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FEMS Microbiology Letters 142 (1996) 301-307
Purification and characterization of LasR
as a DNA-binding protein
Zhiying You a, Jun Fukushima ‘, Tetsuyoshi Ishiwata a, Baotong Chang a,
Minoru Kurata ‘, Susumu Kawamoto a, Paul Williams b, Kenji Okuda aj*
a Department of Bacteriology, Yokohama City University School
b Department
of Pharmaceutical
Sciences,
of Medicine, 3-9 Fukuura,
University of Nottingham,
Kanazawa-ku.
University Park, Nottingham
Yokohama 236, Japan
NG7 ZRD, UK
Received 1 July 1996; revised 11 July 1996; accepted 15 July 1996
Abstract
In Pseudomonas aeruginosa, the activator protein LasR and a cognate autoinducer (AI) are required for expression of the
elastase gene (lasB). In the present study, we investigated the binding properties of the P. aeruginosa IasR gene product. The
LasR protein was overexpressed and purified as a glutathione S-transferase (GST) fusion protein. Using gel retardation and
UV cross-linking analysis, we demonstrated that the GST-LasR could bind to a separate site in the ZasB upstream operator
regions 1 and 3 in the presence of the autoinducer. Regions 1 and 3 are located at 105 and 42 base pairs upstream, respectively,
from the IasB transcriptional start site. Our present results clearly demonstrate that LasR is a specific DNA-binding protein
that regulates the transcription of the IasB gene in the presence of an autoinducer.
Keywords:
LasR; Autoinducer; DNA-binding protein; Transcriptional activator;
1. Introduction
In some homologous regulatory systems such as
the control of bioluminescence
in Vibrio [l], the transcriptional activator protein might be activated by
binding to a small diffusible molecule referred to as
an autoinducer
or N-acylhomoserine
lactone to induce expression of different target genes [2]. The
elastase gene (lasB), encoding extracellular
pathogenic factors [3] has been cloned and sequenced [46], and its expression was shown to require the intact
* Corresponding author.
Tel.: +81 (45) 787 2511; Fax: +81 (45) 787 2509;
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IasB gene;
Pseudomonas
aeruginosa
IasR gene and the P. aeruginosa autoinducer identified as N-(3-oxododecanoyl)homoserine
lactone
(OdDHL)
[7,8]. According
to current models of
LasR, as well as of LuxR [9], the LasR polypeptide
is presumed to consist of two domains [lO,ll]. For
activation of transcription,
it has been suggested that
the conjugation
of OdDHL to LasR protein is important [2,7].
However, there is no report which directly demonstrates binding of LasR and OdDHL complexes to
the 1asB DNA fragment. This represents an obstacle
hindering a better understanding
of the mechanism
by which LasR activates
transcription
of the
1asB gene in vitro. Thus, it is important to clarify
the binding of LasR and the ZasB gene. During
Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PZlSO378-1097(96)00286-8
Z. You et ul. I FEMS
302
h4icrohiolog.r Letters 142 (1996) 301-307
the course of LasR overproduction
experiments in
coli, an insoluble
inclusion body appeared in the cells. In this study, we have overcome
this difficulty by constructing
a plasmid that overproduces the 1asR gene product using the prokaryotic expression vector pGEX in E. coli and by developing a procedure for purifying this fusion protein.
Using the purified LasR protein, we established that
the LasR protein exhibits DNA-binding
activity in
the presence of OdDHL. We also identified the two
1asB operators that are involved in transcriptional
activation of 1asB.
Escherichia
2. Materials and methods
2. I. Bacterial strains, plasmids, and culture conditions
P. aeruginosa IF03455
[3] was obtained from the
for Fermentation,
Osaka, Japan. The P.
aeruginosa non-elastase-producing
strain, PA103 [3],
was donated by P.V. Homma (University of Louisville School of Medicine, Louisville, KY). E. coli
JMlOl was used as the host strain for the plasmid
that overproduced
LasR protein. E. coli cultures
were grown in 2XYT or agar at 37°C with ampicillin (100 ltg ml-‘) as needed. Isopropyl-P-p-thiogalactoside (IPTG, 100 uM), synthetic OdDHL (10 pM)
and protease inhibitors (aprotinin 2 ug ml-‘. leupeptin 1 pg ml-‘, PMSF 50 lrg ml-‘) were added as
indicated.
The procedure
used for synthesis of
OdDHL has been previously described [12].
40) containing protease inhibitors and placed in an
ice bath followed by two 30-s sonication steps. For
fusion protein recovery using glutathione-Sepharose
4B (Pharmacia Biotech), GST-LasR extracts were
resuspended in 2 ~01s. of EBC-DTT buffer (EBC
buffer containing
5 mM DTT). Aliquots (15 ml)
were rocked for 30 min at 4°C with 2.5 ml of equilibrated glutathione-Sepharose
4B beads which had
been previously washed three times and resuspended
(final concentration
1: 1, v/v) in EDC-DTT
buffer
containing 0.075% SDS. The beads were then mixed
gently with 500 pl of glutathione elution buffer (10
mM reduced glutathione, 50 mM Tris-HCl, pH 8.0)
and incubated at 4°C for 30 min to elute the fusion
protein from the matrix. Purified LasR fusion proteins were collected by centrifugation
for 10 s at
12000 rpm and stored at -70°C.
2.3. DNA-binding experiments
Institute
2.2. Expression and purification of glutathione
S-transferase
(GSTJ-LasR
fusion proteins
1asR genes derived from P.
and PA103 was amplified by
PCR. PCR products were ligated in frame into the
BamHI site present within the pGEX-3X (Pharmacia
Biotech) polylinker.
Plasmids
encoding
pGEXLasR3455 and pGEX-LasR103,
respectively, were
introduced into E. coli JMlOl containing
the tat
promoter for overproduction
of LasR protein. Extracts were prepared
from cells harvested
at
Asa = 1.5 after IPTG induction.
The cell pellets
were resuspended in 8 ml of sonication EBC buffer
(50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-
The DNA encoding
aeruginosa
IF03455
Interaction
of LasR with the regulatory regions of
and UV crosslinking tests. The probes were generated by PCR and
consisted of sequences from - 118 to - 12 bp containing regions 1 and 3 upstream of 1asB and forming the region 1 probe sequence from - 118 to -92
(+ 1 designates the transcription
start site[l3]). The
region 3 probe has a 20 base pair oligonucleotide
from the -52 to -33 bp region of the 1asB promoter. The DNA-binding
assay was performed according to established procedures [14]. The 20 l.rl reaction
mixture, containing approx. 1 ng of a (5’-32P)-endlabeled DNA fragment (10 000 cpm) and the protein
extracts in 10 mM Tris-HCl, pH 7.8, 50 mM KCl,
1 mM DTT, 1 mM EDTA, 1 mM MgCls, 5% glycerol, 50 pg ml-’ BSA, 5 yg l,rl’ salmon sperm
DNA, was incubated for 30 min at 25°C. The sample
was loaded onto a 5% polyacrylamide
gel and electrophoresed in 0.5 X TBE buffer (45 mM Tris-borate,
1 mM EDTA) at 8 V/cm for the gel retardation
assay. Alternatively,
an equal volume of 2X SDSPAGE gel loading buffer was added, and the same
reaction products were resolved on a 420% SDSPAGE gel by UV cross-linking.
1asB was studied using gel retardation
Z. You et al. IFEMS Microbiology Letters 142 (1996) 301-307
2.4.
SDS-polyacrylamide
gel electrophoresis
303
and
Western blotting
A 4-20% resolving SDS-polyacrylamide
gel was
prepared and stained with Coomassie blue R-250.
Western blotting was performed following published
procedures [ 151 with anti-GST antibody (Pharmacia
Biotech) and anti-LasR antibody. Anti-LasR serum
was obtained from rabbits immunized with synthesized multi antigenic peptide methods [16]. Antibody
titers could still be observed in the 211 diluted samples by ELISA using LasR peptides as coating antigen.
3. Results and discussion
3.1. Overproduction, purcfkation and identt&ation of
the fusion protein as GST-LasR
To establish the existence of a LasR protein capable of regulating 1usB regions, we first carried out
purification of LasR fusion protein. Due to the insolubility of the inclusion bodies in the bacteria, initial efforts to express LasR protein in these cells
proved difficult. However, when LasR protein was
fused to the carboxyl-terminal
of GST, it became
soluble. We generated a series of GST-LasR fusion
proteins (Fig. 1) which were purified using a glutathione-Sepharose
4B column.
Fig. 1. Glutathione S-transferase-LasR
fusion proteins. Diagram
of the pGEX encoded LasR protein described in this report.
LasR is shown with the proposed C-terminal domain responsible
for DNA binding and activation of the IasB gene. The N-terminal domain binds to the autoinducer (AI) which then binds directly to its transcriptional
activator protein, and DNA binding
and gene activation then occur. IF03455 is a positive elastaseproducing strain and PA103 is a non-elastase-producing
strain.
The arrow indicates an amino acid substitution of R179 to W of
PA103.
1
123456
2
3
kDa
Fig. 2. Expression and purification of glutathione S-transferase
fusion proteins. (A) SDS-PAGE. Whole-cell lysates of bacterial
clones expressing the pGEX-3X-encoded
glutathione
S-transferase leader sequence (lane 4) GST-LasR3455
(lane 5). and GSTLasR103 (lane 6) were prepared as described in Section 2. Each
bacterial sonicate was also prepared and incubated with glutathione-Sepharose
4B (lanes 1-3, respectively). Proteins were resolved by electrophoresis
in a 420%
SDS-polyacrylamide
gel
and visualized by Coomassie blue staining. The arrow indicates
the 53 kDa position of the purified GST-LasR fusion protein.
(B) Western immunoblot using anti-LasR antibody. Lane 1, purified GST protein, Lanes 2,3, purified GST-LasR3455
and GSTLasR103.
Expression and purification
of the LasR fusion
proteins are shown in Fig. 2. Total protein fractions
extracted from E. coli JMlOl carrying the IasR gene
transformed with parental pGEX-3X (lane 4) or with
various pGEX-3X recombinants
(lanes $6) were resolved by SDS-polyacrylamide
gel electrophoresis
and visualized by Coomassie blue staining. Sonicated
samples of these bacterial clones were also prepared
and passed through a glutathione-Sepharose
4B column (lanes l-3). The fusion protein that specifically
bound to glutathione-Sepharose
had a molecular
mass of 53 kDa, consistent with the size of the
LasR, i.e. 27 kDa plus the molecular mass of GST,
26 kDa.
We next examined whether these purified proteins
were GST-LasR fusion proteins by using anti-GST
antibody (data not shown) and anti-LasR antibody.
Each of the 53 kDa GST-LasR fusion proteins was
detected (Fig. 2B). From these results it is clear that
the LasR fusion protein was nearly homogeneous.
2. You ef al. I FEMS Microbiology Letters 142 (1996) 301-307
304
A.
ORF
Fig. 3. (A) Gel retardation assay using an IasB upstream probe and LasR protein. The end-labeled IrsB upstream probe used extends
from -118 to -12 bp which includes regions 1 and 3. The LasR3455 protein used was the GST-LasR fusion protein. A mobility shift assay was performed by 5% polyacrylamide
gel/TBE electrophoresis of a (5’.s’P)-labeled DNA fragment. Region 3 competitor DNA was
used at 140-fold molar excess in lane 3. 1 ul anti-LasR antibody was added to lane 2. (B) Organization
of the upstream region and
probes of IasB.
3.2. LasR binds to the regulatory regions c$lusB
LasR has an amino acid sequence similar to the
DNA-binding
domain of the helix-turn-helix
(H-TH) type of DNA-binding
proteins [17]. We have also
determined the location of sequences required for
efficient promoter activity that exists from -135 to
-86 bp (containing region 1) and from -63 to -27
bp (containing region 3) in the 1asB promoter region
using the CAT assay system with our deletion mutants (Fukushima et al., submitted). We next studied
whether LasR protein binds to the regulatory region
containing regions 1 and 3 upstream of 1usB (Fig.
3B). LasR protein was used for DNA-binding
analyses to determine if DNA binding in vitro is correlated with in vivo transcriptional
activation [13].
A gel retardation assay was performed to determine whether purified LasR binds to the DNA fragment containing regulatory regions 1 and 3 of lusB in
the presence of OdDHL. The electrophoretic mobility of the DNA fragment containing these 1usB regulatory regions was significantly retarded by LasR
(two bands were observed; Fig. 3A, lane 1). To ex-
amine the sequence specificity of binding, a 140-fold
excess of unlabeled oligo DNA containing regulatory
region 3 was added to this reaction mixture. Retardation of a lower band was markedly inhibited by
the unlabeled DNA fragment (Fig. 3A, lane 3). On
the other hand, retardation of this band was inhibited by the addition of anti-LasR antibody, and the
highest super-shift band appeared (Fig. 3A, lane 2).
This retardation was not observed using unrelated
control antibodies
or unrelated
DNA fragments
(data not shown). These observations indicate that
LasR protein binds to the regulatory regions of 1asB
and that region 3 is one of the binding sites.
Binding of LasR protein to the DNA fragment
containing
regions 1 and 3 was further examined
by a UV cross-linking
study. As shown in Fig. 4,
two retarded bands were detected by the simultaneous addition of both LasR fusion protein and
OdDHL (lane 6). Furthermore,
a lOO-fold excess of
unlabeled region 1 or a 140-fold excess of unlabeled
region 3 DNA fragments were added as competitors,
and retardation of the band was inhibited (lanes 3,4
in Fig. 4). Purified GST protein was not detected as
Z. You et al. IFEMS Microbiology Letters 142 (1996) 301-307
a distinct band (lane 7). These data confirm the importance of these two DNA fragment regions when
LasR protein binds to the upstream DNA sequence
of the 1asB gene. Since a complex of LasR and
OdDHL has been reported [7] to be important for
the activation of LasR protein, we tested LasR binding with this purified protein. No shifted band was
detected (Fig. 4, lane 5). On the other hand, the
extent of retardation increased when the concentration of OdDHL was increased (lanes 1,2 in Fig. 4)
indicated that an autoinducer is necessary for LasR
protein to bind to regulatory regions of 1asB. We
hypothesized that the IasR gene contains two domains, an autoinducer
response domain and DNA
binding domain. We assumed that the autoinducer
12345678
Fig. 4. W cross-linking
analysis of the 1asE upstream region
with the LasR fusion protein using regions 1 and 3 DNA as a
probe. The end-labeled 1asB upstream probe used extends from
-118 to - 12 bp. The reaction mixtures were fractionated
on a
12% SDS-polyacrylamide
gel. Lane I,2 contained
10 pM
OdDHL and 100 pM OdDHL; lanes 3,4, addition of 140-fold
excess of region 3 oligo DNA and loo-fold excess of region 1
fragment DNA as competitor;
lane 5, no OdDHL; lane 6, no
competitor; lane 7, only purified GST protein.
12
34
305
56
Fig. 5. Cross-linking assay of the LasR fusion protein with the
IusB upstream region 1 using region 1 DNA as a probe. The
end-labeled IasB upstream probe used was the fragment from
-118 to -92 bp DNA. Lane 1, no competitor;
lane 2, IO-fold
excess of region 1 fragment DNA; lane 3, 150-fold excess of
non-specific salmon sperm DNA; lane 4, 2 fl anti-LasR antibody; lane 5, 140-fold excess of region 3 oligo DNA as competitor.
binds to the N-terminal domains of the regulatory
proteins, thereby stimulating binding of the C-terminal domains to DNA target sites [18]. Therefore,
the active form of LasR regulates expression of lu,sB.
To further examine the binding position of the
upstream 1asB promoter, the 1asB binding region
was more precisely studied using the region 1 DNA
fragment as a probe. Upon mixing the probe with
LasR protein, a single band with retarded mobility
could be clearly recognized (Fig. 5, lane 1). Furthermore, competitive experiments with a lo-fold of unlabeled DNA fragment of region 1 and a 140-fold
unlabeled oligo DNA fragment of region 3 resulted
in inhibition of the retardation band (Fig. 5, lanes
2,5), and retardation was not observed using a 150fold unrelated salmon sperm DNA (Fig. 5, lane 3)
indicating that LasR specifically bound to the 1asB
regulatory region.
306
Z. You et ul. I FEMS
Microbiology
Rust et al. [13] have recently shown that the regulatory region of the 1asB promoter contains two
operator sequences that are involved in LasR- and
OdDHL-mediated
1asB activation. The two operator
sequences reported by these investigators
are included in our binding regions (regions 1 and 3).
The results of DNA-binding
analysis of LasR protein to the upstream region of the 1asB promoter
suggest that the combination
of LasR and OdDHL
is necessary for 1asB transcription,
and control of its
expression. In addition, regions 1 and 3 are shown to
be involved in 1asB activation. The region 3 sequence
is centered 42 bp upstream from the 1asB transcriptional start site which is located 141 bp upstream of
the translation initiation codon [13], containing a lux
box-like 20 bp palindrome.
A similar sequence in
region 1 upstream of 1asB contains a half lux boxlike region as well. Both binding regions are similar
to the reported consensus sequence for other genes
controlled by an autoinducer [l 1] and the two LasRbinding regions contain 14 bp repeats that are 70%
homologous.
LuxR studies have also revealed that
the DNA-binding
region specifically binds to sequences upstream from the lux operator [19,20].
Furthermore, we have shown that conserved amphipathic a-helix sequences in LasR protein precede the
H-T-H DNA binding motif [17]. We predicted that
these motifs including H-T-H contribute
to DNA
binding. This previous discovery accompanied
by
our experimental
results confirms that the LasROdDHL complex interacts with the upstream region
3 (from -52 to -33 bp) and region 1 (from -118 to
-92 bp) of 1asB and that is regulated to activate
transcription
of 1asB expression. This report on in
vitro DNA binding is the first to use purified, full
length transcriptional
activator LasR complexes and
to characterize the binding of LasR protein to 1asB
DNA.
Acknowledgments
We are grateful to Ms. T. Kaneko,
Niikura for technical assistance.
I. Oiji, and K.
Letters 142 (1996)
301-307
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