Pseudomonas duriflava sp. nov., isolated from a
Rui Liu,13 Huan Liu,2,33 Hao Feng,1Xu Wang,1Chong-Xing Zhang,1
Ke-Yun Zhang1and Ren Lai1,2
1Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture,
Life Sciences College of Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China
2Biotoxin Department of Key Laboratory of Animal Models and Human Disease Mechanisms,
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223,
3Graduate School of the Chinese Academy of Sciences, Beijing 100009, China
A Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain HR2T,
was isolated from a soil sample from the Taklimaken Desert in Xinjiang Province, China. Strain
HR2Tgrew optimally at pH 7.0–8.0 and 30–37 6C in the presence of 0–1% (w/v) NaCl. An
analysis of 16S rRNA gene sequences revealed that strain HR2Tfell within the radiation of the
genus Pseudomonas, the highest level of similarity being found with respect to Pseudomonas
luteola IAM 13000T(97.5%); the levels of sequence similarity with respect to other recognized
Pseudomonas species were ,96.4%. DNA–DNA hybridization showed that the genetic
relatedness between strain HR2Tand P. luteola IAM 13000Twas 53.2%. The G+C content of
the genomic DNA of strain HR2Twas 55.2 mol%. The major fatty acids were 18:1, summed
feature 3 and 16:0. The hydroxylated fatty acids 10:0 3-OH, 12:0 3-OH and 12:0 2-OH were
also present. The data obtained in this polyphasic study indicated that this isolate represents a
novel species of the genus Pseudomonas, for which the name Pseudomonas duriflava sp. nov. is
proposed. The type strain is HR2T(5KCTC 22129T5CGMCC 1.6858T).
The genus Pseudomonas sensu stricto originally comprised
only species belonging to Pseudomonas RNA homology
group I (Palleroni, 1984) in the Gammaproteobacteria
(Kersters et al., 1996). However, the genus was subdivided
into two main intrageneric clusters (as reviewed by Anzai et
al., 2000): the first cluster comprised six groups and the
second had only one group. Pseudomonads are abundant
in various environments, such as water, air and soil. In our
search for organisms capable of growing in desert
ecosystems, a bacterial strain, designated HR2T, was
isolated and analysed using polyphasic taxonomy. An
analysis of 16S rRNA gene sequence similarities indicated
that the isolate was closely related to the Pseudomonas
aeruginosa/Pseudomonas stutzeri group within the first
cluster and formed a distinct line. On the basis of
phenotypic and genotypic evidence, it is proposed that
strain HR2Trepresents a novel species of the genus
Strain HR2Twas isolated from a desert soil sample by using
the usual dilution plating technique on Luria–Bertani (LB)
agar (l21: 5 g yeast extract, 10 g tryptone, 10 g NaCl,
pH 7.0) incubated for 2 days at 30 uC under aerobic
conditions. The isolate was cultivated routinely on LB agar
or in LB broth at the same temperature. The strain was
maintained in a glycerol suspension (20%, v/v) at 270 uC.
Amplification and sequencing of the 16S rRNA gene were
performed according to a method described by Cui et al.
(2001), but which was modified slightly by us: the primers
ponding to positions 8–27 and 1521–1540, respectively,
in the 16S rRNA sequence of Escherichia coli (Brosius et al.,
1978). The resultant sequence (1404 bp) of strain HR2T
was compared with sequences available from the GenBank
database, using a BLAST search to determine an approximate
phylogenetic affiliation. Phylogenetic trees were inferred
using PHYLIP (Felsenstein, 1993) and MEGA (version 3.1;
Kumar et al., 2001) after multiple alignment of the data by
CLUSTAL_X (Thompson et al., 1997). Distances (using
distance options according to the Kimura two-parameter
model; Kimura, 1980, 1983) and clustering were based on
3These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain HR2Tis EU046271.
Transmission electron micrographs of strain HR2Tare available as
supplementary material with the online version of this paper.
International Journal of Systematic and Evolutionary Microbiology (2008), 58, 1404–1408
140465716G2008 IUMSPrinted in Great Britain
the neighbour-joining (Saitou & Nei, 1987) and max-
imum-likelihood (Felsenstein, 1981) methods. The boot-
strap resampling method of Felsenstein (1985) (1000
replicates) was used to assess the reliability of the
phylogenetic trees. Comparisons of 16S rRNA gene
sequences showed that strain HR2Tshould be positioned
within the genus Pseudomonas, being most closely related
to Pseudomonas luteola IAM 13000T(97.5% similarity); the
sequence similarities with respect to type strains of other
established Pseudomonas species were less than 96.4%. A
phylogenetic tree based on the neighbour-joining method
indicated that strain HR2Tformed a monophyletic clade
adjacent to P. luteola IAM 13000Twith high bootstrap
support (94%) (Fig. 1).
Chromosomal DNA from strain HR2Twas extracted and
purified according to the procedure of Yoon et al. (1996).
The DNA G+C content was measured according to the
method of Cashion et al. (1997) and was found to be
55.2 mol%. This value is slightly below the range (58–
70 mol%) mentioned by Palleroni (1984) for the genus
Pseudomonas and is close to that of P. luteola IAM 13000T
(54.6 mol%; data from this study). DNA–DNA hybridiza-
tion between strain HR2Tand P. luteola IAM 13000Twas
measured as described by De Ley et al. (1970) with the
modifications described by Huß et al. (1983) and Escara &
Hutton (1980). Renaturation rates were computed with the
TRANSFER.BAS program described by Jahnke (1992). At the
DNA–DNA level, there was 53.2% genetic relatedness
between strain HR2Tand P. luteola IAM 13000T; as the
recommended threshold value for the delineation of
genomic species is considered to be 70% DNA–DNA
relatedness (Stackebrandt et al., 2002), these two strains
could be confirmed as belonging to different species.
Fatty acid methyl esters were prepared and identified
according tothe instructions
Identification system (MIDI), as described by Sasser
(1990), from cultures of strain HR2Tgrown on tryptic
soy agar (Merck) for 2 days at 30 uC. Type strains of the
closely related species P. luteola (IAM 13000T) and
Pseudomonas oryzihabitans (IAM 1568T) were also cultured
under the same nutrient and temperature conditions and
characterized concurrently with strain HR2T. The results of
Fig. 1. Neighbour-joining phylogenetic tree,
based on 16S rRNA gene sequences, show-
ing the positions of strain HR2Tand some
related Pseudomonas species. Bootstrap per-
greater than 50% are shown at nodes. The
maximum-likelihood tree showed essentially
the same topology (data not shown). Bar,
0.005 substitutions per nucleotide position.
Pseudomonas duriflava sp. nov.
the chemotaxonomic analyses are given in Table 1; all of
the strains were shown to have rather similar fatty acid
profiles. The presence of high levels of 18:1, summed
feature 3 and 16:0, along with 10:0 3-OH, 12:0 3-OH and
12:0 2-OH, supported the view that strain HR2Tbelonged
to the genus Pseudomonas (Oyaizu & Komagata, 1983).
Cell morphology and motility were examined using light
microscopy (model BH 2; Olympus) and transmission
electron microscopy (model H-7650; Hitachi) after 48 h
growth on LB agar at 30 uC. For the transmission electron
microscopy observations, cells were negatively stained with
1% (w/v) phosphotungstic acid and air-dried (see
Supplementary Fig. S1 available in IJSEM Online). Gram-
staining and endospore formation were investigated
according to the methods described by Smibert & Krieg
(1994). Growth at different temperatures and pH values
was investigated as described by Xu et al. (2005), using LB
as the basal medium. NaCl tolerance was tested using LB
broth supplemented with 0, 1, 2, 3, 4 or 5% (w/v) NaCl.
Growth was tested on nutrient agar, Simmons’ citrate agar
and MacConkey agar. The production of fluorescent
pigment was tested on King’s medium B (King et al.,
1954). Oxidase, catalase and phenylalanine deaminase
activities and the hydrolysis of casein, gelatin, Tweens 80
and 20, aesculin, DNA, starch and carboxymethylcellulose
were investigated as described by Smibert & Krieg (1994).
Acid production from carbohydrates was determined using
the medium and method described by Yamaguchi & Yokoe
(2000). Studies designed to determine the utilization of
substrates as single carbon sources were carried out as
described by Zhou et al. (2007). Sensitivity to antibiotics
was tested using the diffusion method, as described by Park
et al. (2007), on LB agar at 30 uC with filter-paper discs
(8 mm diameter; Sanofi Pasteur) containing the following
antibiotics: streptomycin (10 mg), penicillin G (10 IU),
ampicillin (10 mg), chloramphenicol (30 mg), erythromycin
(15 mg), tetracycline (30 mg), ofloxacin (5 mg), amoxicillin
(10 mg), cefamezin (30 mg), rifampicin (5 mg), gentamicin
(10 mg), ceftazidime (30 mg), vancomycin (30 mg), kana-
mycin (30 mg), carbenicillin (100 mg) and polymyxin B
(30 mg). All tests were performed at 30 uC and properties
were recorded after 2 days. Additional biochemical features
were tested using API 20E kits according to the instructions
of the manufacturer (bioMe ´rieux). P. luteola IAM 13000T
and P. oryzihabitans IAM 1568Twere included in these
studies andanalysed under
Phenotypic characteristics of strain HR2Tare given in the
species description and in Table 2. As shown in Table 2,
our isolate can be readily differentiated from its closest
phylogenetic relatives in the genus Pseudomonas sensu
stricto on the basis of several phenotypic properties.
the same conditions.
The data presented indicate that strain HR2Tis distin-
guishable from all recognized species of the genus
Pseudomonas. Thus HR2Trepresents a novel species of
the genus Pseudomonas, for which the name Pseudomonas
duriflava sp. nov. is proposed.
Description of Pseudomonas duriflava sp. nov.
Pseudomonas duriflava (du.ri.fla9va. L. adj. durus hard; L.
adj. flavus yellow; N.L. fem. adj. duriflava hard yellow).
Cells are Gram-negative, non-motile, non-spore-forming,
round-ended, aerobic rods approximately 0.6–0.860.7–
1.1 mm in size. Colonies on LB agar are dry, yellow,
circular, convex, translucent, hard, wrinkled and approxi-
mately 1.0–2.5 mm in diameter within 2 days at 30 uC.
Table 1. Fatty acid content (%) of strain HR2Tand the type strains of phylogenetically related species of the genus Pseudomonas
Strains: 1, HR2T; 2, P. luteola IAM 13000T; 3, P. oryzihabitans IAM 1568T; 4, Pseudomonas psychrotolerans LMG 21977T; 5, Pseudomonas oleovorans
IAM 1508T; 6, P. stutzeri CCUG 11256T. For 1–3, data are from this study; for 4 and 5, data are from Hauser et al. (2004); for 6, data are from
Manaia & Moore (2002). tr, Trace (,1.0%); –, not detected.
Summed feature 3*
*16:1v7c and/or iso-15:0 2-OH.
R. Liu and others
1406International Journal of Systematic and Evolutionary Microbiology 58
Fluorescent pigments are not produced on King’s medium
B. The temperature, pH and NaCl ranges for growth are 5–
42 uC (optimum, 30–37 uC),
pH 7.0–8.0) and 0–2% (optimum, 0–1%). Good growth
occurs on nutrient agar, MacConkey agar and Simmons’
citrate agar. Casein, starch, DNA, Tween 80 and aesculin
are hydrolysed, but gelatin, Tween 20 and carboxymethyl-
cellulose are not hydrolysed. Positive for catalase, lysine
decarboxylase, arginine dihydrolase and b-galactosidase
acitivities. Negative for oxidase, arginine decarboxylase,
ornithine decarboxylase, urease, phenylalanine deaminase,
indole and hydrogen sulphide production, nitrate reduc-
tion and in the Voges–Proskauer reaction. Acid is
dextrin, but not from D-galactose, D-mannose, maltose,
D-fructose, trehalose, D-mannitol, myo-inositol or salicin.
The following substrates are utilized (with no acid
production): D-ribose, D-galactose, L-arabinose, sorbose,
cellobiose, melezitose, melibiose, aesculin and amygdalin.
Lactose, sucrose, maltose, D-fructose, trehalose, raffinose, D-
mannose, turanose, glycerol, D-erythritol, D-arabitol, D-
mannitol, sorbitol, D-xylitol, adonitol, myo-inositol, inulin,
salicin, N-acetylglucosamine, gluconate, acetate and methyl
a-glucoside are not utilized. Sensitive to streptomycin,
penicillin G, ampicillin, chloramphenicol, erythromycin,
tetracycline, ofloxacin, amoxicillin, cefamezin, rifampicin,
gentamicin, ceftazidime, kanamycin, carbenicillin and poly-
myxin B, butresistantto vancomycin. DNA G+C contentis
55.2 mol%. The fatty acid pattern is shown in Table 1.
1.6858T), was isolated from a desert soil sample collected
from the Taklimaken Desert in Xinjiang Province, China.
type strain, HR2T
We thank the Chinese National Natural Science Foundation
(BK2005422) for their financial support.
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Tolerance of 5% NaCl
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