From the Institut für Genetik und
Mikrobiologie, Ludwig-Maximilians-Univesitaet,
Maria-Ward-Strasse 1a, 80638 München, Germany and the
¶ Medical Research Council Laboratory of Molecular Biology,
Hills Road, Cambridge CB2 2QH, United Kingdom
Received for publication, January 23, 2001, and in revised form, February 22, 2001
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ABSTRACT |
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The DNA architectural proteins FIS and CRP
are global regulators of transcription in Escherichia coli
involved in the adjustment of cellular metabolism to varying growth
conditions. We have previously demonstrated that FIS modulates the
expression of the crp gene by functioning as its
transcriptional repressor. Here we show that in turn, CRP is required
to maintain the growth phase pattern of fis expression. We
demonstrate the existence of a divergent promoter in the
fis regulatory region, which reduces transcription of the
fis promoter. In the absence of FIS, CRP activates
fis transcription, thereby displacing the polymerase from
the divergent promoter, whereas together FIS and CRP synergistically
repress fis gene expression. These results provide evidence
for a direct cross-talk between global regulators of cellular
transcription during the growth phase. This cross-talk is manifested in
alternate formation of functional nucleoprotein complexes exerting
either activating or repressing effects on transcription.
The rapid reorganization of cellular metabolism in response to
changing growth conditions is a hallmark of bacterial cells. These
metabolic reorganizations involve global regulators, many of which
serve as DNA architectural factors associated with bacterial chromatin
(1, 2). The abundant DNA bending chromatin protein FIS is a global
regulator of bacterial metabolism facilitating the adjustment of cells
to rapid growth conditions (3-6). The expression of fis
positively correlates with the richness of the medium (5), such that
the amount of FIS protein produced is commensurate with the available
metabolic potential. FIS increases the synthesis of stable RNA (tRNA
and rRNA) species (7-9), adjusting the capacity of the translational
machinery to changes in the nutritional supply. FIS is thought to
activate the transcription of stable RNA genes by stabilizing local DNA
architectures in their promoter regions (10). However, at many other
gene promoters, including the fis promoter, FIS acts as a
transcriptional repressor (11-18).
The transcription of the fis operon sharply increases on
subculturing stationary cells in rich medium, then steeply decreases because of autoregulation by FIS, and ceases in late exponential phase
(11, 16). This pattern of expression during the growth phase is
probably optimized, because constitutive fis expression negatively affects the survival of cells under stress conditions (19).
The expression of fis is largely regulated at the
transcriptional level without any significant contribution of
fis mRNA decay rates (11, 16, 20). As expected for a
gene involved in the modulation of cellular physiology, the expression
of fis is subject to a plethora of control mechanisms. The
activity of the fis promoter responds both to the growth
rate-dependent and stringent control systems (16). This
latter abrogates stable RNA production on limitation of amino acid
availability and is mediated by the nucleotide ppGpp (21). However, the
role of ppGpp in the shut-off of fis expression has been
questioned in the study that showed that deletion of the genes for both
ppGpp synthases (relA and spoT) does not change
the growth phase expression pattern of fis (11). The fis promoter is also exquisitely sensitive to changes in the
superhelical density of DNA and is thought to respond to growth
phase-dependent fluctuations in DNA topology (22). In
addition, the fis promoter activity is dependent on the
availability of the initiating nucleoside triphosphates (23) and is
positively regulated by the DNA bending chromatin protein IHF (20).
The CRP protein is another global regulator that, on exhaustion
of glucose or its analogues, switches the cellular metabolism to the
utilization of alternative carbon sources, such as lactose, maltose,
and arabinose (24, 25). As glucose is consumed the intracellular levels
of cAMP rise, and an active cAMP-CRP complex forms, which then
modulates the activity of numerous genes at the transcriptional level
(24, 26, 27). In growing bacterial cells the level of cAMP sharply
increases on transition to stationary phase (28). Thus, the majority of
the effects of CRP and FIS on gene expression are probably exerted
under different growth conditions. Nevertheless, there are many
examples of genes regulated by both FIS and CRP (13, 18, 29, 30). The
expression of most of the FIS-regulated genes involved in the
catabolism of sugars and nucleic acids is also dependent on the
cAMP-CRP complex (4). Notably, the activity of the crp
promoter itself is modulated by both the cAMP-CRP complex and FIS
during the growth phase (15, 31, 32). This control of crp
expression is thought to operate by changing the composition of
transcriptional complexes formed in the crp promoter region
in response to the physiological state of the cell (15).
In this study we investigated the effect of CRP on the expression of
the fis gene. We demonstrate that CRP modulates the
fis promoter activity, supporting the transient mode of
fis expression during the growth phase. In analogy to
crp regulation, this modulation involves alterations in the
composition of functional nucleoprotein complexes formed by FIS, CRP,
and RNA polymerase in the fis regulatory region.
Chemicals and Enzymes--
Chemicals and enzymes used in this
work were obtained from commercial sources.
Bacterial Strains, Growth Conditions, and
Plasmids--
Bacterial strains used in this study were
Escherichia coli K12 derivatives. The genotype of CSH50 is
ara
The construct pFP2 is described by Ninnemann et al. (16).
For construction of pWN1 (fis promoter lacZ
fusion), a fis promoter fragment (positions
A two-step PCR method using mutagenic oligonucleotides and
Pfu DNA polymerase (Stratagene) was employed for
site-directed mutagenesis (35). To construct pWN-75A/C, the primer
pfis-87op comprising positions Proteins--
FIS and RNA polymerase were isolated as described
previously (36, 37). Purified CRP was kindly provided by Dr. A. Kolb.
RNA Isolation and Northern Analysis--
Northern analyses were
performed as described in Schneider et al. (17). The
concentration of RNA in different samples was detected
spectrophotometrically, normalized for each sample, and verified on a
reference gel, using rRNA as loading control. For Northern analysis 15 µg of RNA/lane was separated on a 1% agarose gel in the presence of
glyoxal and transferred to Hybond N+ filters (Amersham Pharmacia
Biotech). Filters were hybridized overnight with
[ Western Analysis--
Western blotting was carried out
essentially as described earlier (39), except that 18%
SDS-polyacrylamide gel electrophoresis was used for the detection of
FIS and methanol (10%) was included in the buffers during
electrotransfer of proteins onto polyvinylidendifluoride membranes
(Immobilon-P, Millipore). FIS was detected by using the FIS-specific
polyclonal (rabbit) antibody (39). The DNase I Footprinting--
The conditions of DNase I footprinting
were as described earlier (39). The fis promoter region
( Potassium Permanganate Reactivity Assay--
The reactions on
linear templates were assembled and processed similarly to those used
for DNase I footprinting. The reactions were carried out as described
by Schneider et al. (17), except that 100 µM
each of CTP and GTP were included in the incubation mixtures.
For the reactions with supercoiled templates, the plasmid DNA (500 ng)
and proteins as indicated were incubated in 50 µl of a buffer
containing 10 mM Tris-HCl, pH 8, 200 mM NaCl,
0.2 mM dithiothreitol, and 100 µM each of CTP
and GTP. After footprinting the plasmid DNA was used for five cycles of
PCR (41) with the 5'-radiolabeled primer ORF1. The reaction products
were analyzed on 6% sequencing gels. The signals caused by
permanganate reactivity of bases were visualized and quantitated as
described above.
In Vitro Transcription--
Supercoiled plasmid pWN1 and
pWN-75A/C were used for in vitro transcription and primer
extension reactions according to Lazarus and Travers (42). The mRNA
obtained after in vitro transcription was divided in three
parts and used for primer extension by reverse transcriptase (Moloney
murine leukemia virus reverse transcriptase, RNase H minus, Fermentas)
with radioactively end-labeled primers ORF1 for fis
mRNA, DMS Footprinting--
In vivo DMS footprinting
was performed essentially as described by Schneider et al.
(43). DMS (0.2 or 0.4%) was added in 1 ml of cell cultures on
transition to stationary phase (A600 = 1.5-2) grown at 37 °C and incubation continued at 32 °C for 5 min. The purified plasmid DNA (500 ng) was used as a template for five
cycles of PCR (41) with 5'-radiolabeled primer 5'-CTGAGCTGATATTGTCCG-3' priming about 60 bp downstream of the fis operon
transcription initiation site.
The DMS footprinting reaction in vitro was performed by
adding 1 µl of DMS to supercoiled pWN1 plasmid DNA (500 ng) and
proteins as indicated in a buffer containing 10 mM
Tris-HCl, pH8.0, 100 mM NaCl, 0.2 mM
dithiothreitol, 0.2 mM cAMP for 1 min at room temperature
in a 100-µl volume. The DNA pellets were dried and used for PCR as
described above. The PCR products were analyzed on 6% sequencing gels
and visualized by autoradiography. A dideoxy sequencing ladder was used
to identify the position of protected guanines.
Effect of crp Deletion on Cell Growth--
In our experiments we
used the E. coli strain CSH50 and its derivative
CSH50 Deletion of crp Alters fis Expression--
To evaluate the effect
of crp on fis expression, total cellular RNA was
isolated at intervals after the shift-up from wild type and
crp cell cultures having similar optical densities. The amount of fis mRNA was quantitated by Northern analyses.
In wild type cells the typical pattern of fis expression was
observed, with an initial sharp increase followed by a steep decrease
and an almost complete shut-off in 120 min (Fig.
2, A and B). In
crp mutant cells the amount of fis message was
elevated at the earliest time point measured (5 min) but achieved its
maximum levels later than in wild type. Most remarkably, in
crp cells substantial amounts of fis transcript
were detectable in late exponential phase (after 120 min), suggesting
that the down-regulation of fis is impaired. During the
analyzed time period both strains grew equally well (Fig.
1A), and in addition, we did not observe any significant differences between the strains in the expression pattern of
eno mRNA (Fig. 2A, lower panel).
Consistent with the Northern data, the crp cells transformed
with the fis promoter-lacZ fusion construct pWN1
(containing the extended fis promoter region from position
To test whether the impaired down-regulation of fis
expression is due to a lower concentration of FIS in mutant cells, we compared the content of FIS in wild type and crp cell
extracts. However, by quantitative Western analyses we detected
increased amounts of FIS protein in crp cell extracts 60 and
120 min after the shift-up (Fig. 2C). Thus higher
fis mRNA and higher FIS protein levels persist in
exponentially growing crp cells. Taken together, these data
suggest that crp is involved in the maintenance of the
growth phase expression pattern of fis.
CRP and FIS Compete with RNAP for Binding at the fis
Promoter--
The fis promoter regulatory region contains sequences
matching the CRP binding site consensus (11, 24). To test whether CRP
directly binds at the fis promoter, we carried out DNase I footprinting studies. Addition of CRP to linear fis promoter
DNA fragments protected the positions
To investigate the effect of CRP binding on the interaction of RNAP
with the fis promoter, we first footprinted the
fis promoter with RNAP alone. Binding of RNAP increased
DNase I cleavage at two positions (
Addition of CRP abolished both the DNase I cleavage around
To gain more information on the interactions between CRP, FIS, and
polymerase at the fis promoter, we footprinted FIS alone and
also in combination with CRP and RNAP. On addition of FIS the first
site occupied was the high affinity binding site II centered at
Similar experiments were carried out using potassium permanganate as a
chemical probe. Potassium permanganate preferentially targets the
pyrimidine residues in the untwisted regions of DNA and thus allows the
extent of promoter opening to be measured. On addition of RNAP, we
observed two regions of enhanced KMnO4 reactivity, one
within the
From these data we infer that binding of CRP in the fis
regulatory region displaces RNAP from upstream DNA site but facilitates the polymerase binding and open complex formation at the fis
promoter. By contrast, binding of FIS predominantly displaces RNAP from the fis promoter. Furthermore, CRP appears to stabilize the
interaction of FIS with binding site II implicated in repressing
fis promoter activity (20). Finally, FIS and CRP cooperate
in repressing the interaction of RNAP with the upstream binding site.
CRP Activates the fis Promoter in the Absence of FIS--
To
evaluate the functional importance of the observed interactions, we
first investigated the effect of CRP on the open complex formation at
the fis promoter. KMnO4 footprinting was carried out using linear fis promoter fragments with RNAP and
different CRP concentrations. Addition of CRP enhanced the reactivity
of the bases within the FIS and CRP Synergistically Repress the fis Promoter--
We next
asked how the simultaneous addition of CRP and FIS affects the
fis promoter activity. For this purpose we monitored fis transcription using pWN1 DNA with FIS and CRP proteins
added either alone or in combination. As expected, addition of
increasing FIS concentrations gradually decreased the transcription of
the fis promoter (Fig. 5,
upper panel, compare lanes 1-4), whereas under
the reaction conditions CRP alone had no detectable effect (Fig. 5,
compare lanes 1, 7, and 8). However,
when these same CRP concentrations were combined with a low FIS
concentration (corresponding to that in lane 2), which alone
had only marginal effect on transcription, the activity of the
fis promoter was strongly inhibited (Fig. 5, lanes
5 and 6). Under the same conditions the transcription
of the reference bla promoter was not noticeably affected
(Fig. 5, lower panel). Thus, a combination of FIS and CRP at
concentrations that themselves have little, if any, effect on
transcription, strongly inhibit fis promoter activity. We
infer that FIS and CRP synergistically repress the fis
promoter in vitro.
CRP Facilitates FIS Binding at Site II both in Vitro and in
Vivo--
To clarify the mechanism of synergy between FIS and CRP in
repressing the fis promoter activity, we investigated the
effect of CRP on the binding of FIS at site II by DMS footprinting. To obtain signatures for site II occupation by FIS, the DMS footprinting reactions were carried out in vitro with the fis
promoter construct pWN1. Most characteristic signatures obtained for
FIS binding at site II were an increased reactivity of G
We next investigated whether CRP similarly modulates the interaction of
FIS with binding site II in living cells. In vivo DMS
footprinting was carried out with wild type, crp, and
fis cells containing pWN1 and grown to similar optical
densities. The footprinting of the fis promoter was
performed on transition to stationary growth phase, i.e.
under the conditions of a shut-off of fis expression in wild
type cells. The comparison of the DMS footprint obtained in
crp mutant cells with that obtained in cells lacking
fis (used as a negative control) revealed in the former a
shift in the reactivity of G The Upstream RNAP Binding Site Represents a Divergent
Promoter--
Finally, we addressed the question on the role of the
upstream polymerase binding site in the regulation of fis.
Previous studies could not detect any transcription driven by RNAP from this site. To test whether this RNAP molecule transcribes in the direction opposite to that of the fis operon, we carried out
primer extension reactions with total RNA isolated from CSH50 cells. Two divergent transcripts were detected with start points at
We generated the construct pWN-75A/C in which a "down" mutation was
introduced impairing the match of the putative
To evaluate the effect of CRP on the activity of the divergent promoter
in vivo, we measured the The central finding of this study is that the cAMP-CRP complex is
required to maintain the growth phase pattern of fis
expression. We demonstrate that CRP is involved in the formation of
distinct nucleoprotein complexes that can either activate or repress
fis transcription. More specifically, we show that a synergy
between FIS and CRP in repressing fis promoter activity
enables an efficient shut-off of fis expression in mid
exponential phase. Because FIS is itself regulating crp
transcription (15), these data indicate a direct cross-talk between two
global transcriptional regulators in coordinating cellular gene
expression with varying growth conditions. RNAP holoenzyme is also
implicated as a potential regulator of fis expression.
The Regulatory Role of the Divergent Promoter--
We have shown
here that the upstream polymerase binding site in the fis
regulatory region represents a promoter transcribing in a direction
opposite to fis operon. We could not identify any meaningful
ORF downstream of the divergent promoter, and no genes transcribed in
this direction have been reported so far. The adjacent panF/prmA operon located upstream is transcribed
in the same direction as fis (45). In agreement with earlier
observations (11), our in vitro data indicate that
polymerase binds more tightly at the divergent than at the
fis promoter. In addition, polymerase forms open complexes
readily at the divergent promoter, whereas detectable open complex
formation at the fis promoter requires magnesium ions and
high concentrations of initiating nucleoside triphosphates.2 Furthermore,
the inactivation of the divergent promoter leads to an activation of
the fis promoter in vitro, the effect being more
pronounced at low RNAP concentrations. One implication of these
observations is that under conditions of RNAP limitation, the divergent
promoter is competing with the fis promoter for RNAP
binding. Thus, it is possible that the divergent promoter has primarily
a regulatory role. Whether this competition for RNAP binding is
involved in the control of fis gene expression is not clear
at present. The reported activation of the fis promoter after the deletion of the region comprising the divergent promoter (20)
would be consistent with the competition model. Morever, we observe a
2-fold increase of Growth Phase Regulation of fis Expression--
Recently we
demonstrated that increasing levels of negative supercoiling strongly
activate the fis promoter both in vivo and in vitro (22). The minimal fis promoter is
sufficient for activation by negative supercoiling, but the presence of
the GC-rich discriminator sequence between the
Previous studies identified six FIS binding sites in the fis
promoter region and demonstrated that autoregulation of the
fis promoter is primarily responsible for the steep decrease
of fis expression during exponential phase (11, 16, 19). The
main FIS binding site implicated in autoregulation is the high affinity site II centered at The Mechanism of Synergy between CRP and FIS in Repressing
fis--
After the shift-up the modulation of fis promoter
activity by CRP occurs concomitantly with the accumulation of FIS
protein. We assume that as soon as sufficient FIS is produced, the
CRP-dependent facilitation of FIS binding to site II (and
perhaps site III) will compete with and override the
CRP-dependent activation of the fis promoter.
Indeed, we observe that in vitro CRP greatly augments the
repressing effect of low FIS concentrations on fis promoter
activity (Fig. 5). Furthermore, addition of polymerase does not impair
the stabilizing effect of CRP on FIS binding to site II (Fig. 6).
Finally, increased binding of FIS at site II is observed even at high
CRP concentrations, which abolish the CRP-dependent
enhancement of RNAP binding at the fis promoter (Fig.
3).
Our finding that in late exponential phase the crp cells
have a higher FIS protein content than wild type yet fail to reduce fis expression efficiently is consistent with a requirement
for a co-repressor at this growth stage. The results showing lower occupancy of FIS site II in the absence of CRP both in vitro
and in vivo indicate that CRP acts synergistically with FIS
in repressing fis promoter activity. We propose that this
synergy results primarily from the formation of a nucleoprotein complex
involving CRP and affecting FIS binding at the repressing site II. In
conclusion, our observations strongly support the notion that CRP and
FIS cooperate in the shut-off of fis expression during the
exponential growth phase (Fig. 8). We
presume that in crp cells the expression of numerous genes
that are under the control of both FIS and CRP will be perturbed. This
will be especially prominent in cases where the regulation involves
cooperative interactions. Our study provides a first example of
synergistic repression by FIS and CRP, which contrasts the synergistic
activation reported for the RpoS-dependent proP
P2 promoter and also, for the malEp promoter (30, 50). Such
cross-talk between FIS and CRP could provide selective advantage by
allowing flexibility of transcriptional control and also increasing the
precision of the coordination of cellular gene expression with changing
growth conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(lac pro) thi rpsL (33). The construction
of CSH50
fis is described elsewhere (34). This
strain carries a kanamycin resistance gene substituted for fis. CSH50
crp is described in
González-Gil et al. (15). This strain carries a
streptomycin resistance determinant. All strains were grown in 2× YT
medium (16 g of tryptone, 10 g of yeast extract, 5 g of
NaCl/liter, pH 7.4). Single colonies were isolated on YT plates (8 g of
tryptone, 5 g of yeast extract, 5 g of NaCl, 15 g of
agar/liter).
308 to +106)
was PCR1 amplified with the
primers LfispRI5' (5'-CGGAATTCCGGTCGTAAGAATTAACCTTC-3') and ORF13'
(5'-AGGTCTGTCTGTAATGCCAG-3') using E. coli CSH50 chromosomal DNA as a template. For the construction of pWN3 (divergent promoter lacZ fusion), a fis promoter fragment
encompassing positions
308 to +106 was PCR amplified with the primers
pfis-308op-NruI (5'- GCCTATCGCGACGTAAGAAATTAACCTTCGCAT-3') and
ORF1-EcoRI (5'-CGGAATTCAGGTCTGTCTGTAATGCCAG-3') using
E. coli chromosomal DNA as a template. These fragments were cloned in EcoRI/NruI sites of ptyrTlac (17), and
thereby the tyrT promoter was replaced by these
fis promoter DNA fragments.
87 to
64 and containing an A-to-C
base substitution at
75 on the top strand
(5'-CATTTTAAATGCCATTCTTTGATC-3') and primer ORF13' were used to
initiate the first round PCR on the pWN1 template. The resulting PCR
product was then used as a "megaprimer" together with the primer
LfispRI5' for the second round PCR on the pWN1 template and the product
cloned in EcoRI/NruI sites of ptyrTlac. For
construction of pWN3-75A/C, the pWN1-75A/C DNA was PCR amplified with
the primers pfis-308op-NruI and ORF1-EcoRI and cloned in
EcoRI/NruI sites of ptyrTlac.
-32P]dCTP-labeled probes made by Megaprime DNA
labeling system (Amersham Pharmacia Biotech). As template DNA for the
Megaprime reactions served a BglII/HindIII
fis fragment cut out of pADfis (38) and an eno
fragment, prepared by PCR amplification of a 1283-bp internal fragment
of the E. coli enolase gene, using the primer eno5
(5'-AGTCTTATGCCTGGCCTTG-3') and eno3 (5'-CATCGGTCGTGAAATCATCG-3'). The
signals obtained in three separate experiments were detected by
phosphorimaging (PhosphorImager Storm 840, Molecular Dynamics) and
quantified using the IMAGE-QUANT software, averaged, and normalized to
the value obtained for the 5-min time point for one of the strains.
subunit of RNAP was detected
by using a RNAP
subunit-specific polyclonal (goat) antibody (a
generous gift of Hermann Heumann). Horseradish peroxidase-conjugated
goat (Promega) and rabbit (Sigma) IgG-specific secondary antibodies
were used for detection with an ECL+Plus kit (Amersham Pharmacia
Biotech). The autoradiographs were scanned and quantified by NIH-IMAGE software.
185 to +65) was PCR-amplified using the primers fis3
(5'-GATTTCTGAGCTGATATTGTC-3') and fis5 (5'-AAAGAAAAATTGAGAACTTACTC-3')
and the pFP2 DNA as a template. The primer fis3 was uniquely
end-labeled by [
-32P]ATP and T4 polynucleotide
kinase. The fragments obtained were purified by polyacrylamide gel
electrophoresis using a neutral 0.5× TBE gel. The incubation mixture
contained 10 mM Tris-HCl, pH 7.9, 75 mM NaCl, 1 mM dithiothreitol, 0.2 mM cAMP (whenever necessary), and proteins as indicated in a 20-µl volume. After incubation for 30 min at 37 °C, DNase I and MgCl2 were
added to 2 µg/ml and 10 mM final concentrations,
respectively. The reaction was terminated after 10 s by adding 80 µl of the solution containing 0.5% SDS and 50 mM EDTA.
After digestion by proteinase K for 45 min at 45 °C and phenol
extraction, the aqueous phase was precipitated with ethanol. The
pellets were washed with 70% ethanol, dried, dissolved in loading dye,
and analyzed on a 6% denaturing polyacrylamide gel. Protected and
hypersensitive bands were identified by using the Maxam-Gilbert
G-ladder (40) of the same DNA fragment as reference.
257op (5'-GAGCAAGCTCACAAAAGGC-3') for divergent transcript
and bla3B4 (5'-CAGGAAGGCAAAATGCCGC-3') for the bla transcript. The extension with primers ORF1,
257op and bla3B4 yields
106-, 149-, and 100-bp fragments, respectively. After primer extension
the samples were loaded on 6% denaturing polyacrylamide gels and
analyzed by phosphorimaging. The length of the transcripts was
estimated by using corresponding dideoxy sequencing reactions as a
reference. The amount of the fis and div
transcript produced was quantitated and normalized to that of
bla.
-Galactosidase Determinations--
Overnight cultures were
diluted 1:30 in fresh 2× YT medium. Samples taken at the indicated
times were assayed for
-galactosidase activity following the
protocol of Sadler and Novick (44).
-Galactosidase units were
multiplied by 1000 to make them equivalent to those of Miller (33).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
crp lacking the crp gene. After
subculturing cells in rich medium (a procedure that hereafter we term a
nutritional shift-up) the wild type and crp cells grew
similarly until the late exponential phase. Although the transition to
stationary growth phase occurred at lower optical densities in the
crp mutant, both cultures achieved similar densities in late
stationary phase (Fig. 1A).
However, when the mutant and wild type strains were grown in mixed
cultures, the wild type demonstrated a clear advantage, because the
crp mutant cells were rapidly selected out (Fig.
1B).
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Fig. 1.
Growth characteristics of CSH50 and
CSH50 crp strains.
A, growth curves of CSH50 and CSH50
crp cells.
The overnight cultures were diluted 1:100 in fresh 2× YT medium, and
the absorbance at 600 nm (ordinate, arbitrary units) was measured after
the time intervals indicated. The zero point on the abscissa
indicates that the measurement was done immediately after the dilution.
B, the growing CSH50
crp cells are rapidly lost
from mixed cultures. The overnight cultures of CSH50 and
CSH50
crp cells were diluted 1:10.000 in fresh 2× YT
medium and mixed (zero point on the abscissa). After 24, 48, 72, and 96 h, aliquots were plated on streptomycin-containing
medium, and the number of survivors was scored (see "Materials and
Methods" for details). wt, wild type.
308 to +106) reproducibly demonstrated a slow decrease of
-galactosidase activity during the growth phase (see Fig.
7C).
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Fig. 2.
Effect of crp deletion on
fis expression. A, a representative
example of Northern hybridization analysis of fis mRNA
is shown. eno RNA is shown as a control. Total RNA was
isolated from CSH50 and CSH50 crp cells at intervals after
shift-up as indicated. We note that the ratio of the two fis
transcripts changes in crp cells, but we did not explore
this phenomenon further. B, quantitative Northern analysis
of fis expression in CSH50 and CSH50
crp cells.
The data were derived from values obtained in three independent
experiments. C, Western analysis of FIS content in CSH50 and
CSH50
crp cells is shown. The amount of total protein was
500 ng for 15 and 120 min and 200 ng for 60 min. The immunodecoration
of the
subunit of RNAP was carried out with the same filter.
wt, wild type.
57/
58 from DNase I cleavage and increased DNase I cleavage around positions
60,
70, and
110
with respect to the start point of transcription at +1 (Fig. 3A, lanes 5 and
6). Inspection of this region revealed two sequences matching the CRP binding site consensus (24), one (site I) centered at
position
62.5 (5'-TTTGAtccatcTCAGA-3') and another (site II) at
position
103.5 (5'-TATGAgtaattaTCGCA-3') (Fig. 3C).
Because only DNase I hypersensitivity indicative of DNA distortion but no extensive protection of these putative sites by CRP was observed, it
appears that CRP does not bind tightly. However, we confirmed CRP
binding in both these regions by DMS footrpinting experiments, showing
a CRP-dependent modification of Gs at
67,
77,
97,
106, and
108 (Fig. 3C; data not shown).
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Fig. 3.
Footprinting analysis of the fis
promoter. A, DNase I footprinting of FIS, CRP and
RNAP binding at the fis promoter. The used 250-bp
fis promoter fragment (~5 nM) was uniquely 5'
end-labeled at the bottom strand. The protein concentrations used are
indicated. The regions binding FIS, CRP, and RNAP are indicated by
thick, thin, and dashed lines,
respectively. The stars indicate hypersensitivities induced
by binding of CRP. The arrowheads indicate
hypersensitivities induced by binding of both FIS and CRP or both CRP
and RNAP. B, KMnO4 reactivity of the
fis promoter (bottom strand) and the divergent
promoter (top strand) on linear templates (0.2 nM). The concentration of RNAP was 20 nM; the
concentration of FIS was 20 nM in lanes 2 and
6 and 100 nM in lanes 7 and
8; the concentration of CRP was 20 nM in
lanes 2 and 3 and 10 nM in lane
4. Lane 5, free DNA. C, sequence of the
fis promoter. The binding sites for proteins are indicated
as in A. The RNA polymerase binding elements ( 10 and
35
hexamers) of the fis and divergent promoters are
boxed, and the start points of transcription are shown by
arrows. The down mutation introduced in the
35 hexamer of
the divergent promoter is indicated by C above the
substituted A in the
35 region. The thick,
thin, and dotted lines indicate the binding
regions for FIS, CRP, and RNAP, respectively. The filled and
open circles mark the G residues whose methylation by DMS is
diminished in the presence of low (10 nM) and high (100 nM) concentrations of cAMP-CRP complex, respectively.
37 and
46) near the
35 element
of the fis promoter and protected from DNase I cleavage the
upstream region between
65 and
125. An additional
DNase-hypersensitive site was induced around position
88 (Fig. 3A,
lane 7). The observed pattern confirms a previous report on
the binding of two closely spaced RNAP molecules with different
affinities at the fis promoter (11).
88 and the
protection of the upstream region by RNAP (Fig. 3A, compare
lanes 7-10), indicating that CRP precludes the binding of
RNAP to the upstream DNA. By contrast, the protection of the fis core promoter by RNAP was enhanced (Fig. 3A,
compare lanes 7-9), suggesting that binding of CRP
stabilizes RNAP at the fis core promoter. However, at higher
CRP concentrations the protection of the fis promoter
by RNAP was abolished (compare lanes 9 and 10).
This latter effect of high CRP concentrations is most probably due to
nucleation of binding in the region overlapping the
10 hexamer of the
fis promoter, as observed by DMS footprinting (Fig. 3C; data not shown).
42
and overlapping the
35 region of the fis promoter (Fig.
3A, lane 1). When FIS and CRP were added in
combination, the DNase I hypersensitivity around positions
38/
39
induced by binding of FIS at site II and the protection of FIS site III were noticeably enhanced (Fig. 3A, compare lanes
13-15). However, CRP did not increase the FIS protection in the
downstream region (e.g. position
31) observed at high FIS
concentrations (compare lanes 1-3 with
lanes 13-15). This suggests that CRP may specifically facilitate the binding of FIS at sites II and III. When FIS and RNAP
were added in combination, FIS prevented the binding of polymerase at
the fis promoter, as judged by the disappearance of the
DNase I hypersensitive sites at
37 and
46 attributable to RNAP
binding (Fig. 3A, compare lanes 7 and
11). However, the RNAP-dependent protection of
the upstream region and the DNase I hypersensitive site around
88
remained largely unaffected.
10 element of the fis promoter and another
upstream at positions
101 and
102 (Fig. 3B, lane
1). We attribute this latter to the binding of the second RNAP
molecule, because deletion of the fis core promoter region
to position
42 does not affect this upstream reactivity pattern (data
not shown). Addition of CRP substantially decreased the
KMnO4 reactivity of the bases at
101 and
102, whereas
the reactivity in the
10 region of the fis promoter was
noticeably increased (Fig. 3B, compare lanes 1,
3, and 4). By contrast, addition of FIS reduced more strongly the KMnO4 reactivity of the fis
promoter
10 region than that of the bases at
101 and
102 (compare
lanes 1 and 6). This latter reactivity was
substantially reduced only at higher FIS concentration (compare
lanes 6 and 7). When FIS and CRP were added in
combination, the KMnO4 reactivity of the fis
promoter
10 element and especially that of bases at
101 and
102
was strongly reduced, suggesting a cooperative effect (Fig.
3B, compare lanes 2, 3, and
6).
10 region of the fis promoter to
permanganate, but as observed by DNase I footprinting experiments
described above, increasing CRP concentrations abolished this enhancing effect (Fig. 4A), probably
because of binding of CRP at a low affinity site overlapping the
10
region (Fig. 3C). Next we performed in vitro
transcription reactions using CRP with supercoiled pWN1 DNA. The
results demonstrated a similar dependence of fis promoter activity on CRP concentration (Fig. 4B, upper
panel), whereas the transcription of the bla promoter
located on the same plasmid was not noticeably affected (Fig.
4B, lower panel). We thus conclude that in the
absence of FIS, CRP can activate the fis promoter by
facilitating open complex formation.
View larger version (37K):
[in a new window]
Fig. 4.
CRP modulates the fis
promoter opening and transcriptional activity in
vitro. A, potassium permanganate
footprinting of the 250-bp fis promoter fragment (~0.5
nM) incubated with RNAP (30 nM) and CRP. The
concentrations of CRP (dimer) in lanes 2, 3,
4, 5, and 6 were 10, 20, 40, 100, and
100 nM, respectively. B, in vitro
transcription from supercoiled pWN1 (0.5 µg) in the presence of RNAP
(10 nM) and CRP. The results of primer extension reactions
of the generated fis mRNA are shown. Halves of the same
mRNA preparations were used to measure bla transcription
as an internal control (lower panel). The concentrations of
CRP (dimer) in lanes 2, 3, 4, and
5 were 4, 10, 15, and 80 nM, respectively.
View larger version (127K):
[in a new window]
Fig. 5.
Effect of CRP and FIS on transcription of the
fis promoter in vitro. The
result of primer extension of the fis mRNA generated by
in vitro trancription from plasmid pWN1 in the presence of
25 nM RNAP is shown. The used concentrations of CRP and FIS
are indicated. Halfs of the same mRNA preparations were used to
measure bla transcription as an internal control
(lower panel). The data were normalized to the
bla transcript.
37 relative
to G
38 and strongly decreased reactivities of G
48 and G
49
(Fig. 6, A and B,
compare lanes 1 and 2). Addition of CRP alone
caused only slight decrease in the reactivity of G
48 (compare
lanes 1 and 10), whereas simultaneous addition of FIS
and CRP yielded essentially the same pattern as with FIS alone.
However, consistent with DNase I footprinting data in the presence of
CRP, the reactivity signatures attributable to FIS binding at site II
were substantially increased (Fig. 6, A and B,
compare lanes 2 and 3). Addition of polymerase
did not change the pattern observed with FIS and CRP alone (compare
lanes 3 and 4).
View larger version (41K):
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Fig. 6.
CRP facilitates the interaction of FIS with
site II at the fis promoter. A, the
DMS reactivity of the fis promoter construct pWN1 treated
either in vitro or in vivo is shown. The
positions of modified guanines and the concentration of proteins used
for the in vitro reactions are indicated. The DMS
concentration used for the in vivo modification was 0.2% in
lanes 5-7 and 0.4% in lanes 11-13. The strains
are indicated above. B, densitometric scans of the FIS
binding site II. The numbers 1-4 correspond to the
lanes in A. Arrows indicate the
positions of guanines whose DMS reactivity is modified by FIS.
37 relative to G
38, and a suppression of the reactivities of G
48 and G
49 (Fig. 6, A, compare
lane 5 with lane 7 or lane 11 with
lane 13; B, compare fis and
crp, right panel). The pattern obtained is almost
identical to that observed in vitro and indicates that FIS
interacts with the binding site II in crp mutant cells.
However, in wild type cells the interaction of FIS with site II was
notably increased in comparison to crp mutant cells, as
judged by the same reactivity signatures (Fig. 6, A, compare
lane 5 with lane 6 or lane 11 with
lane 12; and B, compare crp and
wt, right panel). We thus infer that CRP is required to stabilize the interaction of FIS with binding site II
in vivo.
108 and
around
87 (Fig. 7A,
lane 1). To discriminate whether these divergent transcripts
result from true initiation events or represent processing products, we
carried out in vitro transcription reactions with pWN1.
Three divergent transcripts were produced in vitro only one
of which (initiating at
108) coincided with that observed in
vivo (Fig. 7A, lanes 2-4). This latter is
the only transcript reproducibly observed with different fis
promoter constructs in vitro (data not shown). Inspection of
the region around the common initiation site at
108 identified both
in vivo and in vitro, revealed two sequences
5'-TTGCAT-3' and 5'-GATAAT-3' with reasonable matches to the
70 consensus
35 and
10 elements, respectively (Fig.
3C).
View larger version (33K):
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Fig. 7.
A divergent promoter is located in the
fis regulatory region. A, primer
extension analysis of total RNA (10 µg) extracted from CSH50 cells
(lane 1) and of the transcripts produced from pWN1 in
vitro (lanes 2-4) with the primer pfis-257op. The
concentration of RNAP in lanes 2-4, was 15, 30, and 50 nM, respectively. The dideoxy sequencing ladder performed
with the same primer on pWN1 is shown. B, effect of the down
mutation in the 35 element of the divergent promoter. In the
left panel the result of primer extension of RNAs produced
in vitro from the divergent, fis, and
bla (used as an internal control) promoters on pWN1 and
pWN-75A/C is shown. The amount of the fis and div
transcript produced was quantitated and normalized to that of
bla. In the right panel the effect of the
mutation on the KMnO4 reactivity of the divergent promoter
(bottom strand) and fis promoter (top
strand) after binding of RNAP is shown. Lanes 1,
2, 5, and 6 show the reactions with
pWN1. Lanes 3, 4, 7, and 8 show the reactions with pWN-75A/C. C, the time course of
-galactosidase expression driven from the fis and
divergent promoters using the pWN1 and pWN3 constructs in growing wild
type and crp cells. All values were normalized by setting
the amount of
-galactosidase produced from pWN1 in each overnight
(time 0) wild type and crp cell cultures at 100%. The data
were derived from values obtained in three independent
experiments.
35 element of the
divergent promoter to the consensus sequence (TTGCAT
TgGCAT, see
legend to Fig. 3C) and investigated the effect of the
mutation on promoter function in vitro. Consistent with the prediction, in pWN-75A/C both the initiation of the divergent transcript at
108 and the permanganate reactivity of bases at
101
and
102 were substantially reduced (Fig. 7B, compare
lanes 1 and 2 with lanes 3 and
4 in the left panel, and lanes 5 and 6 with lanes 7 and 8 in the
right panel). Furthermore, the
35 down mutation increased
the activity of the fis promoter on the pWN-75A/C template
about 5-fold with 20 nM and 2-fold with 50 nM
RNAP, respectively (Fig. 7B, compare lane 1 with
lane 3 and lane 2 with lane 4). This
mutation also increased 2-fold the
-galactosidase expression of the
fis promoter from pWN-75A/C measured in 3 h after the
shift-up (2150 Miller units for pWN-75A/C versus 1025 Miller
units for pWN1). The activity of the divergent promoter construct pWN3
containing the extended fis promoter region from position
308 to +106 in an inverted orientation with respect to
lacZ reporter gene was also about 2-fold higher (262 Miller units) than that of the mutant construct pWN3-75A/C (150 Miller units). From these data we conclude that the upstream polymerase binding site in the fis regulatory region acts as a
divergent promoter that interferes with transcription initiation at
the fis promoter.
-galactosidase activity produced
from pWN3 and the fis promoter construct pWN1 in wild type
and crp cells. We observed that the
-galactosidase
activity of cells containing pWN3 rapidly decreased during first 15 min after the shift up in both strains, whereas the
-galactosidase expression from pWN1 increased as expected (Fig. 7C). In
3 h after the shift-up the
-galactosidase levels decreased
further 4-fold in comparison to the 15 min value in wild type but only
slightly in crp cells. Thus, slower decrease in the
activities of both the fis and divergent promoters are
observed in crp cells in comparison with wild type. These
results are consistent with the cooperation of FIS and CRP in
repressing the function of both the fis and divergent
promoters observed in vitro (Figs. 3B and 5).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase production from the fis
promoter when the divergent promoter is inactivated by mutation. The
inverse correlation of changes in the fis and divergent promoter activities observed during the first 15 min after the nutritional shift-up (Fig. 7C) provides further
circumstantial evidence for the regulatory role of the divergent
promoter. However, the interpretation is complicated by the fact that
the data were obtained with fis promoter constructs located
on multi-copy plasmids. A more thorough analysis is required to
understand the link between the growth phase regulation of
fis expression and divergent promoter activity. A divergent
promoter has also been implicated in the regulation of crp
expression, but in this case an overlapping divergent promoter blocks
the occupation of the crp promoter by RNAP (32).
10 region and the
start point of transcription is absolutely required. This latter
element presents a barrier to promoter opening, especially when the
superhelical densities are suboptimal (46). The linkage of
fis expression to growth phase-dependent
fluctuations in DNA topology remains to be elucidated. However, because
the superhelical density of DNA increases after the nutritional
shift-up and CRP might be involved in this increase by activating
gyrA (47, 48), it is conceivable that this indirect effect
would contribute to the rapid attainment of maximal fis expression levels in wild type cells (Fig. 2). In addition, CRP could
directly activate the fis promoter by binding at putative site I centered at
62.5, which is close to the location typical for
the CRP-activated class I promoters (49), thereby displacing the
polymerase molecule from the divergent promoter.
42 and overlapping the
35 region of the fis promoter, whereas the additional binding sites appear to
either have no role in negative autoregulation or support repression to
different extents (20). Consistent with previous reports we observed
that in vitro the binding of FIS at site II displaces the
polymerase from the fis promoter. However, because the
intracellular concentration of FIS decreases rapidly with successive
rounds of cellular division, an additional negative regulatory system has been implicated in keeping the fis promoter in a
repressed state during stationary phase (11). Nevertheless, it is
apparent that at this stage of growth cycle there is enough FIS protein in the cells to repress certain genes (6, 18). Alternatively, at low
intracellular FIS concentrations the FIS effect might be reinforced by
synergistic interactions with other regulators, including CRP.
View larger version (16K):
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Fig. 8.
Model of fis promoter
regulation by FIS, CRP, and RNAP. A, the schematics of
the organization of protein binding sites in the fis
promoter region. The FIS sites I, II, III, and IV are indicated in
roman type, the proposed CRP binding sites I and II in
italic bold type. The 10 and
35 regions of the
fis and divergent promoters are indicated by white
boxes, and DNA is represented as a thin line.
Bent arrows indicate the direction of transcription.
B, changes in the composition of nucleoprotein complexes
modulate the transcriptional activity of the fis promoter.
Step 1, in the absence of FIS and CRP, two polymerase
molecules are bound at the fis and divergent promoters. The
tight binding of RNAP at the divergent promoter reduces the activity of
the fis promoter under conditions of RNAP limitation.
Step 2, binding of CRP displaces RNAP from the divergent
promoter and activates the fis promoter. The relevance and
particular role of each of the putative CRP binding sites I and II in
this effect have not been evaluated at present. Step 3,
after the nutritional shift-up binding of FIS at site II competes with
the binding of RNAP at the fis promoter and reduces promoter
activity. Step 4, simultaneous binding of CRP and FIS
stabilizes FIS binding at site II and perhaps III (see Fig. 3). This
complex synergistically represses the fis promoter activity
during mid exponential growth phase. DNA is represented as a thin
curved line. The changes in the shape of the curve are to indicate
the transitions in local geometry of DNA. The fis transcript
is indicated by a waved line. We note that this model does
not consider the role of IHF in the activation of the fis
promoter.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Annie Kolb for purified CRP protein,
Hermann Heumann for purified RNA polymerase and anti- subunit
antibodies, Regine Kahmann for generous support and critical reading of
the manuscript, and Andrea Schultz for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Département des Sciences de la Vie du CNRS (to W. N.) and by the Deutsche Forschungsgemeinschaft through Grant SFB 190.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Wellcome/Cancer Research Campaign Institute, Tennis Court Rd., Cambridge CB2 1QR, UK.
To whom correspondence should be addressed. Present address:
Max-Planck-Institute for Terrestrial Microbiology,
Karl-von-Frisch-Strasse, D-35043 Marburg, Germany. Tel.:
49-89-2180-6161; Fax: 49-89-2180-6160; E-mail:
Georgi.Muskheli@lrz.uni-muenchen.de.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M100632200
2 W. Nasser and G. Muskhelishvili, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); ORF, open reading frame; DMS, dimethyl sulfate.
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REFERENCES |
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