Botanical Studies (2009) 50: 11-20.
*
Corresponding author: E-mail: jcchou@mail.ndhu.edu.tw;
Tel: 886-3-863-3645; Fax: 886-3-863-3630.
INTRODUCTION
Indole-3-acetic acid (IAA) is the most studied auxin
and plays many important roles in plant physiology.
In addition to its regular function in plant growth and
development, IAA may work in concert with growth
regulators such as cytokinins and gibberellins during
plant-microbe interactions. The best-known example is
the the crown-gall disease in plants, which is thought to
be caused by the overproduction of IAA and cytokinins
by invading bacteria such as Pseudomonas, or by DNA-
transformation of the host by Agrobacterium. Plant tissues
infected with the crown-gall disease suffer changes in
their morphogenesis that lead to the growth of tumor-like
crown-galls. Many rhizobacteria are also believed to elicit
changes in root metabolism through the involvement of
plant growth regulators.
Recent studies have concluded that IAA conjugation
and IAA-conjugate hydrolysis may play important roles in
aspects related to IAA physiology and metabolism. IAA
conjugation (both amide-linked and ester-linked) may be
involved in functions such as homeostatic control of free
IAA levels (Bandurski, 1980), storage and subsequent
reuse of IAA (Cohen and Bandurski, 1982), protection
of IAA from other oxidase attack (Cohen and Bandurski,
1978), transport of IAA (Norwacki and Bandurski, 1980),
IAA non-decarboxylative oxidation (Tuominen et al.,
1994), and adaptation to high temperature environment
(Oetiker and Aeschbacter, 1997). Because of the wide
range of processes involving conjugation and hydrolysis,
the study of IAA conjugate metabolism and its impact on
IAA homeostatic control is important for the understanding
of the biochemistry and physiology of IAA in plants.
Earlier studies on IAA conjugate biochemistry include
the discovery of IAA conjugate synthetases such as the
N-indole-3-L-£`-lysine (IAA-Lys) synthetase and its gene
iaaL from Pseudomonas savastanoi (Glass and Kosuge,
1996; 1998; Hutzinger and Kosuge, 1968a; 1968b), the
1-O-indole-3-acetyl-£]-D-glucose (IAA-Glc) synthetase
and its gene iaglu from maize (Kowalczyk and Bandurski,
1991; Szerszen et al., 1994), and the GH3 of Arabidopsis
in IAA-Asp formation (Staswick et al., 2005). In addition,
many hydrolases have been found to target IAA-conjugates
for hydrolysis, such as a large protein complex of 200 kDa
from carrot (Daucus carota) that can hydrolyze several
IAA- amide conjugates (Kuleck and Cohen, 1992), an IAA
amidohydrolase gene family from Arabidopsis thaliana
Gene cloning, nucleotide analysis, and overexpression in
Escherichia coli of a substrate-specific indole-3-acetyl-
L-alanine hydrolase from Arthrobacter ilicis
Sio San LEONG, Wen-Chih CHIU, and Jyh-Ching CHOU*
Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Shou-feng, Hualien 97401,
Taiwan
(Received March 26, 2008; Accepted July 4, 2008)
ABSTRACT.
Indole-3-acetic acid (IAA) is an essential plant hormone and plays many important roles
in plant growth and development. In plants IAA is found mostly as conjugates, thus studies of IAA-
conjugated hydrolases may provide clues on the function and metabolism of IAA during plant growth and
development. We studied an alanine-conjugated IAA hydrolase in bacteria Arthrobacter ilicis in order to
clone IAA conjugate hydrolase genes., and a gene coding for an IAA-Ala hydrolase was successfully cloned
and sequenced without the need of protein preparation and analysis. The procedure involved the design
of universal degenerate PCR primers for IAA amidohydrolases with similar sequence alignment to IAA
amidohydrolases found in the GenBank database. Then, more pairs of specific PCR primers were generated
based on degenerate PCR products. Real time PCR was performed to determine which PCR products were
inducible under specific conditions. A colony screening procedure was performed later to screen a partial
genomic DNA library. Based on this study, a gene with an ORF of 1218 nucleotides was found and then
overexpressed in E. coli. The enzyme activity assay confirmed this gene as IAA-Ala hydrolase gene.
Keywords: Amidohydrolase; Arthrobacter; Auxin; Gene cloning; Hydrolase; IAA; IAA conjugate; Plant
growth regulator; Rhizobacteria.
mOLeCULAR BIOLOGy
pg_0002
12
Botanical Studies, Vol. 50, 2009
(Bartel and Fink, 1995; Davies et al., 1999; LeClere et al.,
2002), and a substrate-specific indole-3-acetyl-L-aspartic
acid hydrolase and its gene iaaspH from Enterobacter
agglomerans (Chou et al., 1996; 1998). Most recently, a
series of Mt IAR3 genes from Medicago truncatula were
reported to carry hydrolase activity against IAA-Asp,
IAA-Ala, IAA-Gly, IAA-Ile, and IAA-Glc (Campanella et
al., 2008).
To facilitate studies on IAA metabolism, several
bacteria carrying IAA-related activities have been studied
since bacterial materials are much easier to handle and
prepare (Chou et al., 1996). The IAA-Asp hydrolase from
E. agglomerans was the first bacterial IAA amidohydrolase
reported and yielded the first purified IAA-amidohydrolase
specific for the hydrolysis of IAA-Asp (Chou et al.,
1998) . This IAA-amidohydrolase has the potential to
become a molecular tool for plant studies because IAA-
Asp has been found to play many important physiological
and biochemical roles in plants. For instance, IAA-Asp
levels can increase dramatically in plants when high
doses of active auxins are applied (Andreae and Good,
1955). In most plants IAA-Asp is also reported as the last
intermediate that retains the indole ring in the IAA non-
decarboxylative oxidation pathway (Normanly, 1997;
Tuominen et al., 1994). In henbane cell cultures IAA-
Asp has been reported to be the main IAA conjugate to
accumulate in high temperature-resistant lines (Oetiker
and Aeschbacher, 1997).
IAA-Ala is another well characterized IAA amide
conjugate (Hangarter et al., 1980) . In earlier reports, IAA-
Ala was considered a storage form of IAA in tomato,
tobacco, and pea and was proposed to function as a
slow-release agent for IAA through regulated enzymatic
hydrolysis (Hangarter and Good, 1981; Hangarter et al.,
1980). Additional in vivo studies have provided evidence
of IAA-Ala hydrolysis in a variety of plants including bean
(Vicia faba) (Bialek et al., 1983), A. thaliana (Campanella
et al., 1996), and Lemna gibba (Slovin, 1997). A novel
amidohydrolase from wheat was also found to cleave
to IBA-Ala instead of to IAA-Ala (Campanella et al.,
2004). The enzyme catalyzing IAA-Ala hydrolysis has
been identified in A. thaliana (LeClere et al., 2002), and
a gene homolog to ILR1, ILL2 (which has strong IAA-
Ala hydrolase activity) has been described. However, as
in many other plant IAA hydrolases, this enzyme is not
highly specific for IAA-Ala. For instance, it was observed
that in E. coli GST-ILL2 overexpression lines ILL2 can
efficiently hydrolyze IAA conjugates such as IAA-Lys,
IAA-Met, IAA-Pro, IAA-Phe, IAA-Ser, IAA-Thr, IAA-
Tyr and IAA-Val, with hydrolase activities higher than 100
nmol IAA per mg of crude extract per minute. Therefore it
was important to search for a substrate-specific enzyme for
studies on IAA-Ala hydrolysis.
Previously we found that bacterial amidohydrolases
exhibited a more restricted substrate requirement and this
specificity suggested a fundamental difference between
the better-studied plant enzymes and their microbial
counterparts (Chou et al., 1996; Chou et al., 2004). Based
on these results, we set to use bacteria as a promising
source of highly substrate-specific amidohydrolases.
In an earlier report, we successfully developed an
inexpensive and efficient method to screen rhizobacteria
for IAA-amino acid hydrolase activities (Chou and Huang,
2005). By using N-acetyl-L-alanine (a common peptide
synthesis precursor) as an inducer we reported for the
first time IAA-Ala hydrolase activity in Arthrobacter
ilicis. Since IAA amidohydrolases are not expressed
in regular bacterial growth conditions, the molecular
analyses of these bacterial IAA amidohydrolases remained
difficult. The standard approach for cloning a gene such
as amidohydrolase requires the purication of the enzyme,
obtention of partial amino acid sequences, analysis of
partial DNA sequences based on PCR and southern
blotting, and finally, DNA sequencing of the entire gene
(Chou et al., 1998; Lin et al., 2007). The first two steps
involving protein purification and sequence analysis are
the most difficult and time-consuming since the enzyme
is inducible and may not be stable. In this paper, we
adopted a molecular approach in order to clone IAA
amidohydrolase genes without the protein preparation
steps mentioned above. By employing this approach, we
successfully identified and cloned the bacterial IAA-Ala
hydrolase from A. ilicis.
mATeRIALS AND meTHODS
Bacterial strains and plasmids
The A. ilicis strain D-50 and the conditions for its
culture have been described previously (Chou and Huang,
2005). The E. coli strain NovaBlue was used as host for
plasmid constructs derived from pRSET-C (Invitrogen,
Carisbad, California, United States), pGEM-T (Promega,
Madison, Wisconsin, United States), and pETBlue-1 based
DNA cloning (Novagen, Darmstadt, Germany). The E. coli
strain KRX (Promega, Madison, Wisconsin, United States)
was used as the host for pETBlue-1-iaalaH-His for protein
expression.
Genomic DNA extraction
Genomic DNA of A. ilicis D-50 was purified based
on a modified miniprep protocol described by Wilson
(1989). Bacteria from a single colony of A. ilicis D-50
were transferred to a 5 ml LB medium and cultured at
37¢XC with aeration for 16 h. A 1.5 ml aliquot of bacterial
cells was pelleted in a microcentrifuge. The pellet was
then resuspended in 570 £gl of TNE buffer (10 mM Tris-
HCl at pH8 with 10 mM NaCl and 10 mM EDTA), 20
£gl of 20% sodium dodecyl sulphate (SDS), and 20 £gl
lysozyme (30 mg ml
-1
) and incubated at 37¢XC for 1 h. After
1 h incubation for 1 h, 160 £gl of 3 M NaCl was added,
followed by 80 £gl of CTAB (cetyl trimethyl ammonium
brombide) in 0.7 M NaCl solution. The sample was
incubated at 65¢XC for 10 min to lyse the cells. The genomic
DNA was extracted with an equal volume of phenol. The
pg_0003
LEONG et al. ¡X Bacterial IAA-Ala hydrolase gene cloning
13
solution was mixed by gentle vortexing and centrifuged
at 9,400 ¡Ñg for 5 min in a microcentrifuge at room
temperature to separate the aqueous and organic phases.
The upper aqueous phase containing the genomic DNA
was transferred to a fresh tube and extracted with equal
volume of phenol/chloroform (1:1, v/v) to remove all the
protein contaminants and membrane debris. The solution
was mixed by vortex and then spun in a microcentrifuge
for 5 min. The upper aqueous phase was transferred to a
fresh tube and the DNA was precipitated with 0.6 volume
isopropanol at -20¢XC overnight. Later, the DNA was
isolated by centrifugation, air-dried, and redissolved in 50
£gl of TE buffer. To obtain RNA-free genomic DNA, 50 £gl
of genomic DNA was treated with 1 £gl DNase-free RNase
(500 ng £gl
-1
). The concentration and purity of the DNA
sample was determined by spectrophotometric ratio assay
at 260 nm and 280 nm.
Bacterial induction and total RNA extraction
Bacteria from a single colony of A. ilicis D-50 were
transferred to 100 ml LB medium and cultured at 37¢XC to
OD
600
=0.8. The cells were pelleted in a microcentrifuge
and resuspended in 100 ml BSM (Chou et al., 1996). The
resuspended cells were divided equally into four parts.
The first part of bacteria was added N-acetyl-L-alanine
as an inducer to the final concentration at 10 mM. The
same treatment was applied to the second and third parts
of bacteria, but with different inducers, IAA-alanine and
acetamide, respectively. The last part of bacteria was not
treated with any inducer. The induction was performed at
room temperature for 4 h and the bacteria were subjected
for total RNA extraction.
For total RNA extraction, a 1.5 ml bacterial culture
was centrifuged at 8,000 rpm, 4¢XC for 5 min to collect the
pellet. A 250 £gl protoplasting buffer (15 mM Tris-HCl at
pH8 with 0.45 M sucrose and 8 mM EDTA) was added
to the pellet and completely mixed by pipetting. Another
500 £gl protoplasting buffer and 6 £gl lysozyme (50 mg
ml
-1
) were then added. The sample was mixed well, put
in ice for 15 min, and then centrifuged at 7,000 rpm, 4
o
C
for 5 min to collect the pellet. A 38 £gl lysing buffer (10
mM Tris-HCl at pH 8 with 10 mM NaCl, 1 mM sodium
citrate, and 1.5% SDS) was added to resuspend the pellet
with pipeting. A 1.2 £gl DEPC (diethylpyrocarbonate) was
then added and mixed gently. The sample was centrifuged
for a few seconds and incubated at 37¢XC for 5 min. After
incubation, the sample was transferred to the ice for 5 min.
Another 19 £gl saturated NaCl was added and continued to
incubate on ice for 10 min. The sample was centrifuged
at 13,000 rpm, 4¢XC for 10 min; then, the supernatant was
transferred to a new tube. A 150 £gl 100% EtOH was added
to the sample and the sample was, then, stored at -20¢XC
overnight. After the overnight cold treatment, the sample
was centrifuged at 14,000 rpm, 4¢XC for 15 min to collect
the pellet. The pellet was washed with 150 £gl of 70%
EtOH, centrifuged at 13,000 rpm, 4¢XC for 5 min, and air-
dried before being redissolved in 15 £gl of DEPC-treated
H
2
O. The concentration and purity of the RNA sample was
determined by spectrophotometric ratio assay at 260 nm
and 280 nm.
Protein alignment and preparation of the
universal primers
The IAASPH (IAA-Asp hydrolase) amino acid
sequence was used to search the NCBI GenBank database
for similar IAA-amidohydrolases. A total of 26 protein
sequences were chosen for protein sequence alignment
based on the CLC Free Workbench software (CLC
bio, Cambridge, Massachusetts, United States). Three
conserved domains (domain 1 through domain 3 in the
order from N-terminus to C-terminus) were chosen to
design a degenerate primer pair for PCR. The forward
primer (IAALA-F1) was designed based on the domain
1 sequence and the reverse primer (IAALA-R1) was
designed based on the domain 3 sequence. The domain 2
was used to confirm the PCR products.
Degenerate PCR and PCR product analysis
The two degenerate oligonucleotides, IAALA-F1 (5¡¦-
MGN GYN GAY ATG GAY GCN YT-3¡¦) and IAALA-R1
(5¡¦-CCY T CY TCN GCN GGY TGR AA-3¡¦), were used
as PCR primers and the genomic DNA of A. ilicis D-50
as the PCR template to synthesize the DNA fragments.
The PCR temperature profile was one cycle at 94¢XC (5
min) followed by 30 cycles of 55¢XC (30 s): 72¢XC (15 s):
95¢XC (30 s). DNA products of approximately 200 to 230
bp were expected for this PCR experiment. DNA products
obtained from the degenerate PCR were cloned into the
DNA cloning vector pGEM-T, maintained in the E. coli
NovaBlue strain, and extracted later for DNA sequencing
(Seeing Bioscience Co., Ltd, Taipei, Taiwan). Based on the
DNA sequencing information, two pairs of non-degenerate
PCR primers (IAALA-F3/IAALA-R3 and IAALA-F4/
IAALA-R4) were successfully designed based on two
PCR products for reverse transcription and real time PCR
experiments.
Reverse-transcription experiments
Based on the study of degenerate PCR primers, two
PCR products were found and sequenced. To obtain
the first strand of cDNA for the following real time
PCR experiments the following sequences were used:
IAALA-R3 (5¡¦-GGT TGG AAC ATG AGT ACG AC-3¡¦)
based on the first DNA product sequence, IAALA-R4 (5¡¦-
AAG ACT GCG ATC AGG GTG-3¡¦) based on the second
DNA sequence, and the 16s rRNA lower primer (5¡¦-ACG
GCT ACC TTG TTA CGA CTT-3¡¦) as an internal control.
Total RNA samples from different induction treatments
were used as templates. Each experiment mix contained
2 £gl of 1 £gM 16s rRNA lower primer, 2 £gl of 1 £gM
IAALA-R3 or IAALA-R4, and 8 £gl DEPC-treated-H
2
O to
the final volume of 12 £gl. The reaction mix was incubated
at 70¢XC for 5 min and then transferred to ice for another
2 min. Another 2 £gl 5 mM dNTP mix, 1 £gl 100 mM DTT,
pg_0004
14
Botanical Studies, Vol. 50, 2009
4 £gl 5X first strand synthesis buffer, and 1 £gl reverse-
iT RTase blend (ABgene, Taipei, Taiwan) were added to
the reaction mix. The reverse-transcription reaction was
performed at 47¢XC for 50 min and then inactivated by
75¢XC for 10 min. The first strand cDNA samples were
stored at -20¢XC for real time PCR experiments.
Real time PCR
Real time PCR was performed to determine whether
the IAA amidohydrolase candidate DNAs were inducible
under the above induction conditions or not. Each
induction treatment was tested with three sets of PCR
primers, including IAALA-R3/IAALA-F3 for the first
DNA product, IAALA-R4/IAALA-F4 for the second
DNA product, and 16s rRNA-F (5¡¦-TCG AAC GAT GAT
CCC AGC TT-3¡¦)/16s rRNA-R (5¡¦-TCC GGT ATT AGA
CCC AGT TTC C-3¡¦) as internal controls. The real time
PCR mix contained 2 £gl of the above reverse transcription
product as template, 1.8 £gl of each appropriate primer pair,
12.5 £gl of Absolute QPCR SYBR Green Mix (ABgene,
Taipei, Taiwan), and addition of H
2
O to a final volume of
25 £gl. The reaction condition was 1 cycle 50¢XC for 2 min;
1 cycle 95¢XC for 15 min; 40 cycles 95¢XC (15 sec): 65¢XC (1
min). In addition, the dissociation protocol was set at 60¢XC
(ABI prism 7000 sequence detection system, Applied
Biosystems, Foster City, California, United States).
IAA-Ala hydrolase gene cloning
The genomic DNA of A. ilicis D-50 was digested by
various restriction enzymes and analyzed under a 1%
agarose TAE gel electrophoresis. The DNA from three
agarose gel blocks containing different DNA sizes of 1-3
kb, 3-5 kb, and 5-7 kb was gel extracted. The extracted
DNA was tested to see whether or not it contained the
inducible DNA of the earlier degenerate PCR by regular
PCR with IAALA-R3/IAALA-F3 primer set. (The first
DNA product was confirmed inducible from the real time
PCR experiments). The DNA fragments with positive
PCR results were cloned to a plasmid vector pRSET-C and
transformed into E. coli NovaBlue.
The transformed E. coli NovaBlue colonies were
colony-screened by PCR with IAALA-R3/IAALA-F3
primer set. The temperature profile used was one cycle at
94¢XC (5 min) followed by 30 cycles of 58¢XC (30 s): 72¢XC
(15 s): 95¢XC (30 s). The colonies that yielded a 161 bp
product were subjected to further DNA sequencing and
analysis.
Construction of plasmid DNA with IAA-Ala
hydrolase (IAALAH) gene
To clone the IAA-Ala hydrolase gene (iaalaH) into
a protein expression vector, the IAALAH-U-ATG
primer (5¡¦-ATG ACC ATC GCC GCT GAC GC-3¡¦)
and IAALAH-L-SpeI primer (5¡¦-ACT AGT GTT GGC
GGC GAG GG-3¡¦) were used as gene cloning primers
to amplify iaalaH from A. ilicis D-50 genome by PCR.
The PCR product was cloned into the pETBlue-1 vector
and transformed into E. coli NovaBlue. For fast protein
purification, a His-tag linker was generated by annealing
of His-linker-U/SpeI-EcoRI (5¡¦-CTA GTC ATC ACC
ATC ATC ACC ACT-3¡¦) and His-linker-L/SpeI-EcoRI
(5¡¦-AAT TAG TGG TGA TGA TGG TGA TGA-3¡¦)
primers and was incorporated into the EcoRI and SpeI
treated pETBlue-1-iaalaH by ligation. The plasmid was
transformed into E. coli KRX for protein expression. All
cloned DNA were subjected to DNA sequencing analysis.
Protein expression and enzyme activity assay
In order to confirm whether or not the cloned gene is
the IAA-Ala hydrolase gene, the E. coli KRX containing
pETBlue-1-iaalaH-His was cultured in LB medium and
induced based on the manufactural protocol (Promega,
Madison, Wisconsin, United States). Qualitative enzyme
assays were performed by incubating 95 £gl of crude
extract with 5 £gl of 20 mM IAA-Ala at 30¢XC for 30
min. The reaction was terminated by the addition of 20
£gl 85% H
3
PO
4
, and extracted by 200 £gl EtOAc. The
EtOAc extract was analyzed by silica gel 60-F
254
thin-
layer chromatography (TLC) using a solvent system of
chloroform:methanol:H
2
O (85:14:1, v/v/v) (Chou and
Huang, 2005). The TLC plate was expressed by immersion
in Ehmann¡¦s reagent (Ehmann, 1977) for about 5 s and
was incubated at 100¢XC for 1 min. Both IAA and IAA-Ala
were identified by their bright-blue color.
ReSULTS
The universal PCR primers for IAA
amidohydrolase gene cloning
Based on the three highly conserved domains of 26
representative IAASPH homologs, a pair of degenerate
PCR primers, IAALA-F1 and IAALA-R1, were generated
(Figure 1). The IAALA-F1 (5¡¦-MGN GYN GAY ATG
GAY GCN YT-3¡¦) was derived from the conserved domain
1, Arg-Ala/Val-Asp-Met-Asp-Ala-Leu, and IAALA-R1
(5¡¦-CCY TCY TCN GCN GGY TGR AA-3¡¦) was derived
from conserved domain 3, Phe-Gln-Pro-Ala-Glu-Glu-
Gly (M represent A and C; N represent A, C, T and G; Y
represent C and T; R represent A and G).
Two DNA fragments were found based on a
degenerate PCR
A degenerate PCR was performed with IAALA-F1/
IAALA-R1 as PCR primers and genomic DNA of A .
ilicis as template. Two PCR products located around
200 bp and 230 bp were found and named as DNAa
and DNAb, respectively (Figure 2A). Both DNAs were
subjected for DNA sequencing and found both containing
the conserved domain 2, Met-His-Ala-Cys-Gly-His-Asp,
which was used for confirming the DNAs as candidates
of IAA amidohydrolase genes (Figure 2B). Two pairs of
non-degenerate primers were designed according to the
sequence information of DNAa and DNAb and labeled
as IAALA-F3 (5¡¦-CCC GTC CAG GAA ACA ACC-3¡¦) /
pg_0005
LEONG et al. ¡X Bacterial IAA-Ala hydrolase gene cloning
15
Figure 1. Partial alignment of 26 selected IAA-Asp hydrolase homologs (A) and strategy for generation of universal degener-
ate PCR primers for IAA am idohydrolase genes based on the conserved domains 1 and 3 (B). The protein assignments, organ-
ism names and accession numbers for the 26 protein sequences are listed as follow: 1, IAASPH (IAA-Asp hydrolase) from E.
agglomerans (AF006687); 2, a putative am idohydrolase from E. coli (AE000231); 3, a probable amino acid amidohydrolase
from Clostridium perfringens (AP3189); 4, a putative hydrolase from Haemophilus influenzae (U32740); 5, an unknown protein
from Pasteurella multocida (AE006168); 6, a hypothetical protein from Halobacterium sp. (AE004999); 7, a theoretical N-acyl-
L -am ino acid amidohydrolase from Synechocystis sp. (D90917); 8, a theoretical N-acyl-L -ami no acid am idohydrolase from
Fusobacterium nucleatum (AE010570); 9, a theoretical N-acyl-L-am ino acid amidohydrolase from Nostoc sp. (AP003598); 10,
a putative hippurate hydrolase from Agrobacterium tumefaciens (AE009220); 11, an IAA-amino acid hydrolase homolog from
P yrococcus furiosus (AE010182); 12, a hypothetical amino acid amidohydrolase from Pyrococcus horikoshii (AP000003); 13,
an amino acid hydrolase from Pyrococcus abyssi (AJ248287); 14, a thermostable carboxypeptidase from Sulfolobus solfatari-
cus (AE006750); 15, a putative amino acid amidohydrolase from E. coli (AP002567); 16, a putative hippurate hydrolase from
Sinorhizobium meliloti (AL591784); 17, a probable hydrolase from Pseudomonas aeruginosa (AE004718); 18, a putative N-acyl-
L -amino acid amidohydrolase from Deinococcus radiodurans (AE001894); 19, a thermostable carboxypeptidase from S. solfa-
taricus (AE006803); 20, a hypothetical amidohydrolase from P. horikoshii (AP000004); 21, an N-acyl-L-amino acid amidohy-
drolase from Staphylococcus aureus (AP003359); 22, ILL1 from A. thaliana (U23795); 23, a putative IAA-Ala hydrolase from
Oryza sativa (AP003924); 24, ILL2 from A. thaliana (U23796); 25, an IAA-amino acid hydrolase from A. thaliana (AF081067);
26, an IAA-amino acid hydrolase from A. thaliana (U23794).
pg_0006
16
Botanical Studies, Vol. 50, 2009
IAALA-R3 (5¡¦-GGT TGG AAC ATG AGT ACG AC-3¡¦)
and IAALA-F4 (5¡¦-TTG CCC GTC CAA GAG GC-3¡¦) /
IAALA-R4 (5¡¦-AAG ACT GCG ATC AGG GTG-3¡¦). The
IAALA-R3 and IAALA-R4 were also used to generate
first strand cDNA as the templates for real time PCR
experiments.
One DNA product was confirmed inducible by
N-acetyl-L-alanine or IAA-L-alanine based on
real time PCR analysis
For the real time PCR experiments, we used the
IAALA-F3/IAALA-R3 primer set for detection and
quantification of DNAa, and the IAALA-F4/IAALA-R4
primer set for DNAb. The PCR templates were prepared
from four different induction treatments, including 10 mM
N-acetyl-L-alanine, 10 mM IAA-L-Ala, 10 mM acetamide,
and without inducer as a control. Results show that DNAa
was significantly inducible by N-acetyl-L-alanine and
IAA-L-Ala (Figure 3A), but DNAb was not (Figure 3B).
When we set the non-induced sample as 1, we could
quantify the relative induction of DNAa by acetamide,
values for N-acetyl-L-Ala and IAA-L-Ala were 1.09¡Ó0.10,
1.43¡Ó0.02, and 3.30¡Ó0.46, respectively, and 1.46¡Ó0.34,
1.02¡Ó0.12, and 1.29¡Ó0.35, respectively, for DNAb. Based
on these results, DNAa was concluded to be an inducible
gene and a candidate for an IAA-Ala hydrolase gene.
Gene cloning and nucleotide sequence analysis
of IAA-Ala hydrolase from A. ilicis
To clone and analyze the full-length sequence of
gene containing DNAa, we made a partial DNA library
which contained 1~3 kb DNA fragments derived from
the BamHI/KpnI double digestion of A. ilicis genomic
DNA. The DNA fragments were tested positive by PCR
with IAALA-F3/IAALA-R3 as primers indicating these
DNA fragments contained DNAa. The DNA fragments
were ligated to pRSET-C treated with BamHI/KpnI
double digestion. A PCR-based colony screening using
IAALA-F3/IAALA-R3 as primers was performed to the
subgenomic library. One of 96 colonies was tested positive
and picked up for further analysis for IAA-Ala hydrolase
gene. From sequence analysis of the plasmid from this
colony we found a partial open reading frame (Figure 4).
Figure 2. The degenerate PCR results analyzed on a 2% TAE
agarose gel elec trophoresi s (A) a nd the DNA se quencing
results of the PCR products (B). Two PCR products at 200 bp
(DNAa) and 230 bp (DNAb) were identified and sequenced,
respect ively. Th e speci f ic pr i mer pai rs of I AA LA-F3/
IAAL A-R3 and IAALA-F4/IAALA-R4 were designed a nd
ge ne rate d for fur the r real t ime PCR a nd colony screeni ng
based on DNAa and DNAb sequences.
Figure 3. Expression of genes containing DNAa (A) and DNAb (B) under various induction conditions based on real time PCR
analysis with IAALA-F3/IAALA-R3 for detection of DNAa and IAALA-F4/IAALA-R4 for DNAb, respectively.
pg_0007
LEONG et al. ¡X Bacterial IAA-Ala hydrolase gene cloning
17
Therefore, another subgenomic library was made from a
BamHI/NcoI double digestion of A. ilicis genomic DNA.
Based on the same screening procedure, we obtained
the rest of the DNA sequence of the gene containing
DNAa. The complete open reading frame contains
1218 nucleotides and encodes a protein of 405 amino
acids (Figure 5) with the calculated molecular weight
of 43044.69. The complete DNA sequence of this gene
was deposited to GenBank database under the accession
number "EU400596" and named as "indole-3-acetyl-L-
alanine hydrolase gene".
Overexpression and enzyme activity assay of
bacterial IAA-Ala hydrolase
To confirm that the gene product was capable of
hydrolyzing IAA-Ala, we cloned this gene into a protein
expression vector pETBlue-1 as pETBlue-1-iaalaH-His
which contains the full ORF of this gene and a His-tag
for easy protein purification under the control of a T7
promoter (Figure 6). The protein was over-expressed in
E. coli KRX strain under the induction of 0.1% rhamnose
and purified through His-bind nickel column (Novagen,
Darmstadt, Germany). The purified protein was then
subjected for the enzyme activity assay. The results
showed that IAA-Ala was the major subtrate while IAA-
Phe and IAA-Asp were weakly hydrolyzed by this protein
(Figure 7). The enzyme activity profile completely agreed
with our earlier report (Chou and Huang, 2005) and
strongly supported that the gene containing DNAa is the
gene of IAA-Ala hydrolase.
DISCUSSION
As part of efforts to better understand the metabolism
of IAA-conjugates we have isolated the gene encoding a
bacterial IAA-Ala hydrolase from A. ilicis, strain D-50.
The gene was identified by DNA sequence analysis and
confirmed by the assay of enzyme activity, that is IAA-Ala
hydrolysis in cell-free extracts of E. coli transformed with
pETBlue-1-iaalaH-His.
Degenerate oligonucleotides derived from two highly
conserved peptide domains of amidohydrolases were
used to generate another pair of specific DNA primers
for first strand cDNA generation, real-time PCR, and
colony screening of target DNA. There were no protein
purification steps involved during the gene identification
and cloning which greatly reduced the difficulty of
Figure 4. The restriction endonuclease map of a 4.4 kb DNA
containing iaalaH ORF from A. ilicis ge nom ic DNA. The
"B", "K", and "N" denote BamHI, KpnI, and NcoI, respec-
t i ve l y.
Fi gure 5. Nucleotide sequence and deduced amino acid
seq uence of the iaalaH f rom A. ilicis. The nu cleo tide
sequence has been deposited in the GenBank database under
the accession number EU400596.
Figure 6. The pETBlue -1-ia ala H-His con st ruct based on
pETBl ue-1 expr essio n vecto r w it h iaalaH and His-tag
sequences for overexpression in E. coli KRX under T7 pro-
moter cont rol.
pg_0008
18
Botanical Studies, Vol. 50, 2009
protein preparation when dealing with a novel and
inducible enzyme like the bacterial IAA-Ala hydrolase
reported in this report. We consider this approach as a
fast, and powerful for cloning genes from similar IAA
amidohydrolases.
The deduced amino acid sequence of iaalaH from
A. ilicis was subjected to GenBank Blastp search and
similarities were found to other proteins. Most of the
hits came out as hypothetical peptidases, such as metal-
dependent amidases, amino hydrolases, amidohydrolases,
hippurate hydrolases, carboxypeptidase, and N-acetyl-L-
amino hydrolases. Surprisingly, when pairwisely aligned
the IAA-Ala hydrolase (IAALAH) of A. ilicis with IAA-
Asp hydrolase (IAASPH) of E. agglomerans, we found
only 26% of identities and 42% of positives. The two
bacterial IAA-amino acid hydrolases shared relatively
small sequence similarity indicating a much complex
diversity in molecular characteristics within bacterial
IAA amidohydrolases. The protein sequence was also
confidently predicted carrying a Pfam:Peptidase_M20
domain and a Pfam:M20_dimer domain inside the region
of Pfam:Peptidase_M20 domain based on a Simple
Modular Architecture Research Tool (SMART) at the
website http://smart.embl-heidelberg.de/indicating that
the tertiary protein structure relationship may be involved
in the enzyme activity. Further analysis of this protein
structure in relation with enzyme activity will be of
interest to solve this issue.
The molecular size of this protein is of approximately
43 kDa and is quite different from the major induced
protein at 40 kDa in our earlier report (Chou and Huang,
2005). We examined the 40 kDa protein based on an LC-
MS-MS analysis (data not shown) and found this protein
to be unrelated to IAA-Ala hydrolysis. This result suggests
that several pathways and proteins might be induced
during the induction conditions in bacteria. Thus in this
particular bacterium, A. ilicis, the induction of IAA-
Ala hydrolase might not be the only and most important
induced pathway. Further analysis of the bacterial
induction mechanism is underway to clarify this issue.
In this report we found that IAA-Ala is the strongest
inducer for IAA-Ala hydrolase (Figure 3) and that
N-acetyl-L-alanine is the second strongest one. This
result agrees with the induction pattern of IAA-Asp
hydrolase from E. agglomerans reported in our earlier
paper (Chou et al., 1996) where we determined which
enzyme/substrate combination was optimal for induction
of activity. However, this result conflicted with another
paper (Chou and Huang, 2005), which reported IAA-
Ala is not an inducer for IAA-Ala hydrolase. In current
study, we performed several repeats of this experiment and
concluded that IAA-Ala is a strong inducer for the IAA-
Ala hydrolase induction.
In conclusion, we have cloned the IAA-amino acid
hydrolase gene from bacteria through simple molecular
biology procedures. By using the degenerate PCR primers
and real time PCR for detection of gene expression, we
bypassed the examination of protein induction, enzyme
assays, and protein purification and sequencing. This
technique may greatly reduce the study time and increase
the detection sensitivity for this kind of experiment. We
expect that this experimental procedure will be applied for
the isolation of similar, inducible IAA amidohydrolases
and facilitate studies on IAA metabolism in plants.
Acknowledgements. This work was supported by a
grant from the R.O.C. National Science Council NSC-94-
2311-B-259-002 to JCC. Especially thanks to Mr. Adam
Allen for the English editing.
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