
104
To further characterize strain 8H1T, analysis of cellular fatty acid methyl esters (FAME), polar lipids,
105
respiratory quinones and DNA G+C content were carried out by the Identification Service of DSMZ,
106
Braunschweig, Germany. Fatty acids, polar lipids, respiratory quinones (freeze-dried biomass) and DNA G+C
5
107
content analyses were carried out from cell biomass growing on Trypticase Soy Broth (TSB) agar (30 g TSB l -
108
1
109
Fatty acids were extracted and prepared according to standard protocols described for the MIDI Microbial
110
Identification System (Sasser, 1990). Cellular fatty acid content was analyzed by GC with an Agilent 6890N
111
gas chromatograph, with the MIDI Microbial Identification System using the TSBA6 method (MIDI, 2008) and
112
Microbial Identification Sherlock software package version 6.1. The fatty acid profile of strain 8H1 T contained
113
summed feature 3 (C16:1 ω6c and/or C16:1 ω7c; 38.0 %), C16:0 (35.4 %) and summed feature 8 (C18:1 ω7c; 9.5
114
%), as the major fatty acids and small proportions of C10:0 3-OH (3.0 %), C12:0 (2.4 %), C12:0 2-OH (3.7 %),
115
C12:0 3-OH (3.7 %) and C17:0 cyclo (2.2 %) and other fatty acids traces (
