World Journal of Microbiology & Biotechnology (2006) 22:1061–1064
Springer 2006

DOI 10.1007/s11274-005-4561-1

Auxin, gibberellin, cytokinin and abscisic acid production in some bacteria

_
A. Karadeniz*, Ş.F. Topcuoğlu and S. Inan
Science and Art Faculty, Department of Biology, Akdeniz University, 07058, Antalya, Turkey
*Author for correspondence: Tel.: +90 242 310 24 01, Fax: +90 242 227 89 11, E-mail: asu@akdeniz.edu.tr

Received 5 November 2004; accepted 24 March 2005

Keywords: abscisic acid, auxin, Bacteria, cytokinin, gibberellin

Summary

In this study, auxin (indole-3-acetic acid), gibberellin, cytokinin (zeatin) and abscisic acid production were inves-
tigated in the culture medium of the bacteria Proteus mirabilis, P. vulgaris, Klebsiella pneumoniae, Bacillus mega-
terium, B. cereus, Escherichia coli. To determine the levels of these plant growth regulators, high performance liquid
chromatography (HPLC) technique was used. Our findings show that the bacteria used in this study synthesized the
plant growth regulators, auxin, gibberellin, cytokinin and abscisic acid.

Introduction hormone production. Researchers have also determined

In recent years, secondary metabolites, which are espe-
cially synthesized by bacteria, have become very
important in biotechnology. One group of these sec-
ondary metabolites is plant growth hormones, which are
widely used in the agriculture. Plant growth hormones,
which are synthesized in minute amounts, affect many
activities in plant growth and development. These hor-
mones are not only synthesized by higher plants (Pala-
van-Ünsal 1993; Davies 1995), they have also been
synthesized by lichens (Epstein et al. 1986; Lurie &
Garty 1991, Ergün et al. 2002), mosses (Ergün et al.
2002), fungi (Ünyayar et al. 1996; Yürekli et al. 1999)
and bacteria (Martinez-Toledo et al. 1988; Bottini et al.
1989; Davies 1995; Tuomi (Rosenquist 1995; Martinez-
Morales et al. 2003; Khalid et al. 2004).
It has been reported that different bacterial species
either interact with plants (especially plant pathogens) or
live in the soils, synthesize indole-3-acetic acid (IAA),
gibberellic acid (GA3), zeatin, and abscisic acid (ABA).
Most of the studies on bacterial plant hormone pro-
duction have concentrated on auxins especially IAA. For
example Sequeira ( Williams (1964) reported that IAA
levels were higher in tobacco plants infected with the
vascular pathogen Pseudomonas solanacearum than in
healthy plants. In another study carried out on Azoto-
bacter chroococcum, a soil bacterium living with maize
roots symbiotically, researchers determined that A.
chroococcum synthesizes IAA, GA3 and kinetin in a
nitrogen-free culture media supplemented with maize
root exudates and 0.5% glucose. Bottini et al. (1989)
determined that, when most of the grasses were inocu-
lated with the free-living bacteria Azospirillum ssp., grain
production was increased because of the bacterial plant
GA3, GA1 and iso-GA3 in the aseptic culture of the
Azospirillum lipoferum.
The effects of the tumour-inducing bacteria Agro-
bacterium tumefaciens and Pseudomonas savastanoi and
causal agent of witches broom, Corynebacterium fas-
cians are due to the cytokinin production. Sturtevant &
Taller (1989) have determined cytokinins in the culture
media of the nodule-forming bacteria Bradyrhizobium
japonicum 61A68 strain in soybeans.
There are a few studies on ABA production by bac-
teria. For example, Tuomi & Rosenquist (1995) have
determined ABA, IAA and GA3 in microbial sources.
However, there are no reports on the bacteria commonly
found in the human body which also live in the soil and
in water; Proteus mirabilis, P. vulgaris, Bacillus mega-
terium, B. cereus, Klebsiella pneumoniae, Escherichia coli,
which produce IAA, GA3, zeatin and ABA.
The aim of our investigation was to determine IAA,
GA3, zeatin and ABA in the culture media of the bac-
teria P. mirabilis, P. vulgaris, B. megaterium, B. cereus,
K. pneumoniae and E. coli.
Material and methods
Being able to obtain the exponential and stationary
(bacteria generally produce secondary metabolites at this
stage) growth phases, the growth curve of the bacteria
was determined in optimum growth conditions using the
plate count method and the absorbance method (Tor-
tora et al. 1995). Bacteria were grown in the Brain Heart
Broth [ingredients; brain and heart extract, peptones
(27.5 g/l), D(+) glucose (2.0 g/l), sodium chloride
(5.0 g/l), di-sodium hydrogen phosphate (2.5 g/l)],
1062
aerobic growth medium at 120 rev/min at 37 C and
100 ml samples from the exponential and stationary
growth stages of each bacterium were extracted and
purified.
Hundred millilitre samples (extra cellular culture fil-
trates), containing 1 mg of Butylated Hydroxy Toluene
(BHT) to prevent oxidation of the hormones (Ross et al.
1987), were centrifuged at 959 g for 10 min then the
samples were extracted. Extraction, purification and
quantitative determination of IAA, GA3, zeatin and
ABA were carried out, with minor modifications,
according to the methods of Ünyayar et al. (1996) and
Baydar & Ülger (1997). The extraction procedure is
schematized in Figure 1.
Thin Layer Chromatography (TLC) was developed
with a mixture of isopropanol/ammonia/distilled water
Figure 1. Flow diagram outlining the extracts used in purification of IAA, GA3, zeatin and ABA for HPLC.
A. Karadeniz et al.
(10:1:1 v/v/v). IAA, GA3, zeatin and ABA bands and Rf
values belong to samples visualized under 254 nm u.v.
light according to the standard synthetic IAA, GA3,
zeatin and ABA. Bands detected were scraped from the
TLC plate, dissolved in methanol and analysed by HPLC.
IAA, GA3, zeatin and ABA were separated on a
25 cm · 4, 6 mm, 5 lm reverse phase Supelcosil LC-18
column on a Cecil 1100 Series Liquid Chromatograph.
Samples were analyzed under isocratic conditions with
35% methanol (in 1% acetic acid) for IAA, 30% meth-
anol (adjusted to pH 3.0 with 0.1 M H3PO4) for GA3,
70% methanol for zeatin and 55% methanol (in 0.1 M
acetic acid) for ABA. Wavelengths in the u.v. detector
were 280, 208, 254 and 265 nm for IAA, GA3, zeatin
and ABA respectively. The total run time for separa-
tions was approximately 15 min at a flow rate of 1 ml/
Bacterial production of plant hormones 1063

0.16
0.08
0.13
0.00
0,02
0.02
0.05
0.01
0.15

0.09 ± 0.00
0.09 ± 0.00
min. Hormones were quantified by reference to the peak
area obtained for the IAA (Sigma, I-2886), GA3 (Sigma,

±
±
±
±
±
±
±
±
±
G-7645), zeatin (Sigma, Z-0164) and ABA (Sigma,

1.87
0.73
2.60
0.36
0.76
1.12
0.40
0.02
0.42
9 hc
A-1049) standards.

a
0.06 ± 0.01

0.06 50.68 ± 19.89 2.19 ± 0.25 0.06 ± 0.01

0.04 1.14 ± 0.22 0.70 ± 0.09 0.90 ± 0.12
0.01 1.37 ± 0.51 2.04 ± 0.07 0.63 ± 0.01
0.03 2.51 ± 0.52 2.74 ± 0.06 1.53 ± 0.10
0.73 ± 0.27
0.04 ± 0.02
0.77 ± 0.27
0.08 ± 0.03 0.46 ± 0.07 1.36 ± 0.81
0.13 ± 0.03 0.45 ± 0.03 0.09 ± 0.00
0.21 ± 0.04 0.91 ± 0.10 1.45 ± 1.00
E. coli

4 hb
Results and discussion

Table 1. Amounts of free, bound and total IAA, GA3, zeatin and ABA in culture medium samples of the bacteria used in this study in exponential and stationary growth phases.

0.00 1.12 ± 0.22 2.19 ± 0.25 a

The lowest and the highest total levels (expressed as
equivalent standard synthetic IAA, GA3, zeatin and

17 hc
ABA) of plant growth hormones in 100 ml extracellular

0.06 49.56 ± 19.88 a

0.00 0.03 ± 0.02 a

a

0.44 0.03 ± 0.02 a

culture filtrate samples is given in Table 1.

Any plant growth hormone has been determined in the bacterial culture medium samples. bExponential stages of the growth. cStationary stages of the growth.
K. pneumoniae
The presence of IAA has been indicated in bacteria
by different researchers. For example, in a study, Fett
et al. (1987) have observed 0.03–6.37 lg/ml IAA in

6 hb
different Xanthomonas campestris pv. glycines strains

0.44 a
cultured in liquid culture medium containing 0.05%
tryptophan. Costacurta et al. (1998) found 11.6–

±
±
±
±
±
±
±
±
±
55.3 lg IAA in 400 ml of MPI medium without the

15 hc

0.54
0.25
0.79
0.46
0.38
0.84
0.06
0.47
0.53
addition of tryptophan, in the late exponential phase of

a
a
a
the bacterial growth of the different strains of Xan-

0.02
0.02
0.05
1.88
1.84

0.04 ± 0.02
0.04 ± 0.02
0.03 ± 0.01

0.03 ± 0.01
thomonas axonopodis, pathogen to citrus. In another

B. cereus

±
±
±
±
±
study, obligate and facultative methylotropic bacteria

6 hb

0.18
0.18
0.48
3.03
3.51
were able to produce auxins, particularly IAA, in

a
amounts of 3–100 lg/ml (Ivanova et al. 2001). In a

0.26
0.09
0.35
0.20
0.13
0.22
0.08
1.18
1.15
0.01
0.00
0.00
different study on nitrogen-fixing bacteria and pro-

±
±
±
±
±
±
±
±
±
±
±
±
duction of IAA, Pedraza et al. (2004) found that 12 hc

1.49
0.66
2.15
0.75
0.85
1.60
0.89
1.32
2.21
0.04
0.03
0.07
Azospirillum strains produced the highest concentra-
B. megaterium

tions of IAA (16.5–38 lg IAA/mg protein) whereas

55 ± 0.12
0.37 ± 0.21
0.92 ± 0.10
0.54 ± 0.07
0.66 ± 0.03
1.20 ± 0.07
0.15 ± 0.01
0.12 ± 0.04
0.08 ± 0.00 0.27 ± 0.05
0.04 ± 0.01
0.03 ± 0.00
0.07 ± 0.00
Gluconacetobacter and Pseudomonas stutzeri strains
produced lower concentrations of IAA ranging from 1
6 hb

to 2.9 lg/mg protein in a culture medium supple-

Equivalent amounts of plant growth hormones (lg/100 ml)

mented with tryptophan. Researchers suggested that

0.00
0.03
0.00
0.12
0.17
0.17
0.00
IAA production may enable bacteria to promote a
±
±
±
±
±
±
±

growth-promoting effect in plants, in addition to their

12 hc

6.08
0.41
6.49
0.74
1.00
1.74
0.08

nitrogen-fixing ability. We thought that it was neces-

a

a
a

0.44 ± 0.02 a
sary to carry out more investigation into the ability of
1.47
0.11
1.59
0.15
0.21
0.35
0.02
0.00
0.02
0.02

human gut flora bacteria to produce IAA.

P. vulgaris

There are some researches on gibberellins in bacteria.

±
±
±
±
±
±
±
±
±
±
5 hb

4.75
0.59
5.34
1.29
1.01
2.30
0.07
0.01
0.08
0.44

For example, Bottini et al. (1989) determined the

a

amounts of GA1 and GA3 to be approximately 20–

003 ± 0.03
0.14 ± 0.02
2.21 ± 1.18 0.17 ± 0.03

Bound-Zeatin 0.25 ± 0.04 3.16 ± 0.23

805 ± 0.05

40 pg/ml respectively in A. lipoferum cultures using the

2.29 ± 0.13 11.21 ± 0.2

GC–MS technique.
In another study, Taller & Wong (1989) determined
17 hc

cytokinins as equivalent to 0.75 lg of kinetin per litre in

36.00 ± 0.43 a
34.52 ±1.87 a
70.52 ± 1.87 a

4.20 ± 1.75 a
a

4.20 ± 1.75 a

A. vinelandii culture medium. The same researchers re-

2.21 ± 1.18

2.04 ± 0.13
P. mirabilis

ported that Barea & Brown (1974) determined 20 lg of

Species

cytokinin equivalent per liter for Azotobacter paspali

6 hb

and 50 lg/l for A. vinelandii. In addition, it was deter-

Bound- IAA a

Bound-ABA a

mined that the A. vinelandii culture medium contained

plant growth

Total-Zeatin

1 mg of cytokinin per liter.

Bound-GA3

Free-Zeatin

Total-ABA
Total-GA3
Total-IAA

Free-ABA
hormones

Free-GA3
Free-IAA
Plant growth Forms of

To date, the presence of ABA in the microbial

sources has been shown only by Tuomi & Rosenquist
(1995).
In conclusion, from the results obtained, it may be
suggested that the bacteria used in this study produced
hormones

these plant growth hormones as primary and secondary

Zeatin

ABA
GA3
IAA

metabolites in the peptone-rich growth medium.

1064 A. Karadeniz et al.
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