ORIGINAL ARTICLE
Isolation and characterization of drought-tolerant ACC
deaminase and exopolysaccharide-producing fluorescent
Pseudomonas sp.
Shaik Zulfikar Ali & Vardharajula Sandhya &
Linga Venkateswar Rao
Received: 9 March 2013 /Accepted: 5 June 2013
#
Springer-Verlag Berlin Heidelberg and the University of Milan 2013
Abstract The enzyme 1-aminocyclopropane-1-carboxylate de-
aminase catalyzes the degradation of 1-aminocyclopropane-1-
carboxylic acid (ACC), the immediate precursor of the plant
hormone ethylene, into α-ketobutyrate a nd ammonia. The e n-
zyme has been detected in a limited number of bacteria and plays
a sig nificant role in sustaining plant growth and development
under biotic and abiotic stress conditions by reducing stress-
induced ethylene production in plants. We have screened 32
fluorescent Pseudomonas sp. isolated from rhizosphere and
non-rhizosphere soils of different crop production systems for
drought tolerance using polyethylene glycol 6000 (PEG 6000).
Nine of these isolates were tolerant to a substrate metric potential
of −0.30 MPa (15 % PEG 6000) and therefore considered to be
drought-tolerant. All of these drought-tolerant isolates were
screened for ACC d eaminase activity u sing ACC as the sole
nitrogen source, and one (Sor gP4) was found to be positive for
ACC, producing 3.71±0.025 and 1.42±0.039 μM/mg protein/h
of α-k etobutyrate under the non-stress and drought stress condi-
tion, respectively . The isolate Sor gP4 also showed other plant
growth-promoting traits, such as indole acetic acid production,
phosphate solubilization, siderophore and hydrogen cyanide pro-
duction. The ACC deaminase gene (acdS) from the isolate
SorgP4 was amplified, and the nucleotide sequence alignment
of the acdS gene showed significant homology with acdS genes
of NCBI Genbank. The 16S rRNA gene sequencing analysis
identified the isolate as Pseudomonas fluor escens.Bothse-
quences have been submitted to the NCBI GenBank under the
accession numbers JX88576 7 and KC192771 respectively.
Keywords Pseudomonas fluorescens
.
ACC deaminase
.
Drought stress
.
acdS gene
.
PGP traits
Introduction
The gaseous hormone ethylene (C
2
H
4
) synthesized in plant
tissues from the precursor 1-aminocyclopropane-1-carboxylic
acid (ACC) is involved in multiple physiological and devel-
opmental processes in plants, such as tissue differentiation,
lateral bud development, seedling emergence, leaf and flower
senescence, root hair development and elongation, anthocya-
nin synthesis, fruit ripening and degreening, and the produc-
tion of volatile compounds responsible for aroma in fruits
(Abeles et al. 1992; Frankenberger and Arshad 1995; Spaink
1997; Bleecker and Kende 2000). Ethylene also regulates
plant responses to biotic and abiotic stresses (Abeles et al.
1992; Roman et al. 1995;O’Donnell et al. 1996; Penninckx
et al. 1998). Under ambient conditions, plants produce the
required levels of ethylene, conferring beneficial effects on
plant growth and development; however, in response to biotic
and abiotic stresses the plant often significantly increases
endogenous ethylene production, which has adverse effects
on plant growth and is thought to be responsible for senes-
cence in plants (Abeles et al. 1992; Woltering and Van Doorn
1988;Nayanietal.1998; Ali et al. 2012).
Plant growth-promoting rhizobacteria (PGPR) are a group
of free-living saprophytic bacteria that can be found in the
S. Z. Ali (*)
Department of Microbiology, Agri Biotech Foundation,
Behind College of veterinary Science (old), Acharya N.G Ranga
Agricultural University, Rajendranagar, Hyderabad,
Andhra Pradesh, India 500030
e-mail: skzali28@gmail.com
V. Sandhya
Department of Dryland Cereals, International Crops Research
Institute for the Semi-Arid-Tropics, Patancheru, Hyderabad,
Andhra Pradesh, India 502324
L. Venkateswar Rao
Department of Microbiology, University College of Science,
Osmania University, Hyderabad, Andhra Pradesh, India 500007
Ann Microbiol
DOI 10.1007/s13213-013-0680-3
rhizosphere in association with the root system and which
enhance the growth and development of the plant either direct-
ly or indirectly (Kloepper and Beauchamp 1992; Liu et al.
1995). Interestingly, these PGPR strains also possess the en-
zyme ACC deaminase (Jacobson et al. 1994; Glick et al. 1998;
Shah et al. 1997) which can cleave the plant ethylene precursor
ACC to ammonia and α-ketobutyrate, thereby lowering the
level of ethylene under various biotic and abiotic stresses
(Glick et al. 1998), such as salt stress (Cheng et al. 2007,
Mayak et al. 2004a; Zahir et al. 2009), flooding stress (Grichko
and Glick 2001),droughtstress(Mayaketal.2004b), heavy
metal stress (Belimov et al. 2005; Stearns et al. 2005), and
pathogen attack (Wang et al. 2000). ACC deaminase-contain ing
PGPR lower the level of ACC in the stressed plants, thereby
limiting the amount of stress ethylene synthesis and, consequent-
ly, damage to the plant. These bacteria are beneficial to plant
growth as plants are often subjected to stresses that induce the
production of ethylene. Soil-borne fluorescent pseudomonads
have excellent root colonizing ability and catabolic versatility,
and they produce a wide range of enzymes and metabolites
which favor plant resistance to various biotic and abiotic stresses
(Ramamoorthy et al. 2001; Mayak et al. 2004; V ivekananthan
et al. 2004).
Drought stress is one of the major agricultural problems
limiting crop productivity in most of the arid and semiarid
regions of the world. This form of abiotic stress affects the
plant–water relations at both the cellular and whole-plant
level, causing both specific and non-specific reactions and
damage. Bacteria can survive under stress conditions due to
the production of exopolysaccharide (EPS), which protects
microorganisms from water stress by enhancing water reten-
tion and regulating the diffusion of organic carbon sources
(Wilkinson 1958; Hepper 1975; Roberson and Firestone
1992; Chenu 1993; Chenu and Roberson 1996). EPS also
helps microorganisms to irreversibly attach and colonize the
roots due to involvement of a network of fibrillar material
that permanently connects the bacteria to the root surface
(Bashan et al. 2004). Inoculation of plants with native ben-
eficial microorganisms with drought -tolerant ACC deami-
nase may increase the drought tolerance of plants growing in
arid or semiarid areas. Therefore, we made an attempt to
isolate and characterize EPS and ACC deaminase from
drought-tolerant Pseudomonas strains from cropped soils
of different arid and semiarid natural habitat as a means to
provide the best benefit to drought-stressed plants.
Materials and methods
Isolation of fluorescent Pseudomonas sp.
A total of 16 soil samples (non-rhizosphere and rhizosphere)
were collected from different ecosystems covering arid, semi-
arid and sub-humid zones. Non-rhizosphere soil samples were
collected from a depth of 0–15 cm into flasks containing sterile
normal saline and kept on a shaker for 30 min. For rhizosphere
soil samples, the plants were uprooted and the bulk soil removed
by gently shaking the plants; rhizosphere soil samples were
collected by dipping the roots in containers containing sterile
normal saline followed by shaking for 30 min. The soil suspen-
sion samples (non-rhizosphere and rhizosphere) were serially
diluted, and the appropriate dilutions were spread plated on solid
King’s B medium. The plates were incubated at 28±2 °C, and
fluorescent colonies were selected for further studies.
Screening for drought tolerance and EPS production
Trypticase soya broth with different water potentials
(−0.05, −0.15, −0.30, −0.49, and − 0.73 MPa) was pre-
pared by adding the appropriate concentrations of poly-
ethylene glycol (PEG 6000) (Michel and Kaufmann
1973; Sandhya et al. 2009) and then inoculated with
1 % of bacterial cultures cultivated overnight in TSB.
Six replicates of each isolate at each concentration were
prepared. After incubation at 28 °C under shaking con-
ditions (120 rpm) for 24 h, growth was estimated by
measuring the optical density at 600 nm using a spec-
trophotometer (Thermo Spectronic model 336002; Ther-
mo Fisher Scientific, Waltham, M A). The growth of the
isolates at various stress levels was recorded.
The cultures able to grow at the maximum stress
level were analyzed for their ability to produce EPS
(Fett et al. 1986, 1989) under no stress and at the
maximum stress level (− 0.30 MPa). EPS was extracted
from 3-day-old cultures raised in TSB (15 % PEG 6000
was added to TSB for inducing stress). The culture was
centrifuged at 20,000 g for25minandthesupernatant
collected. Highly viscous cultures were diluted with
0.85 % KCl before centrifugation. The pellet was
washedtwicewith0.85%KCltocompletelyextract
the EPS. The possible extraction of intracellular poly-
saccharides was r uled out by testing for the presence of
DNA in the supernatant by DPA reagent (Burton 1956).
The concentration of protein in t he supernatant was
estimatedbyBradford’s reagent (Bradford 1976). The
supernatant was then filtered through 0.45-μm nitrocel-
lulose membrane and dialysed extensively against water
at 4 °C. The dialysate was centrifuged (20,000 g)for
25 min to remove any insoluble material and then
mixed with 3 volumes of ice-cold absolute alcohol and
kept overnight at 4 °C. The precipitated EPS obtained
by centrifugation (10,000 g,15min)wassuspendedin
water and further purified by repeating the dialysis and
precipitation steps. Total carbohydrate content in the
precipitated EPS was determined according to Dubois
et al. (1956).
Ann Microbiol
Screening for ACC deaminase activity
The ACC deaminase activity of drought-tolerant Pseudomo-
nas isolates was screened based on the ability of the respec-
tive isolate to use ACC as a sole nitrogen source . All nine
drought-tolerant Pseudomonas isolates were grown in 5 ml
of TSB medium at 28 °C for 24 h with shaking (120 rpm).
The cells were harvested by centrifugation at 3,000 g for
5 min, washed twice with sterile 0.1 M Tris–HCl (pH 7.5),
resuspended in 1 ml of 0.1 M Tris–HCl (pH 7.5), and spot
inoculated on petri plates containing modified DF (Dworkin
and Foster 1958) minimal salts medium [glucose, 2.0 g;
gluconic acid, 2.0 g; citric acid, 2.0 g; KH
2
PO
4
, 4.0 g;
Na
2
HPO
4
, 6.0 g; MgSO
4
.
7H
2
O, 0.2 g; micro-nutrient solu-
tion (CaCl
2
, 200 mg; FeSO
4
.
7H
2
O, 200 mg; H
3
BO
3
, 15 mg;
ZnSO
4
.
7H
2
O, 20 mg; Na
2
MoO
4,
10 mg; KI, 10 mg
;
NaBr,
10 mg; MnCl
2
, 10 mg; COCl
2
, 5 mg; CuCl
2
, 5 mg; AlCl
3
,
2 mg; NiSO
4
, 2 mg distilled water, 1,000 ml), 10 ml; distilled
water, 990 ml] supplemented with 3 mM ACC as the sole
nitrogen source. Plates containing only DF minimal salts
medium without ACC were used as the negative control
and those with (NH
4
)
2
SO
4
(0.2 % w/v) were used as the
positive control. The plates were incubated at 28 °C for 72 h.
Growth of isolates on ACC-supplemented plates was com-
pared to the negative and positive controls and was selected
based on growth by utilizing ACC as the nitrogen source.
ACC deaminase activity assay
To measure ACC deaminase activity, Pseudomonas iso-
lates were grown in 5 ml of TSB medium at 28 °C
until they reached the stationary phase. To induce ACC
deaminase activity under non-stress and drought stress
conditions, the cells were collected by centrifugation,
washed twice with 0.1 M Tris–HCl (pH 7.5), suspended in
2 ml of modified DF minimal medium either supplemented
with 3 mM final concentration of ACC without PEG (non-
stress condition) or with PEG 6000 (−0.30 MPa; drought
stress condition) and incubated at 28 °C with shaking for
another 36–72 h.
ACC deaminase activity was determined by measuring
the production of α-ketobutyrate and ammonia generated by
the cleavage of ACC by ACC deaminase (Honma and
Shimomura 1978 ; Penrose and Glick 2003). The induced
bacterial cells were harvested by centrifugation at 3,000 g for
5 min, washed twice with 0.1 M Tris–HCl (pH 7.5), and
resuspended in 200 μl of 0.1 M Tris–HCl (pH 8.5). The cells
were labilized by adding 5 % toluene (v/v) and then vortexed
at the highest speed for 30 s. Each sample of labilized cell
suspension (50 μl) was incubated with 5 μl of 0.3 M ACC in
an Eppendorf tube at 28 °C for 30 min. The negative control
for this assay included 50 μl of labilized cell suspension
without ACC, while the blank included 50 μl of 0.1 M
Tris – HCl (pH 8.5) with 5 μl of 0.3 M ACC. The samples
were then mixed thoroughly with 500 μl of 0.56 N HCl by
vortexing and the cell debris removed by centrifugation at
12,000 g for 5 min. A 500-μl aliquot of the supernatant was
transferred to a glass test tube and mixed with 400 μlof
0.56 N HCl and 150 μl of DNF solution (0.1 g 2,4-
dinitrophenylhydrazi ne in 100 ml of 2 N HCl); the mixture
was then incubated at 28 °C for 30 min. One milliliter of 2 N
NaOH was added to the sample before the absorbance at
540 nm was measured.
The concentration of α-ketobutyrate in each sample was
determined by comparison with a standard curve generated
as follows. A 500-μl aliquot of different α-ketobutyrate
solutions (concentration 0, 0.01, 0.05, 0.1, 0.2, 0.5, 0.75,
1 mM) was mixed with 400 μl of 0.56 N HCl and 150 μl
DNF solution . One milliliter of 2 N NaOH was added to the
mixture and the absorbance read at 540 nm as described
above. The values for absorbance versus α-ketobutyrate
concentration (mM) wer e used to construct a stand ard curve.
Protein concentrations determination
The protein concentration of toluenized cells was determined
by the method of Bradford (1976). A 26.5-μl aliquot of the
toluene-labilized bacterial cell sample used for the ACC de-
aminase enzyme assay was first diluted with 173.5 μlof0.1M
Tris–HCl (pH 8.0) and then boiled with 200 μl of 0.1 N NaOH
for 10 min. After the cell sample was cooled to room temper-
ature, the protein concentration was determined by measuring
the absorbance at 595 nm immediately after mixing the solu-
tion with 200 μlofBradford’s reagent. Bovine serum albumin
was used to establish a standard curve.
Screening of drought-tolerant Pseudomonas sp. for their plant
growth promoting traits
The ACC deaminase-positive drought-tolerant isolates were
tested in vitro for their multiple PGP traits. The method of
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17
OD at 600 nm
Pseudomonas isolates
Fig. 1 Growth pattern of fluorescent Pseudomonas isolates under
drought stress (−0.30 MPa) conditions. Error bars Mean ± standard
deviation (SD) (n=6)
Ann Microbiol
Gordon and Weber (1951) was followed for the estimation of
indole acetic acid (IAA). Luria–Bertani broth (LB) amended
with 5-mmol tryptophan was inoculated with bacterial cultures
cultivated overnight (0.5 OD at 600 nm) and incubated at
28 °C for 48 h. A 1-ml sample of each culture was centrifuged
(3,000 g for 20 min), and the supernatant was collected for
further analysis which involved the addition of 4 ml of
Salkowsky reagent, followed by incubation for 1 h at room
temperature under dark conditions. Absorbance of the pink
color that developed was read at 530 nm. The concentration of
proteins in the pellet was determined (Bradford 1976), and the
amount of IAA produced was expressed as micrograms per
milligrams cell protein. To determine phosphate solubilization,
we first spotted 5 μl of bacterial culture raised overnight on
Pikovskaya’s agar plates containing 2 % tri-calcium phosphate.
The plates were then incubated at 28 °C for 24– 72 h and
observed for the development of a solubilization zone around
the bacterial colonies. For the quantitative analysis, 5 ml of
NBRI-BBP medium (Mehta and Nautiyal 2001) was inocu-
lated in replicates with 50 μl of bacterial culture (0.5 OD at
600 nm) followed by incubation for 7 days at 28 °C on an
incubator shaker (120 rpm). The cells were harvested by
centrifugation at 3,000 g for 10 min and the supernatant thus
obtained was used for the quantitative estimation of phosphate
(Fiske and Subbarow 1925). For siderophore production, 1 μl
of bacterial culture raised overnight in Luria broth was spotted
on Chrome Azurol S agar plates and incubated at 28 °C for
48 h. The plates were observed for the development of an
orange halo around the bacterial colony (Schwyn and
Neilands 1987). For hydrogen cyanide (HCN) production,
Table 1 Exopolysaccharide production by Pseudomonas isolates un-
der non-stress and drought stress conditions
Isolates Non-stress
(mg/mg protein)
Drought stress
(−0.30 MPa) (mg/mg protein)
SorP1 0.89±0.07 a 1.65±0.11 a
SorP3 2.85±0.07 b 3.73±0.09 b
SorP4 2.75±0.06 c 4.16±0.05 c
GnP9 1.28±0.04 d 2.42±0.11 d
Rdgp10 3.22±0.04 e 4.33±0.05 e
SunfP12 2.71±0.10 f 4.63±0.10 f
SunfP13 1.62±0.08 g 2.76±0.11 g
BriP15 2.18±0.25 h 3.34±0.07 h
BriP17 1.74±0.10 I 3.18±0.04 I
Values are presented as the mean of six replicates ± standard deviation
(SD). Values followed by different lowercase letters are significantly
different at P<0.05 in all treatments
Fig. 2 Screening of bacterial
isolates for 1-
aminocyclopropane-1-carboxylic
acid (ACC) deaminase activity. a
DF minimal medium with
nitrogen source (positive
control), b DF minimal medium
without any nitrogen source
(negative control), c DF minimal
medium with ACC as nitrogen
source. Arrows Pseudomonas
isolate SorgP4
Ann Microbiol
the culture was streaked on King’s B medium amended with
0.4 % (w/v) of glycine, and a Whatman no.1 filter paper disc
(Whatman, New York, NY) soaked in 0.5 % picric acid (w/v)
in 2 % (w/v) sodium carbonate was placed in the lid of petri
plate. The plates were sealed with Parafilm and incubated at
28 °C for 4 days for the development of a deep-orange color
(Bakker and Schipper 1987).
Characterization of acdS and 16S rRNA gene
For molecular characterization, bacterial genomic DNA was
isolated (Chen and Kuo 1993), and the acdS gene was ampli-
fied by PCR using the reference primers (Farajzadeh et al.
2010)AccF5′-ATG AAT CTG AAT CGT TTT GAA C-3′
and 5′-TCA GCC GTT GCG GAA CAG-3′. PCR reactions
were carried out in a 25-μl volume of reaction mixture con-
taining 1× reaction buffer, 2.5 mM dNTP mixture, 10 pM of
each primer, Taq DNA polymerase (1 U), and 25 ng of
template DNA. PCR cycling was performed in a DNA
thermal cycler (Eppendorf, Hamburg, Germany) under the
following conditions: one cycle of initial denaturation for
5 min at 94 °C, 35 cycles of denaturation for 1 min at 94 °C,
annealing for 50 s at 54 °C, and elongation at for 2 min at
72 °C, followed by a final extension at 72 °C for 7 min. The
PCR product (approx. 996 bp) was separated by electropho-
resis through 1 % agarose gel, purified, and sequenced
(Xcelris Genomics Ltd, Ahmedabad, India). The 16S rRNA
gene was amplified by PCR using universal forward (5′-
AGAGTTTGATCCTGGCTCAG-3′) and reverse (5′-AAG
GAGGTGATCCAGCCGCA-3′) primers under standard con-
ditions (initial denaturation, 94 °C for 5 min; 30 cycles of
denaturation at 94 °C for 1 min, annealing at 50 °C for 40 s,
extension at 72 °C for 90 s; final extension at 72 °C for 7 min).
The PCR product (approx. 1,500 bp) was purified and se-
quenced (Xcelris Genomics Ltd). The sequences (acdS and
16S rRNA genes) obtained were compared with the existing
database of acdS and 16S rRNA gene and submitted to
GenBank.
Fluorescens of isolate SorgP4under UV light Siderophore production by isolate SorgP4
H
y
dro
g
en c
y
anide
p
roduction b
y
isolate Sor
g
P4 Phos
p
hate solubilization b
y
isolate Sor
g
P4
Negative
Positive
Fig. 3 Plant growth-promoting traits of Pseudomonas isolate SorgP4
Ann Microbiol