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

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

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,

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¡¦) /

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).

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.

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.

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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