WANG Jian-Gang et al.: Regulation of aroP Expression by tyrR gene in Escherichia coli

　

Regulation
of aroP Expression by tyrR Gene in Escherichia coli

WANG Jian-Gang,
FAN Chang-Sheng^*, WU Yong-Qing,
JIN Rui-Liang, LIU Dong-Xin,
SHANG Liang, JIANG Pei-Hong¹

(Department of Microbiology,
School of Life Sciences, Fudan University, Shanghai 200433, China；
¹Department
of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433,
China)

Abstract    tyrR gene encodes a global regulatory
protein (TyrR), which plays an important role in the transcriptional regulation
of eight transcription units (including tyrR gene itself) whose protein
products catalyze key steps in aromatic amino acid biosynthesis and/or transport.
The aroP gene encodes an integral membrane protein (AroP) that transports
aromatic amino acids through the cell membrane. The transcription of aroP
was reported to be repressed by TyrR. In this work, aroP (p) (aroP
gene carrying its own promoter), aroP (aroP gene without promoter)
and tyrR genes were amplified by PCR from genomic DNA of E. coli K12
and introduced into E. coli WT5. The expression of aroP and
tyrR were detected and the activities of AroP and TyrR were determined.
The introduction of either aroP(p) or aroP elevated the strain’s
transport activity by 1.40 or 1.46-fold respectively. Transformant carrying
tyrR gene showed an ATPase activity 1.69-fold compared with the control.
When the genes were linked in tandem and co-expressed in a plasmid, the relative
AroP transport activity of the strain harboring aroP(p)-tyrR (0.95)
was significantly lower than that of aroP-tyrR (1.31). The results
indicated that TyrR might be able to reduce the expression of aroP gene
by binding with the aroP promoter region in E. coli.

Key words      

tyrR; aroP; metabolic
regulation; phenylalanine transport

Biosynthesis of aromatic amino
acids is a complex process［1，2］, which involves bottleneck enzymes in the
pathway, regulators in the metabolic mechanism and permeases in the transport
system［3］. TyrR, a regulator protein of E. coli, regulates the transcription
of a number of genes involved in the biosynthesis and transport of the aromatic
amino acids positively or negatively［4］. These genes, which are distributed
in eight transcription units, comprise the TyrR regulon［5］. When it binds
to DNA of the regulon, TyrR protein recognizes sequences related to the palindrome
TGTAAAN6TTTAC referred as TyrR boxes［6］. In vitro,
when tyrosine and ATP present, TyrR represses the transcription of tyrB, aroP,
aroL-aroM, aroF-tyrA and tyrP［4，7，8］. The aroG and tyrR genes are also subject
to repression by TyrR, but this repression is independent of tyrosine［9，10］.
TyrR protein also activates the expression of two transport genes, tyrP and
mtr［11－14］. It was established that TyrR forms a complex with ATP before it
acquires the ability of transcription regulation. TyrR protein possesses an
ATPase activity［15］, and the hydrolysis of ATP is very important in the TyrR
regulation process［11，12］.

The aromatic amino acids tyrosine,
tryptophan, and phenylalanine are actively transported across the cytoplasmic
membrane of E. coli. A general aromatic transport permease encoded by the
gene aroP, is responsible for the active transport of the three aromatic acids
into the cell, whose transcription is repressed by TyrR protein［16］.

In this paper, tyrR and aroP genes
of E. coli K12 were cloned and expressed, and the related enzyme activities
were assayed. The effects of TyrR protein on the AroP transport activity were
analyzed. The host strain E. coli WT5 was an engineered bacterium for phenylalanine
production and by this work we wanted to cast some light on the mechanism
of phenylalanine biosynthesis regulation.

1 Materials and Methods
1.1 Strains and plasmids
Bacterial strains and plasmids used in this study are listed in Table 1.

Table 1 Strains and plasmids
used in this study

Strains and plasmids	Characteristics and function	Sources and references
E. coli K12	Donor for gene cloning	Stock in this lab
E. coli DH5α	Receptor for cloning	Stock in this lab
E. coli WT5	Engineered strain for phe-producer	This work
pBluescript SK(-)	ApR vector for cloning	Stock in this lab
pλPR	ApR vector for expression	Reference [1]

1. 2 Culture of bacteria
Strains were grown in Luria-Bertani (LB)［17］. When required, ampicillin was
added at the final concentration of 100 mg/L.

1. 3 Preparation and manipulation
of DNA
Preparation and manipulation of DNA were performed as described by Sambrook
et al.[17].

1.4 Construction of recombinant plasmids
Primers were designed according to the DNA sequence of aroP and tyrR described
in references［16,18］ and were synthesized by BioAsia Company in Shanghai.
Primers used in this study are listed in Table 2. Construction of recombinant
plasmids is shown in Fig.1.

Table 2 Primers for PCR used
in this study

Genes	DNA sequences of PCR primer*	Restriction endoenzymes
tyrR	5′-CCCAAGCTTTCCCATGCGTCTGGAAGTC-3′ 5′-AACTGCAGGCATATTCGCGCTTACTCTT -3′	HindIII PstI
aroP	5′-GGAATTCATGATGGAAGGTCAACAG -3′ 5′-CGGGATCCACGGGTGAGGGCGTAGAG- 3′	EcoRI BamHI
aroP(p)	5′-GGAATTCTTAAGCAACTCATCTTCA -3′ 5′-CGGGATCCACGGGTGAGGGCGTAGAG- 3′	EcoRI BamHI

*Bold: recognized by restriction
endonuclease.

Fig.1 The construction of recombinant
plasmids

1. 5 Maxicell methods for AroP
and TyrR expression
The expression of AroP, an integral membrane protein, was detected by the
method of Sancar et al.［19］. AroP was labeled with［³⁵S-methionine.
The TyrR expression was detected like that of AroP

1.6 Enzyme assays
1.6.1 TyrR-ATPase activity assay TyrR-ATPase activity was determined as
described by Cui et al.［15］.
1.6.2 Analysis of the AroP transport activity and the TyrR repression on
AroP transport activity The cells were harvested by centrifugation at
5000 r/min for 10 min at 4 ℃, and washed with buffer X (7.2 mmol/L K2HPO4,
2.8 mmol/L KH2PO4, 100 mmol/L
NaCl, 1 mmol/L EDTA, pH 7.4). A portion of the cells was used to determine
the ratio of wet and dry weight; the rest were weighed wet and suspended by
5 mmol/L L-phenylalanine (in buffer X). The cells were kept at 37 ℃ on a rotary
shaker at 250 r/min for 30 min. Every 3 min, the phenylalanine concentration
in the supernatant was assayed by the method of Cao et al.［20］, and the uptake
of L-phenylalanine per mg dry weight of cells was calculated.
The virtual value of enzyme activities
was obtained by three experiments on average as described methods.

2 Results
2.1 Gene cloning and recombinant plasmids construction
The aroP (p), aroP and tyrR genes were obtained by PCR from the genome of
E. coli K12. The construction of recombinant plasmids was shown in Fig.1.
The obtained strains and recombinant plasmids were shown in Table 3 and 4.
These constructs were used to transform E. coli WT5.

2.2 AroP and TyrR expression
of E. coli transformed with tyrR and aroP constructs
In E. coli WT5 the expression of TyrR protein (60 kD) had been detected by
SDS-PAGE analysis as shown in Fig.2, and that of AroP protein (37 kD) by [³⁵S］-methionine
in Fig.3.

Fig.2 SDS-PAGE analysis of TyrR
protein in E. coli cells
M， marker； 1， E. coli WT5a (plasmid control); 2, E. coli WT5d; 3, E. coli
WT5e; 4, E. coli WT5f; 5, E. coli WT5 (strain control).

Fig.3 SDS-PAGE analysis of AroP
in E. coli cells, the proteins were labeled by [³⁵S]-methionine
1， pλPR (plasmid control)； 2， E. coli WT5f; 3, E. coli WT5b; 4, E. coli WT5c;
5, E. coli WT5e; 6, E. coli WT5c； 7， E. coli WT5 (strain control).

2.3 The ATPase activity of E.
coli transformed with tyrR and aroP constructs
The transformants were assayed for their ATPase activity, as shown in Table
3. Compared with the control, the transformed strain E. coli WT5d showed the
elevation of ATPase activities by 0.69-fold, and WT5e and WT5f were also increased
in the activity， respectively.

Table 3 ATPase activity of TyrR
in recombinant strains

Strains	Plasmids	ATPase activity of TyrR*
		ATPase activity by [32P] (cpm)	Relative activity
E. coli WT5	None (control)	1.361×106	1.00
E. coli WT5a	pλPR	1.334×106	0.98
E. coli WT5d	pλPR-tyrR	2.305×106	1.69
E. coli WT5e	pλPR-aroP-tyrR	2.094×106	1.54
E. coli WT5f	pλPR-aroP(p)-tyrR	1.838×106	1.35

*ATPase activity of E. coli WT5
was used as the standard, i.e. relative activity of 1.

2.4 AroP transport activities of E. coli transformed with tyrR and aroP
constructs
As shown in Fig.4, in the first 3 min, the transformants’ capabilities of
uptaking L-phenylalanine were saturated. So, the transport activity of AroP
is defined as the absorption of phenylalanine in the first three minutes.

Fig.4 The transport activities
of AroP

Table 4 Transport activity of
AroP in recombinant strains

Strains	Plasmids	Transport activity of AroP*
		Transport activity [nmol /(mg dry weight)］	Relative activity*
E. coli WT5	None (control)	9.63	1.00
E. coli WT5a	pλPR	9.38	0.97
E. coli WT5b	pλPR-aroP	14.10	1.46
E. coli WT5c	pλPR-aroP(p)	13.55	1.40
E. coli WT5d	pλPR-tyrR	8.61	0.89
E. coli WT5e	pλPR-aroP-tyrR	12.63	1.31
E. coli WT5f	pλPR-aroP(p)-tyrR	9.22	0.95

As in Table 4, the strain E. coli
WT5b could uptake L-phenylalanine 14.10 nmol per mg dry weight; the strain
E. coli WT5c could uptake L-phenylalanine13.55 nmol per mg dry weight. The
strain E. coli WT5 could uptake L-phenylalanine 9.63 nmol per mg dry weight.
Transport activity of AroP is defined as the absorption of phenylalanine in
the first three minutes. Thus, the introduction of aroP(p) or aroP genes elevated
the transport activity of L-phenylalanine by 1.40 or 1.46-fold respectively
compared with control.

As shown in Table 4, the relative
AroP activity in E. coli WT5e was 1.31, and in E. coli WT5f was 0.95. The
expression of tyrR gene lowered the transport activity of phenylalanine in
E. coli WT5f, but did not lower the transport activity of phenylalanine in
E. coli WT5e. The former carried aroP gene’s original promoter in addition
to the pλPR promoter in the vector, while the latter
only used pλPR promoter. As we know, the two promoters
in tandem sometimes have lower initiative efficiency compared with one promoter
in one plasmid. But in our study this difference was minimal. E. coli WT5b
(harboring pλPR-aroP) and E. coli WT5c ［harboring pλPR-aroP(p)］
possess the almost identical transport capacity. Thus, it was deduced that
tyrR decreased the AroP transport activity, most likely by repress of the
transcription of aroP gene in E. coli WT5f through binding to the aroP gene’s
native promoter. TyrR might not bind to pλPR‘ promoter
and has little effect on the transcription of aroP gene in E. coli WT5e.

3 Discussion
TyrR is a global regulatory protein and represses the transcription of aroP
gene［4，21，22］. The aroP gene encodes a membrane protein responsible for the
active transport of the three aromatic acids into the cell［13，23］. In this
study, we assayed the activities of AroP and TyrR proteins and found that
the introduction of aroP(p), aroP and tyrR could elevate the related enzyme
activities compared with the control strain. The TyrR protein lowered the
L-phenylalanine transport capacity of E. coli WT5f harboring pλPR-aroP(p)-tyrR
plasmid, but did not lower L-phenylalanine transport capacity of E. coli WT5e
harboring pλPR-aroP-tyrR plasmid. This result might
be due to that TyrR protein could bind with the promoter of aroP(p) and repress
the expression of aroP, but it could not repress the promoter of plasmid pλPR.
This result will help us understand the function of the two genes in the aromatic
amino acid biosynthesis. However, further investigations will be needed to
fully understand the regulation mechanism of aroP expression by tyrR gene.

Acknowledgements We appreciate
the kindly help of Professor YU Long, Professor CAO Kai-Ming, Associate Professor
DONG Ai-Wu, and Dr. YAN Qing-Yan in School of Life Sciences, Fudan University.

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Received: June 23, 2003 Accepted:
September 8, 2003
This work was supported by a grant from the National Natural Science Foundation
of China (No. 30070020)
*Corresponding author: Tel, 86-21-65642808; Fax, 86-21-65650149; e-mail, [email protected]

 

Updated at: 12-18-2003