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Journal of Bacteriology, October 2001, p. 5529-5534, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5529-5534.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Promoter Specificity Elements in Pseudomonas
aeruginosa Quorum-Sensing-Controlled Genes
Marvin
Whiteley and
E. P.
Greenberg*
Department of Microbiology, University of
Iowa, Iowa City, Iowa 52242
Received 12 April 2001/Accepted 26 June 2001
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ABSTRACT |
The LasR-dependent and RhlR-dependent quorum-sensing systems are
global regulators of gene expression in Pseudomonas
aeruginosa. Previous studies have demonstrated that promoter
elements of the quorum-sensing-controlled genes lasB and
hcnABC are important in density-dependent regulation. We
have identified LasR- and RhlR-dependent determinants in
promoters of quorum-sensing-controlled genes qsc102, qsc117
(acpP), and qsc131 (phzA to
-G) by in silico, deletion, point-mutational, and
primer extension analyses. Each of these genes (in addition to
lasI and rsaL) is activated by LasR, and
qsc117 and qsc131 also respond to RhlR. Point mutations in the
promoters of the LasR-specific gene, qsc102, relax specificity so that
this promoter can respond to RhlR in addition to LasR. Our findings
indicate that quorum-sensing-controlled promoters in P.
aeruginosa are either specific for LasR or respond to both LasR
and RhlR and that critical bases in the promoter elements determine
specificity.
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INTRODUCTION |
The opportunistic human pathogen
Pseudomonas aeruginosa possesses two quorum-sensing systems:
the LasR-LasI system and the RhlR-RhlI system. These systems are global
regulatory elements that control the expression of approximately 50 to
200 genes (26). LasI catalyzes the synthesis of
N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) (16), and LasR
is a 3OC12-HSL-dependent transcriptional
activator (11). RhlI catalyzes the synthesis of the signal
N-butyryl-L-homoserine lactone
(C4-HSL) (17), and RhlR is a
C4-HSL-dependent transcriptional activator
(3). These two quorum-sensing systems are interrelated in
that LasR activates the expression of the rhlR and
rhlI genes (13, 19, 26).
RhlR and LasR are members of a family of transcription factors that
influence expression of genes with specific palindromic sequences in
their promoter regions. These sequences have been called
lux-box-like sequences. The lux box is a 20-bp
inverted repeat centered at 
41.5 bp from the start of the
Vibrio fischeri lux operon (6). Inverted
repeats similar to the lux box have been identified in the
promoter regions or putative promoter regions of a number of
quorum-sensing-controlled (qsc) genes of P. aeruginosa. lux-box-like elements have been shown to be involved in
P. aeruginosa quorum sensing in only two cases. One case is
lasB, which is controlled primarily by RhlR but which is
also influenced by LasR (15, 26). Mutational analyses have
demonstrated that there are two lux-box-like elements
involved in LasR control of lasB (1, 22). The
other case is the promoter of the hydrogen cyanide (hcn)
operon (20). Both LasR and RhlR can exert control over this operon (20, 26). There are genes that appear to be
controlled solely by 3OC12-HSL-LasR but not
C4-HSL-RhlR, and there are genes that appear to
be controlled primarily by C4-HSL, but these
genes also respond to 3OC12-HSL (5, 24,
26). We do not understand the promoter specificity determinants
for 3OC12-HSL-LasR versus those for
C4-HSL-RhlR.
Some P. aeruginosa qsc genes are regulated by additional
factors. Although they require acyl-HSL signals for activation, they are repressed until stationary phase even in the presence of acyl-HSLs (26). These are termed late qsc genes. Other genes, early
qsc genes, show activation upon addition of acyl-HSL signals even in
early logarithmic phase. We have analyzed the promoters of several
P. aeruginosa early qsc genes. These promoters all possess lux-box-like elements that we show are involved in qsc
transcription. Furthermore, we show that two bases in
lux-box-like elements define LasR-specific promoters.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, growth conditions, and media.
The bacterial strains and plasmids used in this study are listed in
Table 1. P. aeruginosa and
Escherichia coli were grown at 37°C in Luria-Bertani (LB)
broth or LB agar (23) unless otherwise indicated. We used
E. coli DH5
for cloning and plasmid propagation, and
E. coli JM109 was used as a heterologous host for P. aeruginosa gene expression studies. For plasmid selection and
maintenance, antibiotics were added to growth media at the following
concentrations: ampicillin, 100 µg/ml; carbenicillin, 300 µg/ml;
chloramphenicol, 30 µg/ml; HgCl2, 15 µg/ml;
tetracycline, 20 µg/ml for E. coli and 50 µg/ml for
P. aeruginosa. Acyl-HSLs were added at concentrations of 10 µM for C4-HSL and 2 µM for
3OC12-HSL for P. aeruginosa and 100 nM 3OC12-HSL for E. coli.
DNA manipulations.
Standard methods were used to manipulate
plasmids and DNA fragments (2). Restriction endonucleases
and DNA modification enzymes were purchased from New England Biolabs
(Beverly, Mass.). Plasmids were isolated using spin miniprep kits
(Qiagen, Chatsworth, Calif.), and DNA fragments were purified using
Qiaquick PCR purification kits (Qiagen). Chromosomal DNA from P. aeruginosa was prepared using a DNeasy tissue kit (Qiagen). DNA
fragments were excised and purified from agarose gels using GeneClean
spin kits (Bio 101, La Jolla, Calif.), and PCR was performed with an
Expand Long Template PCR system (Roche, Indianapolis, Ind.). DNA was
sequenced at the University of Iowa DNA core facility by standard
automated-sequencing technology.
Primer extension analysis.
Primer extension analysis of the
genes qsc102, qsc117, and qsc131 was according to standard
methods (2). RNA was prepared from P. aeruginosa PAO1 and PAO-MW1 using the Trizol reagent (Life Technologies, Grand Island, N.Y.). RNA was extracted from cultures at
an optical density at 600 nm (OD600) of 2.0. The
initial culture OD600 was 0.1. The extension
primers for qsc102, qsc117, and qsc131 were
5'-GTCAGGCGTGGATAGCTTGTC-3',
5'-TCGCATTCCTCCACGCCGAAC-3', and
5'-GTTAAGGTGCGACAGACGAGG-3', respectively. Each primer was 5' end labeled using [
32-P]dATP and a
KinaseMax kit (Ambion, Austin, Tex.). 32P-labeled
primers were annealed to 5 to 10 µg of P. aeruginosa RNA and extended using a First-Strand cDNA synthesis kit (Amersham, Piscataway, N.J.). DNA sequences were obtained using plasmid templates, and the oligonucleotides were used for the primer extension. Sequencing was with -35S-dATP and a Sequenase, version
2.0, DNA sequencing kit (U.S. Biochemicals, Cleveland, Ohio). DNAs were
resolved on 8 M urea-8% polyacrylamide gels.
Plasmid constructions.
The parent plasmid for all
lacZ fusions was pQF50. Promoter fragments were generated
from P. aeruginosa genomic DNA or plasmid templates by
using PCR. Specific nucleotide changes within promoter fragments were
incorporated into the oligonucleotide primers used in PCR
amplification. For cloning, promoter fragments were end polished with
T4 polymerase and 5' phosphorylated with T4 polynucleotide kinase. The
resulting fragments were blunt end cloned into
SmaI-digested, phosphatase-treated pQF50, and correct
orientations were identified by PCR analysis. After cloning,
PCR-generated promoter fragments were verified by DNA sequencing.
Monitoring promoter activity in P. aeruginosa
and E. coli.
Transformation of P. aeruginosa was as described previously (27). For
lacZ expression studies, mid-logarithmic-phase cultures (OD600 of 0.2 to 0.5) were diluted 1:1,000 in LB
broth and allowed to grow for 17 h at 37°C, at which point
-galactosidase activity was measured as described by Miller
(14). With the acyl-HSL signal generation mutant
P. aeruginosa PAO-MW1, cultures were grown in the
presence and absence of 3OC12-HSL and
C4-HSL. The starting OD600
was 0.1, and -galactosidase activity was measured after 7 h at
37°C.
To monitor expression of qsc promoters in a heterologous host, we used
a two-plasmid system in
E. coli JM109. Overnight cultures
grown in supplemented A medium (
23) were diluted to an
OD
600 of 0.1 in fresh A medium. Cultures were
grown to an OD
600 of 0.2.
At this culture
density, 1 mM
isopropyl-
-
D-thiogalactopyranoside
(IPTG) was
added, and bacteria were grown in the presence and
absence of
3OC
12-HSL or C
4-HSL.
-Galactosidase activities were
assayed using a luminescent
microtiter dish assay (
26).
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RESULTS |
Primer extension analysis of three qsc genes.
To begin our
study of qsc promoter elements, we determined the transcript start
sites for three early genes by primer extension. These genes were
qsc102, which responds only to 3OC12-HSL, and qsc117 and phzA, which respond to either
3OC12-HSL or C4-HSL but which require both for full activation in P. aeruginosa
(26). Primer extension products were obtained with RNA
isolated from the parent strain, PAO1 (Fig.
1), and with acyl-HSL signal generation mutant strain PAO-MW1 grown in the presence of
3OC12-HSL and C4-HSL, but
not with PAO-MW1 grown without autoinducers (data not shown).
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FIG. 1.
Primer extension analysis of three qsc transcripts.
(Top) Polyacrylamide gels of qsc102 (A), qsc117 (B), and
phzA (C). Sequencing ladders are shown on the left, and
primer extension products from P. aeruginosa PAO1
RNA are on the right. (Bottom) DNA sequences of the promoter
regions. Transcription start sites are in boldface,
putative 10 regions are underlined, and
lux-box-like elements are boxed.
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For qsc102, two primer extension products were evident (Fig.
1A). The
start of the longest product mapped to a position 211
bp from the
predicted translation start site of qsc102. The other
product was 11 bp
shorter. The sequence at
10 from the start
of the longer of the two
transcripts was characteristic of a
10
hexamer in a promoter
(TTTAAT). A
lux-box-like element is centered
at
44.5 from the start of the long
transcript.
For qsc117 there was a single primer extension product (Fig.
1B). The
start of this transcript mapped to a position 39 bp
from the predicted
translational start site. A
10-like hexamer
(TTTAGT) is
upstream of the start of the qsc117 transcript, and
a
lux-box-like sequence is centered at
40.5.
There were three transcripts evident in the analysis of
phzA
(Fig.
1C). These started at 334, 335, and 336 bp from the predicted
translation start site. There is a
10-like hexamer
(TTTTAT) positioned
appropriately upstream of the
transcription start site suggested
by the primer extension analysis,
and there is a
lux-box-like
sequence centered at
44 to
46 with respect to the transcript
start
sites.
The
lux-box-like sequences positioned 40 to 60 bp upstream
of each transcript start showed considerable dyad symmetry (16
to 18 bp
of 20 bp) and considerable sequence variation. The three
20-bp
sequences matched the minimal consensus sequence
(NNCT-[N]
12-AGNN)
for a
P. aeruginosa qsc regulatory element (
26), with the
proximal
and distal nucleotide pairs possessing dyad symmetry. To
define
a minimal promoter sequence and to test the hypothesis that the
lux-box-like sequences were
cis-acting qsc gene
regulatory elements,
we carried out deletion and point mutation
analyses.
Definition of promoter elements by analysis of deletion and point
mutations.
We constructed plasmids containing P. aeruginosa DNA extending from 4 bp upstream of the
lux-box-like elements of qsc102, qsc117, and phzA
(pMW308C, pMW302E, and pMW303C). These plasmids contain lacZ
transcription fusions in the qsc gene coding regions. We assessed
promoter strength by measuring -galactosidase activity in
P. aeruginosa with different quorum-sensing
mutations. For all of these reporters, promoter strength in a
P. aeruginosa lasR rhlR double mutant (JP3) was <10%
of the promoter strength in the parental strain, PAO1 (Table
2). This established that the plasmids
contain the P. aeruginosa promoter DNA required for
quorum-sensing-dependent transcription activation.
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TABLE 2.
Activities of the qsc102, qsc117, phzA, and
qsc102 8A-13T promoters in P. aeruginosa quorum-sensing
mutants
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Transcription from the qsc102 promoter was low in a
lasR
mutant (Table
2) but high in an
rhlR mutant. Addition of
3OC
12-HSL
to strain PAO-MW1, a signal generation
mutant, resulted in the
induction of the qsc102-
lacZ fusion.
Addition of both 3OC
12-HSL
and
C
4-HSL did not result in further induction (Table
2). This
is consistent with our previous finding that qsc102
transcription
is regulated by 3OC
12-HSL and not
by C
4-HSL (
26).
For qsc117 and
phzA, promoter activity was reduced by
mutations in
lasR or
rhlR or both (Table
2).
Although reduced compared
to the activity in the parent, the activity
in the
rhlR mutant
was greater than that in the
lasR mutant. One explanation for
this is that
rhlR transcription depends on LasR but that
lasR transcription does not depend on RhlR (
19). Addition of
3OC
12-HSL
to signal generation mutant strain
PAO-MW1 resulted in a small
induction of the qsc117-
lacZ
fusion. Addition of both 3OC
12-HSL
and
C
4-HSL resulted in a much larger induction (Table
2). The
signal addition results are consistent with previous studies of
qsc-
lacZ chromosomal fusions (
26). One
explanation for the results
with the
lasR and
rhlR mutants is that the qsc117 and
phzA
promoters
can interact with either LasR or
RhlR.
Are the
lux-box-like elements in the vicinity of
40 to
60 from the starts of the qsc102, qsc117, and
phzA
promoters required
for quorum-sensing-dependent gene activation? To
address this
question, we generated site-specific point mutations in
the
lux-box-like
elements. Mutations in conserved
nucleotides of minimal sequence
NNCT-(N)
12-AGNN
have been shown to be critical for quorum-sensing
control of
lasB (
1,
22). Thus we changed the conserved C
to T in these promoters and measured quorum-sensing-activated
transcription. Replacement of the C with a T resulted in a significant
decrease in signal activation for all three promoters, and deletion
of
portions of the
lux-box-like elements resulted in a severe
decrease in signal-activated transcription (Fig.
2). These data
support the
hypothesis that the
lux-box-like sequences of qsc102,
qsc117, and
phzA are
cis-acting elements
required for quorum-sensing
control of transcription.
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FIG. 2.
Effects of deletions and point mutations on the
expression of qsc102 (A), qsc117 (B), and phzA (C) in
P. aeruginosa PAO1. The starting bases with respect
to the transcript start site are shown at the left (for
phzA the number is for the longest transcript). The
sites of lacZ insertion are given on the right, also
with respect to the transcription start sites. -Galactosidase
activities are in Miller units (means ± standard errors of the
means [SE]). Numbers in parentheses are percentages of wild-type
promoter activity (pMW308C, pMW302E, and pMW303E in PAO1). In
lasI rhlI signal synthesis mutant
P. aeruginosa PAO-MW1, the activities of the
wild-type qsc promoters (Table 2) were indistinguishable from the
activities of the promoters with point mutations or deletions and were
<10% of the activities in wild-type P. aeruginosa
PAO1. Thus the differences in promoter strengths shown are not a
reflection of changes in the basal transcription levels as monitored in
the signal synthesis mutant.
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LasR specificity determinants in P. aeruginosa
qsc promoters.
Palindromic lux-box-like sequences can
be found in the putative promoter regions of a number of early qsc
genes (26). The available evidence is consistent with the
hypothesis that some of these genes, the class I genes (qsc102, for
example), respond to LasR specifically and that others, the class III
genes (qsc117 for example), require a functional LasR and a functional
RhlR for full induction. An alignment shows that the class III genes qsc117 and phzA have an A at position 8 and a complementary
T at position 13 of the lux-box-like sequence and that the
class I gene qsc102 does not have the 8A-13T
lux-box-like sequence motif (Fig.
3). The palindromic sequences of other
class III promoters for which transcript start site information exists
also show the 8A-13T motif (1, 20, 22).
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FIG. 3.
An alignment of the NNCT-(N)12-AGNN elements
of qsc102, rsaL, lasI, qsc117,
phzA, lasB, and hcnA.
Nucleotides in black background represent bases present in all qsc
elements, and those boxed are putative specificity determinants.
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To test the hypothesis that the bases at positions 8 and 13 are
specificity determinants for class I promoters, we used site-directed
mutagenesis to construct reporter plasmids. We constructed a plasmid
with base 8 of the qsc102
lux-box-like element converted
from
a C to an A, a plasmid with the base at position 13 converted
from
a G to a T, and a plasmid with an A at position 8 and a T
at position
13. Neither of the promoters with single base substitutions
showed an
altered specificity. Induction depended solely on LasR
(data not
shown). The promoter with both the 8A and 13T mutations
(called qsc102
8A-13T) responded in a fashion similar to class
III promoters such as
the qsc117 promoter (Table
2). The promoter
was stronger than the
parent qsc102 promoter, and activity was
reduced not only in a
lasR mutant but also in an
rhlR mutant.
We monitored qsc102, qsc117, and qsc102 8A-13T promoter activity in
E. coli to test two hypotheses. The first hypothesis is
that
quorum sensing controls qsc102, qsc117, and qsc102 8A-13T
directly, and
the second hypothesis is that qsc102 responds to
LasR but not RhlR,
whereas qsc117 and qsc102 8A-13T are less specific
and can respond to
either LasR or RhlR. We monitored promoter
activity in
E. coli containing a p
tac-lasR plasmid (pPCS11) or
a
p
tac-
rhlR plasmid (pECP11) plus the
qsc102-
lacZ reporter plasmid.
The qsc102 promoter was
activated by LasR and 3OC
12-HSL but was
not
activated by RhlR and C
4-HSL (Table
3). The qsc117 and qsc102
8A-13T
promoters were activated by either LasR and
3OC
12-HSL or
RhlR and
C
4-HSL (Table
3). The LasR- or RhlR-dependent
activation
in recombinant
E. coli supports the conclusion
that activation
is direct. The lack of qsc102 activation by RhlR
supports the
conclusion that the qsc102 promoter responds to LasR but
not RhlR.
The activation of qsc117 and qsc102 8A-13T by both LasR and
RhlR
further substantiates that these promoters are less specific than
qsc102 and that the 8C
A and 13G
T mutations in the qsc102 promoter
converted it from a class I (LasR-controlled) to a class III (LasR-
and
RhlR-controlled) qsc gene.
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TABLE 3.
Activation of qsc102, qsc117, and qsc102 8A-13T
promoter-lacZ fusions by the las and
rhl systems in E. coli
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We were unable to convert the class III qsc117 promoter into a class I
promoter. Changes in the
lux-box-like element base
8 from an
A to a C or base 13 from a T to a G resulted in very
low promoter
activity. In fact, when we modified the qsc117 promoter
in pMW302E by
site-specific mutagenesis so that the 20-bp inverted
repeat matched
that of qsc102, the promoter was inactive (data
not
shown).
Predictive value of the minimal consensus sequences for
lux-box-like elements in P.
aeruginosa early qsc genes.
Previous studies have
established that lasI, which codes for the
3OC12-HSL synthase, is positively autoregulated
by 3OC12-HSL and LasR and is a class I
gene (24). The lasI gene is adjacent to
and divergently transcribed from rsaL, another qsc gene
(5). Prior to our ability to define a minimal consensus
for the lux-box-like element of P. aeruginosa qsc promoters, NNCT-(N)12-AGNN,
with 16 or more bases showing dyad symmetry, LasR-binding elements were
predicted for lasI and rsaL, but they did not
match the consensus. We hypothesized that there should be at least one
NNCT-(N)12-AGNN motif in the lasI-rsaL
intergenic region, and a search of the region revealed such a sequence
(Fig. 3). This element is centered at 40.5 from the lasI
transcription start site (24). The sequence does not show
the 8A-13T motif characteristic of promoters that respond to both LasR
and RhlR. Thus we predict that the promoter is LasR specific.
The transcription start site of
rsaL has not been mapped,
but the
lux-box-like element is nearly equidistant from the
rsaL and
lasI open reading frames (56 and 55 bp,
respectively). Thus
it is quite conceivable that the
lux-box-like element that we
have identified serves in the
bidirectional quorum activation
of both
lasI and
rsaL. To test our hypothesis that the predicted
lux-box-like sequence serves in quorum activation of both
lasI and
rsaL, we constructed
lacZ
reporter plasmids. One plasmid,
pMW312, contains a
rsaL-lacZ
fusion and begins 63 bp upstream
of the
rsaL coding region.
The other plasmid, pMW313, contains
a
lasI-lacZ fusion
and begins 62 bp upstream of the
lasI coding
region. The
lacZ gene was induced by addition of
3OC
12-HSL, but
not C
4-HSL,
to the
lasI rhlI signal generation mutant PAO-MW1,
containing either the
lasI reporter (4-fold) or the
rsaL reporter
(25-fold) (Table
4). Thus the minimal promoter elements
present
on pMW312 and pMW313 are sufficient for activation by
quorum-sensing
signal 3OC
12-HSL. The experiment
is also a confirmation that
rsaL and
lasI do not
respond to C
4-HSL-RhlR.
To test the hypothesis that the
lux-box-like sequence in the
lasI-rsaL intergenic region is involved in the activation of
lasI and
rsaL, we generated a point mutation in
the third base
of the element with respect to
lasI (pMW313B)
and a point mutation
in the third base with respect to
rsaL
(pMW312B). The point mutations
severely decreased
3OC
12-HSL-LasR-dependent expression of the
rsaL-lacZ and
lasI-
lacZ fusions to 4 and 30% of parental levels,
respectively. Thus we believe that there
is now sufficient information
to make predictions about promoter
elements of early qsc genes
and whether these genes will respond to
LasR specifically or to
either RhlR or
LasR.
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DISCUSSION |
qsc genes have been grouped depending on whether they are
fully activated by 3OC12-HSL, whether they
require both 3OC12-HSL and
C4-HSL for full activation, and whether the
acyl-HSL signals can activate them in logarithmic phase (early genes)
or stationary phase (late genes) (26). To begin to
understand promoter elements of qsc genes, we have examined several
early genes. We studied highly activated
3OC12-HSL-dependent early gene qsc102, and we studied two highly activated genes that require both signals for full
induction, qsc117 and phzA. The transcript start sites were mapped by primer extension (Fig. 1), and we identified palindromic lux-box-like sequences in the 40 to 50 regions with
respect to the start sites of the promoters (Fig. 3). Our genetic
dissections of the promoter regions of these genes indicate that DNA
including the palindromic region and a few base pairs upstream is
sufficient for quorum signal activation. They also indicate that the
specific sequences of the palindromic regions are important for quorum control (Fig. 2; Tables 1 and 2). We refer to the quorum control element as the NNCT-(N)12-AGNN element to signify
the minimal conserved unit.
The positions of the NNCT-(N)12-AGNN elements,
centered in the 40 regions, suggest that they serve as activator
binding sites and that the activators are the ambidextrous type,
functioning at these promoters by making contact with the RNA
polymerase C-terminal domain and with some other part of RNA
polymerase (6, 8, 21, 25). We cannot conclude that all
P. aeruginosa qsc genes contain
NNCT-(N)12-AGNN elements in the 40 region. We
suspect that some of the qsc genes showing small signal responses (for example, qsc104, which shows about 8-fold induction compared to qsc102,
which shows about 400-fold induction) (26) may have different promoter arrangements.
Our promoter analysis revealed that the qsc117 and phzA
NNCT-(N)12-AGNN elements showed an additional
similarity, an A at position 8 and a T at position 13. These genes
require both acyl-HSL signals for full induction, and we have shown
that they can respond to either of signal receptors LasR and RhlR
(Tables 2 and 3). The qsc102 gene responds only to LasR and does not
have the 8A-13T motif (Tables 2 and 3; Fig. 3). When we placed an
8A-13T motif in the qsc102 promoter, its behavior was altered such that
it responded to either LasR or RhlR. Activity was similar to the activity of the phzA and qsc117 promoters (Tables 2 and 3). The 8A-13T element appears to relax the specificity of the qsc102 promoter so that it will respond to either LasR or RhlR. Our attempt to
convert qsc117 into a LasR-specific promoter failed. Even replacement of the NNCT-(N)12-AGNN element of qsc117 with
that of qsc102 did not yield a LasR-specific promoter. Rather it
resulted in the inactivation of the promoter. Thus it is clear that
there are elements that remain to be defined in P. aeruginosa qsc promoters.
The analysis of qsc102, qsc117, and phzA afforded us an
ability to learn about other promoters. We tested the predictive power of our analysis by examining the rsaL-lasI intergenic
region. These two genes are early qsc genes activated solely by
3OC12-HSL (5, 24). Previous
examinations of the intergenic region between rsaL and
lasI were prior to the definition of the
NNCT-(N)12-AGNN minimal consensus. The previous
studies suggested that another region might represent the
LasR-binding region (5). There is a sequence matching
the NNCT-(N)12-AGNN consensus, and, as expected of a LasR-specific promoter, it does not possess an 8A-13T
element (Fig. 3). Our genetic analysis of the promoter region suggests that the NNCT-(N)12-AGNN motif is in fact an
element required for bidirectional qsc control of rsaL and
lasI by LasR. This is very similar to the bidirectional
quorum-sensing control of the divergently transcribed
traA and traC genes in Agrobacterium
tumefaciens (9).
Our current view is that early qsc genes can respond to LasR. Some of
these genes, for example, qsc102, are LasR specific, and, if there is a
response to RhlR, we cannot detect it. Other genes, for example, qsc117
and phzA, respond to either RhlR or LasR. We believe
these less-specific genes respond primarily to RhlR in P. aeruginosa. LasR is required for induction of the RhlR system
(19, 26). Thus both systems are required for full
induction of genes such as qsc117 and phzA, but, because of
the loose specificity, there is some response to either LasR or RhlR
alone. We believe that there may be RhlR-specific genes, for example,
qsc132, which unlike qsc117 and phzA show no detectable
response to 3OC12-HSL alone (26).
The qsc132 gene is a late gene, and a detailed analysis of its promoter
has not been carried out.
| |
ACKNOWLEDGMENTS |
This research was supported by a grant from the National
Institutes of Health (GM59026). M.W. was supported by a National Science Foundation Research Training grant (DBI9602247) and by a United
States Public Health Service Training grant (732 GM8365).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319)
335-7775. Fax: (319) 335-7949. E-mail:
everett-greenberg{at}uiowa.edu.
| |
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Journal of Bacteriology, October 2001, p. 5529-5534, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5529-5534.2001
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Top
Abstract
Introduction
