From the Department of Cellular Biochemistry and the
§ Department of Molecular Biology, Hebrew
University-Hadassah Medical School,
Ein Kerem, Jerusalem, 91120 Israel
Received for publication, September 27, 2000, and in revised form, October 31, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Escherichia coli mazEF system is
a chromosomal "addiction module" that, under starvation conditions
in which guanosine-3',5'-bispyrophosphate (ppGpp) is produced, is
responsible for programmed cell death. This module specifies for the
toxic stable protein MazF and the labile antitoxic protein MazE.
Upstream from the mazEF module are two promoters,
P2 and P3 that are strongly negatively
autoregulated by MazE and MazF. We show that the expression of this
module is positively regulated by the factor for inversion
stimulation. What seems to be responsible for the negative
autoregulation of mazEF is an unusual DNA structure, which
we have called an "alternating palindrome." The middle part,
"a," of this structure may complement either the downstream
fragment, "b," or the upstream fragment, "c". When the
MazE·MazF complex binds either of these arms of the alternating
palindrome, strong negative autoregulation results. We suggest
that the combined presence of the two promoters, the alternating
palindrome structure and the factor for inversion stimulation-binding
site, all permit the expression of the mazEF module to be
sensitively regulated under various growth conditions.
In Escherichia coli programmed cell death is mediated
through unique genetic elements called "addiction modules." These
consist of two genes, where the second gene specifies for a stable
toxin, and the first gene specifies for a labile antitoxin. Addiction modules were first discovered in a number of extra-chromosomal elements
where they were found to be responsible for the post-segregational killing effect, that is, the death of cells from which these
extra-chromosomal elements have been removed. In other words, these
cells are "addicted" to the continuous presence of a labile
antitoxic element. Among the best studied addiction modules of this
kind are ccdAB borne on factor F, pemIK borne on
plasmid R100, and phd-doc borne on bacteriophage P1
(reviewed in Refs. 1-4).
All known extra-chromosomal addiction systems have been shown to be
negatively autoregulated at the level of transcription. For example,
such modules as ccdAB of the F factor (5-7),
parD of the plasmid R1 (8), pemIK of the plasmid
R100 (9), or phd-doc of the plasmid P1 (10, 11). Magnuson
and Yarmolinsky (11) suggested that the autoregulation of addiction
modules might prevent fluctuations in the levels of the antidote and
the toxin that would result in the activation of the toxin. During autoregulation, both the toxin and the antidote bind to a palindrome sequence in their own promoter region thereby decreasing their own
transcription. In a few cases, the binding of antitoxin by itself
resulted in a low level of autoregulation; the concomitant binding of
the toxic element increased the level of binding (8, 10). In more
complicated cases, as has been found for the pemIK (9) and
phd-doc (11) modules, the promoter region of addiction module contains two separate palindrome sequences. Based on the stoichiometry and dynamics of binding, Magnuson and Yarmolinsky (11)
suggested a model in which the palindrome sequence binds the antidote
dimer independently but cannot bind the toxin. When the toxin interacts
with the antidote it increases the binding affinity of the antidote to
the palindrome sequence, and thus increases half-life of the complex.
Pairs of genes homologous to some of the extra-chromosomal addiction
modules have also been found on the E. coli chromosome (12-16). As we have reported previously, the E. coli mazEF
system is the first known regulatable prokaryotic chromosomal addiction module (12). This system consists of the two genes, mazE and mazF, that are located in the rel operon
downstream from the relA gene (16). We found (12) that the
mazEF gene pair has all the properties required for an
addiction module. MazF is toxic and long lived, while MazE is antitoxic
and short lived. MazE and MazF are coexpressed and they interact. In
addition, the mazEF system has a unique property: its
expression is regulated by guanosine-3',5'-bispyrophosphate (ppGpp),
which is synthesized by the RelA protein under conditions of amino acid
starvation (17). Furthermore, overproduction of ppGpp induces
mazEF-mediated cell death (12, 18). These properties suggest
that the mazEF module may be responsible for programmed cell
death under conditions of nutrient starvation (12).
Here we studied the regulation of the expression of the
mazEF system. The promoter region of the chromosomally borne
mazEF addiction module is partially homologous to that of
the promoter of the pemIK plasmid borne addiction module
(14). The promoters of both modules contain similar palindrome
sequences, although, like the promoter of the phd-doc module
(11), the pemIK promoter includes two separated palindromes
(9) and the mazEF promoter was found to include only one
(14). Since pemIK is autoregulated, Masuda and colleagues
(14) hypothesized that mazEF might also be autoregulated.
Results of our previous in vitro work (12) revealed that the
chromosomal addiction module mazEF can be expressed from two promoters, P2 and P3, which are located 13 bp1 apart. In in
vivo studies, we found that, P2, the upstream
promoter, was active in exponentially growing cells. Here we showed
that the in vivo activity of the P3 promoter is
only one-tenth of that of the P2 promoter.
We found that the mazEF system is weakly autoregulated by
the antitoxic component MazE, and efficiently autoregulated by the combined action of the antitoxic component MazE and the toxic component
MazF. In this respect, the chromosomal promoter of the mazEF
system is regulated as are most of the previously studied promoters of
extra-chromosomal addiction modules. However, the mazEF
promoter has two unique properties: it has an unusual DNA structure
that we call an "alternating palindrome," and it carries a binding
site for the factor for inversion stimulation (FIS). In the following
discussion, we shall consider the relevance to mazEF
regulation of these two sites.
Media
The media used were LB broth or LB agar (Bio 101, Inc.)
supplemented with the appropriate antibiotics at the following final concentrations: 100 µg/ml ampicillin, 34 µg/ml chloramphenicol, 50 µg/ml kanamycin, or 20 µg/ml tetracycline.
Bacterial Strains
The bacterial strains used in this work are listed in Table
I. The bacterial strain MC4100 and its
derivatives bearing either the mazEF null allele or a
fis
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
allele were constructed by P1 transduction
using strains bearing corresponding mutations as we have described
previously (12, 19).
Bacterial strains and plasmids
Plasmid Construction
Construction of Plasmids Bearing a mazEF Promoter-lac'Z Gene
Fusion--
Using the EF-1 and EG-3 oligomer primers (Table
II), we synthesized a PCR fragment
bearing the mazEF promoter. This fragment contained the end
of the relA gene, the mazEF promoter, and the first 17 codons of the mazE gene. After cutting with
BamHI, we cloned this fragment into the
SmaI-BamHI sites of plasmid pSK106 that bears
the lacZ gene lacking both its promoter and its first eight
codons, that is lac'Z. We called this new plasmid
pSK10
6-pef. Clones of pSK10
6-pef were
selected on 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) plates to which
ampicillin had been added. Plasmids from the selected clones were
purified and sequenced.
|
Construction of Plasmids Bearing mazE or mazEF under the Control of ptac-- Using appropriate DNA primers (EE-1 and EF-2 for mazE gene, or EE-1 and FG-1 for mazEF genes, see Table II), we synthesized PCR fragments bearing the open reading frame of the mazE or the mazEF genes. These PCR products were used for cloning the corresponding genes under the tac promoter present on the expression vector pKK223-3. We called the resulting plasmids pKK-mazE and pKK-mazEF (Table I).
We also cloned these PCR fragments into the modified pSK106
compatible plasmid pLex1 that bears the IPTG inducible promoter ptac (20). We could not use the plasmids of the pKK set for testing the influence of MazE and MazF proteins on their promoter because both pKK223-3 and pSK10
6 are derivatives of the plasmid pBR322 and they are not compatible. Using pLex1 as our parent plasmid,
we constructed the plasmids pLex-mazE and
pLex-mazEF by introducing the promoter ptac, the
chloramphenicol resistance gene, the p15A replication origin, and
either mazE or mazEF such that they were under
control of the ptac promoter. We used these plasmids to
study the influence of the proteins MazE and MazE-MazF on the
mazEF promoter when it was present on the
pSK10
6-pef plasmid.
Crude Extracts of Cellular Proteins
With the exception of the wild type N99 strain, we made crude
cell extracts of all E. coli strains mentioned in Table I. We grew all of the strains at 37 °C to A600
0.2 in LB medium supplemented with appropriate antibiotics. We
transformed strain MC4100mazEF with plasmids
pKK-mazE or pKK-mazEF. We induced the expression of the genes cloned under the ptac promoter by adding 1 mM IPTG, and then allowing growth to continue for 1 more
hour. No plasmids were added to the control bacteria that were also
grown to A600 0.2. Cells were harvested,
sonicated, and centrifuged, and the supernatants were dialyzed
overnight against 10 mM HEPES, 50 mM NaCl, 1 mM dithiothreitol, and 50% glycerol at pH 8.0. The amount of protein in the samples was estimated by use of the Bradford assay
(Bio-Rad). Dialyzed supernatants were stored at
80 °C.
Proteins expressed either from the ptac promoter
bearing plasmid pKK223 or from the chromosome of E. coli
strain MC4100 were analyzed by electrophoresis on denaturing and native
gels and by Western blot analysis using antibodies raised against MazE (Fig. 1, A and C) and MazF (Fig. 1B).
As a control, we tested the proteins expressed from the chromosome of
MC4100mazEF. As we found previously (12), we also found
here that MazE and MazF interact directly. We observed no bands of the
proteins from the cell extracts of the addiction module
mazEF expressed from the cell chromosome, presumably because
under such conditions these proteins were expressed at very low
physiological concentrations. When both MazE and MazF were present on
native gels, we observed a complex between the toxin and its antidote
(indicated by an arrow on the Fig. 1C). We used
these crude protein extracts to study the influence of MazE and MazF on
their own promoter.
Preparation of DNA Fragments
DNA fragments for the gel mobility shift assays and DNase I footprint analysis were obtained by PCR with appropriate primers (Table II) and purified with a gel extraction kit (Qiagen). Short fragments (about 20-30 bp) were obtained by slow annealing of complementary primers in the presence of 100 mM NaCl and 1 mM EDTA. The primers that were used are listed in Table II.
The fragments obtained were end labeled by polynucleotide kinase (New
England Biolabs, Inc.) with [-32P]ATP (Amersham
Pharmacia Biotech) and purified on the Sephadex G-50 columns (Roche
Molecular Biochemicals, Germany).
DNA Sequencing
DNA sequencing was done by the dideoxy method (21) using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech).
Gel Mobility Shift Assay and DNase I Footprint Analysis
Ten-microgram samples of crude protein extracts were diluted with the binding buffer (0.1 M Tris HCl, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl2, 5% glycerol) to a final volume of 10 µl. To inactivate the nucleases, the samples were then heated at 65 °C for 3 min. When the mixtures had cooled to room temperature, 2 µg of poly(dI-dC) (Roche Molecular Biochemicals) and 2 µl of labeled DNA fragments were added. The binding reactions were conducted at room temperature for 10 min, after which they were loaded onto 6% native polyacrylamide gels and run in TAE buffer (22) at 200 V. DNase I footprinting analyses were done according to Giladi et al. (23).
-Galactosidase Assays
-Galactosidase assays were done according to Miller (24).
Gel Filtration Analysis
Crude cellular extract of E. coli
MC4100mazEF strain was loaded on Sephadex G-100 (Amersham
Pharmacia Biotech) column (1.5 × 75 cm, Bio-Rad) equilibrated
with binding buffer, and supplemented with 0.4 M NaCl to
prevent unspecific adsorption. The column was initially calibrated with
standard proteins having established molecular weights. Collected
fractions were analyzed by the gel mobility shift assay.
RNA Extraction and Primer Extension
RNA extraction was carried out using the RNeasy Mini kit
(Qiagen). Primer extension experiments were carried out with avian myeloblastosis virus reverse transcriptase (U. S. Biochemical Corp.) according to Gafny and colleagues (25). The oligonucleotide primer used for the pSK106-pef construct and its mutant
derivatives was "
40 M13 forward" (Amersham Pharmacia Biotech)
from the lacZ gene to the transcription start sites of the
mazEF promoter. The primer was end labeled as a DNA
fragments (see above). Reaction products were resolved on a 6%
sequencing gel. A DNA sequencing reaction was performed with the same
primer and run on the gel parallel to the primer extension reaction. To
quantify the RNA levels, the gels were analyzed and the bands were
quantified using the Fujix BAS100 PhosphorImager.
The Mutagenesis of the Promoter Fragment
Point mutations were introduced into mazEF promoter
region by PCR-based site-directed overlap extension mutagenesis (26) using appropriate primers (Table II). All introduced mutational changes
were verified by DNA sequence determination.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mazEF Promoter Is Negatively Autoregulated--
Like most
addiction modules, sequence analysis of the promoter region of
mazEF suggests that it is autoregulated at the
transcriptional level (15). To test whether mazEF was indeed
autoregulated we chose lac'Z as a reporter gene and fused
it to the mazEF promoter region, where the beginning of
mazE is fused to the eighth codon of lacZ. We
introduced this gene fusion into plasmid pSK106-pef. We
used pSK10
6-pef to transform the
MC4100
mazEF strain and then measured the cellular levels
of
-galactosidase (Fig. 2A). Under optimal growth
conditions at A600 0.2-0.3, we found cellular
levels of
-galactosidase around 7000-8000 Miller units. This high
level of lac'Z expression suggests that the
mazEF promoter is very strong, similar to the promoters of
other addiction modules that have been studied (5, 8-10).
Strain MC4100mazEF, already harboring
pSK10
6-pef, was transformed with the compatible plasmids
pLex-mazE or pLex-mazEF in which the
mazE or mazEF genes were under control of the
IPTG inducible tac promoter. The levels of
-galactosidase
activity were measured at mid-log phase. Inducing plasmid
pLex-mazE to produce MazE led to moderate inhibition (about
40%) of mazEF promoter activity, as reflected by the
reduction in
-galactosidase activity; however, inducing
pLex-mazEF to produce both MazE and MazF led to a much higher level (up to 90%) of inhibition (Fig. 2A). Using
Western blot analysis, we found increased cellular levels of protein
MazE when plasmid pLex-mazE was induced, and similarly,
increased cellular levels of MazE and MazF when pLex-mazEF
was induced (data not shown). Thus, the activity of mazEF
promoter is about five times more inhibited by the combination of MazE
and MazF then by MazE alone.
To verify that the regulation of the mazEF promoter took
place at the transcriptional level, we performed a series of primer extension experiments using plasmid pSK106-pef as the
template. Using RNA extracted from cells carrying this plasmid, we
estimated the relative efficiency of transcription from the two
promoters P2 and P3 (Fig. 2B).
Transcription initiated from promoter P3 was about 10-fold
weaker then that from P2, located 13 bp upstream (Fig.
2B). This explains why we were unable to observe initiation from P3 by primer extension on RNA transcribed from a
chromosome borne mazEF module, which is present in only one
copy per cell (12).
Primer extension experiments under the same experimental conditions
revealed that induction by IPTG led to repression of transcription from
both P2 and P3 by MazE or by the MazE·MazF
complex. Twenty minutes after induction, MazE repressed P2
expression by 53% compared with the activity of the unrepressed
promoter; MazE·MazF complex repressed P2 by 92% (Fig. 2,
B and C). We believe that these two promoters are
inhibited similarly; however, after repression, the levels of the
P3 transcript may have been so low that we could not
measure them (Fig. 2B). These results from our primer
extension experiments (Fig. 1,
B and C) confirmed the data that we obtained in
our assays for -galactosidase activity (Fig.
2A). Thus, we concluded that
the mazEF addiction module is autoregulated at the
transcriptional level.
|
|
Overexpressing the MazE·MazF Complex Leads to a Gel Mobility
Shift of the mazEF Promoter Fragment--
To further investigate the
mechanism of the action of MazE-MazF on the promoters, we studied how
MazE and MazF bind to the promoter region of the mazEF
module (see map in Fig. 3A).
As the source of proteins for these assays we used crude cell extracts enriched for either MazE or MazE and MazF (Fig. 1). For our
electrophoretic mobility shift assay we used the 74-bp fragment of the
mazEF promoter that extends from the multi-linker to the
residue +2 of the P2 promoter (Fig. 3A). This
DNA fragment was labeled and exposed to every one of the cell extracts
that we had prepared (defined in the legend to Fig. 1). We found that
the mazEF promoter was bound by the MazE·MazF complex
(Fig. 4A, lane 4),
confirming our hypothesis that mazEF is negatively
autoregulated (Fig. 2). The crude extract containing only MazE but
lacking MazF also bound the promoter fragment (Fig. 4A,
lane 3). Although MazE was present in approximately equal
amounts when by itself or in the presence of MazF, here the shift was
much weaker (Fig. 1A, compare lanes 3 and
4). Thus, MazE could bind to its own promoter, but, like the
antidotes from most other addiction modules of plasmid origin, the
binding affinity of MazE to its promoter was very low. The cooperative
binding of the toxic protein, here MazF, greatly enhanced the binding
of MazE (Fig. 4A, lane 4).
|
|
We also found that exposing the promoter fragment to the extract of
MC4100mazEF, that contained neither MazE nor
MazF, resulted in an additional retarded band with a very
low shift (Fig. 4A, lane 2). This same band was
present when the promoter fragment was exposed to the extracts that
either contain no MazF or contained it at very low level (Fig.
4A, lanes 1 and 3). This unexpected result suggested that in addition to the binding by its own proteins MazE and MazF, the promoter region of mazEF also bore the
binding site of another as yet unidentified protein. The presence of
MazF together with MazE and this unknown protein in a cell extract caused an overshift of the promoter fragment (Fig. 4A,
lane 4) (see below).
Footprint Analysis of the mazEF Promoter Revealed Protection
against DNase I by the MazE·MazF Complex--
We used DNase I
footprint analysis to define the binding sites in the mazEF
promoter. The 180-bp DNA fragment contained the whole region of the
mazEF promoter from the relA stop codon to the
start codon of mazE, and an additional 100 bp of the
mazE gene. Before digestion by DNase I, we exposed each
strand of this fragment to crude cell extracts: (i) E. coli
strain MC4100mazEF (Fig. 5,
lanes 4); (ii) MC4100
mazEF with MazE
overexpressed from a plasmid (Fig. 5, lanes 3); (iii)
MC4100
mazEF with the MazE·MazF complex overexpressed
from a plasmid (Fig. 5, lanes 2). As seen in the schematic
diagram (Fig. 5, bottom), the protein complex MazE·MazF
protects the large area including (
10) and (
35) elements of both
promoters (P2 and P3). Unfortunately, we were
not able to show DNA protection by MazE alone, probably because of the low affinity of MazE for the promoter region (Fig. 5, lanes
3).
|
Further Characterizing the mazEF Promoter Region by Dividing It
into Two Overlapping Fragments--
To define the binding sites of the
mazEF promoter more precisely, we divided it into two
overlapping fragments: the upstream fragment (72 to
19) and the
downstream fragment (
38 to +2) (Fig. 3B). These two
overlapping fragments shared a common element (
38 to
19). When we
exposed this common element to each of our cell extracts, we observed
no shift in its electrophoretic mobility (data not shown), suggesting
that the sequence that it bears is not long enough to permit binding.
In contrast, using this same set of cell extracts to expose each of the
overlapping fragments, the upstream fragment and the downstream
fragment, caused each of them to be shifted very differently (Fig. 4,
B and C). The upstream element was shifted
identically by every extract used; it was not at all affected by the
presence of MazE or of MazE and MazF (Fig. 4B). In fact, the
upstream element appeared to have bound only the unidentified protein
with which some of the molecules of the whole 74-bp promoter fragment
were observed to bind (Fig. 4A). In contrast, the
retardation of the downstream fragment revealed a strong dependence on
the presence of MazE and MazF (Fig. 4C). As shown above
(Fig. 4A), high concentrations of the MazE·MazF complex
caused an overshift of this DNA probe (Fig. 4C, lane
4) compared with the retardation level obtained with the cell
extract containing only MazE (Fig. 4C, lane 3). When the fragment of the whole promoter region was exposed to a
cellular extract containing high levels of MazE and MazF, we observed
both a supershift and an additional band above it. However, this
highest band was missing from the gel when this same cellular extract
was used to retard the downstream fragment (compare Fig. 4,
A and C, lanes 4). We propose that
this overshifted band may have been formed by the mazEF
promoter fragment binding to all three proteins: MazE, MazF, and the
unidentified protein factor. The mobility pattern of the downstream DNA
fragment exposed to the cell extract of E. coli strain from
which the mazEF genes had been deleted and (Fig.
4C, lane 2) looked like that of the probe alone
(Fig. 4C, lane C), that is, there was no binding.
It did not seem to matter if the downstream fragment was exposed to MazE and MazF expressed from the E. coli chromosome (Fig. 4C, lane 1) or to MazE alone expressed from a plasmid (Fig. 4C, lane 3). We propose that because the promoter region binding affinity of the MazE·MazF complex is much higher than that of MazE alone, high concentrations of MazE result in retardation of the mazEF promoter DNA fragment to the same extent as do low amounts of the MazE·MazF complex (Fig. 4D). High concentrations of the MazE·MazF complex caused an overshift of the whole promoter fragment, although this was not observed in the presence of low concentrations of these proteins (Fig. 4D). These dynamics probably indicate that there are several available binding sites in the promoter region.
FIS Is the Cellular Protein Responsible for the Gel Mobility
Shift of the Upstream mazEF Promoter Fragment--
We showed above
that an unidentified E. coli protein bound the upstream
mazEF promoter fragment (72 to
19) (Fig. 4B).
To determine the size of this protein, we loaded the crude extract of
E. coli MC4100
mazEF on a Sephadex G-100
column. The fractions collected were subjected to a gel mobility shift
assay with the upstream fragment of the mazEF promoter (data
not shown). In these fractions, the factor that bound the promoter
corresponded to a protein with a molecular mass of 20-25 kDa.
To identify this promoter-binding protein, we used our broad collection
of E. coli mutants. We prepared crude cell extracts (see
"Experimental Procedures") of the mutants defective in the
following DNA-binding factors: the FIS, integration host factor, rpoS
(stationary phase
factor), gyrase, and histone-like proteins H-NS
and HU. Gel mobility shift assays on the upstream DNA fragment of the
mazEF promoter exposed to each of these extracts (Fig.
6A) revealed retardation in
every case except when FIS was absent (Fig. 6A, lane
2). The addition to that same DNA fragment of increasing amounts
of the pure protein FIS (kindly provided by G. Muskhelishvili)
(Fig. 6B, lanes 9-11) resulted in retardation at
the same level as that observed by the unidentified protein factor from
the crude extract of E. coli MC4100
mazEF (Fig.
6B, lane 8). FIS, a protein of molecular mass 12 kDa that in vivo forms homodimers of molecular mass 25 kDa (27) corresponded perfectly to the protein obtained in our gel filtration experiment (see above). Thus, we concluded that the unidentified protein was in fact FIS.
|
That the effect of the addition of FIS on the mobility of the upstream fragment represented a specific DNA binding event was further supported by the results of two additional sets of experiments. (i) The character of the gel shift was not changed (data not shown) when we cut the upstream mazEF promoter fragment with the enzyme SalI whose restriction site is in the multi-linker (see map in Fig. 3B). (ii) On the other hand, when we used the enzyme HaeIII, whose restriction site is within the promoter region, neither of the two resulting fragments were retarded by the addition of FIS (data not shown).
We determined the extent of the influence of the FIS on
mazEF promoter expression by primer extension experiments.
Wild type E. coli strain MC4100 and its
fis mutant derivative were transformed with
the plasmid pSK10
6-pef bearing the lac'Z
gene under the control of the mazEF promoter. We measured
the levels of transcription in these transformants at the entry to
logarithmic growth (Fig. 6, C and E). The level of RNA transcription from this plasmid in the wild strain was about
1.6-fold higher then that in the mutant strain, indicating that FIS
activates the mazEF promoter as it acts on most other promoters that it regulates (28). A mutation in the putative FIS-binding site of the mazEF promoter, in which the T
residue at position (
40) is replaced by a G residue, caused
the mazEF promoter to be insensitive to activation by FIS
(Fig. 6, D and F).
Analysis of the Autoregulation Region of the mazEF
Promoter--
Since we were able to show that the downstream
fragment of the mazEF promoter was responsible for
mazEF autoregulation (Fig. 4C), the next step was
to analyze its sequence (Fig.
7A). The autoregulation
regions of all known addiction modules contain palindrome structures,
and mazEF was no exception: the palindrome sequence
"a-b," analogous to the palindrome of pemIK, had already been predicted by Masuda and colleagues (14).
|
Sequence analysis of the area protected by the MazE·MazF complex revealed an unusually complicated structure (Fig. 7): (i) fragment "a" of the palindrome could be complemented not only by fragment "b," but also by fragment "c." We called this "c-a-b" component an alternating palindrome, (ii) the center of an additional palindrome, "d-e," is located 4 bp upstream from the center of the "c-a" palindrome (Fig. 7).
We present a possible alignment of the palindrome fragments (Fig.
7B) and a possible base pairing among them (Fig.
7C). The "d" and "e" parts of the palindrome show a
perfect complementation (Fig. 7, B and C). The common
element (38
19) of the overlapping upstream and downstream
fragments, tested before, contained almost the whole "d-e"
palindrome, except for its last nucleotide T (
18). In the gel shift
mobility assay the cell extracts contain either MazE alone or
MazE·MazF complex did not show retardation with this DNA fragment
(data not shown).
Within the alternative palindrome the homology was high, especially
between parts "c" and "b". We hypothesized that at any given
moment the alternating palindrome might exist in one of two possible
configurations: "c-a" or "a-b" (Fig. 7C). To test our model, we performed gel mobility shift experiments using the two
overlapping parts of the double palindrome, "a-b" (20 to +6) and
"a-c" (
34 to
7) and of their common fragment "a" (
23 to
7) alone. The MazE·MazF protein complex bound each of the two
palindrome sequences ("a-b" or "a-c"), while none of the cell extracts caused a shift of the middle fragment "a" alone (Fig. 8A).
|
To verify our model further, we constructed a variation of the
downstream promoter fragment "c-m-b" (38 to +6), in which we
replaced the middle part "a" with an unrelated multilinker sequence
that we called "m". After exposing this "c-m-b" fragment to our
standard set of crude cellular extracts, we ran the mixtures in a gel
mobility shift assay. As we observed for fragment "a" alone, none
of the cell extracts led to retardation of the "c-m-b" fragment
(Fig. 8A). In these experiments, we observed no binding of
MazE by itself to any other fragment than to the complete "c-a-b" fragment (Fig. 8A).
We asked: what was the factor that was important for binding? Was it
simply the sequence of the double palindrome or was the secondary
structure of the promoter region required? To distinguish between these
two possibilities we introduced several mutations into the sequence of
the mazEF promoter region (indicated by the black points in
Fig. 7C and specified in Table II). Every mutation we
introduced destroyed a hydrogen bond in the proposed structure, and in
the case of mutations (25,
26, and
27) three such bonds were
destroyed simultaneously. Each mutated fragment was exposed to cell
extracts enriched for the MazE·MazF complex and was run on a gel
mobility shift assay. It appears that introducing these mutations
caused no changes in the mobility of the promoter fragment (data not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In previous studies we have shown that the chromosomal genes
mazE and mazF, located in the E. coli
rel operon, have all the properties required to be an addiction
module (12). Along with properties shared with other known addiction
systems, the mazEF addiction module has two additional
properties: (a) it is directed from two promoters,
P2 and P3, located 13 nucleotides apart; and (b) under conditions of amino acid starvation,
mazEF expression is regulated by the cellular level of
ppGpp, the product of the RelA protein. Here we have shown the
following: (i) the P2 promoter is about 10-fold stronger
then is the P3 promoter; (ii) expression from both
P2 and P3 is repressed by MazE and is highly
repressed by the MazE·MazF complex; (iii) MazE and MazF could bind to
an alternating palindrome that we found in the promoter region (34 to
+6). This alternating palindrome, which in fact is the operator of
mazEF, could exist in one of two alternative states: its
middle part a complemented with either of the outer parts b or c (Fig. 7); (iv) expression from mazEF promoters is activated by
FIS.
When -galactosidase was expressed under the control of the
mazEF promoters, we observed as much as 7000-8000 Miller
units at mid-logarithmic growth (Fig. 2A). This high level
of synthesis indicated that the mazEF promoter was very
strong, as are the promoters of most addiction modules (5, 8-10).
Moreover, the presence in trans of the gene products of the
mazEF module led to negative autoregulation, and about
10-fold repression of transcription (Fig. 2).
In further experiments, we obtained cell extracts containing the MazE and MazF proteins, either transcribed from the chromosome or overexpressed from a plasmid under the control of the tac promoter. When MazE and MazF are present together in a cell extract they form a complex. Here, and in other studies, we observed the formation of such a complex in native gel electrophoresis with [35S]methionine-labeled proteins (12), with antibodies against MazE (Fig. 1C), and also in an E. coli two-hybrid system.2
By gel mobility shift assays we clearly showed that the retardation of the promoter fragment depended on exposure to either the MazE or MazE·MazF complex. We were surprised to find that this DNA fragment could also be retarded by an initially unidentified protein present in the crude cellular extract of a strain from which mazEF had been deleted (Fig. 4A). On the basis of our results reported above, we have concluded that this regulation protein is FIS. FIS is one of the nucleoid-associated proteins that regulate various processes, including transcription, recombination, and replication (28, 29). Here we found that FIS increased the activity of the mazEF promoter by 1.6-fold (Fig. 6, C and E). However, it is possible that under certain specific stressful conditions the effect of FIS on mazEF could be more profound. In this regard, we suggest that FIS may affect the role of mazEF in programmed cell death. Under various physiological conditions, the cellular levels of FIS vary over a large interval (up to 100-fold) and they depend on both the growth phase and nutritional conditions (29, 30). In rich medium, the concentrations of FIS are very high in the early exponential phase, but sharply decrease toward stationary phase. FIS is known to act as a homodimer (27); the molecular weight that we calculated for the initially unidentified protein from the results of our gel filtration experiments corresponded to the molecular weight of FIS as a homodimer. It has been shown that by binding to the DNA region upstream from promoters, this homodimer causes the DNA to bend, thus increasing the binding efficiency of the RNA polymerase (31). Thus, positive regulation of the mazEF promoter by FIS must be maximal under conditions of rapid growth on rich media.
We found a high level of conformity between the sequence of the upstream fragment of the mazEF promoter and the consensus sequence of the known FIS-binding sites (28). When we introduced a point mutation in the FIS-binding site of the mazEF promoter, the influence of FIS on the promoter was abolished (Fig. 6, D and F), further confirming that FIS participates in mazEF gene regulation. We were able to ascertain the precise location of the FIS-binding site in the mazEF promoter (underlined in the schematic diagram in Fig. 3C). FIS regulation of the promoter of this addiction module seems to be a unique feature of the mazEF module.
While FIS caused positive regulation of the mazEF promoter, autoregulation of mazEF promoter was strongly negative as it is for most known addiction modules. We found that the autoregulation site of mazEF was longer and more complicated than would have been predicted by the results of previous studies (14). In our DNA footprint experiments, we found that an area more extended then the a-b palindrome was protected against DNase I digestion by the MazE·MazF protein complex (Fig. 5). In addition to this a-b palindrome, which is similar to the palindrome in the pemIK promoter (14), we discovered an unusual structure that we have called an alternating palindrome. Thus, our results suggest that the middle fragment a may complement not only the downstream fragment b, but also fragment c located upstream from a (Fig. 7C). Comparing fragments b and c revealed that they were highly similar (Fig. 7B). The "alternating binding model" that we have proposed here is supported by the results of our gel mobility shift experiments: the MazE·MazF complex could bind both alternate structures, c-a and a-b, but not the central a fragment by itself. Moreover, the MazE·MazF complex could not bind the c-m-b fragment in which the a fragment was replaced by an unrelated sequence (Fig. 8A). Based on mutational analyses, we propose that the mazEF promoter can exist in two possible alternate states (Fig. 8). The MazE·MazF complex can bind either of these structures, resulting in strong negative autoregulation (see below).
The role of the additional d-e palindrome is not yet clear. The regulation areas of the promoters of many known addiction modules contain a palindrome sequence (9-11, 32). Moreover, some addiction modules, like phd-doc and pemIK, also contain two palindromes (9, 10, 11, 14). It has been suggested that in the regulation of the phd-doc addiction module, the toxic, and the anti-toxic proteins bind to the two palindromes cooperatively. This binding process is accompanied by an increased affinity of the protein for the DNA, and hence an increasing stability of the DNA-protein complex (11).
The numerous mutations that we introduced into the alternating palindrome did not at all affect the binding efficiency of the Maze·MazF complex, suggesting that the secondary structures of the regulating region is more important than its DNA sequence per se. The alternating palindrome that we have described seems to be a unique feature of the mazEF promoter. In this structure, it is as though the two palindromes often found in the promoters of other addiction modules (9-11, 14) have been collapsed, thus minimizing the space required for the regulating elements without losing efficiency.
We suggest that the combined presence of two promoters, a complicated palindrome structure, and the FIS-binding site permits regulation of expression that is simultaneously safe and dynamic, enabling quick responses to changes in physiological conditions. The duplication of the structural elements (promoters or binding sites for autoregulation) assures that mazEF regulation will be adequate even in the case that one of these elements may be destroyed. The action of two promoters, P2 and P3, is additive; during exponential growth they are repressed by auto-regulation to 10-12% of their full capacity. On the one hand, cellular levels of the positive regulator FIS are high in cells growing in rich medium; on the other hand, cellular levels of the negative regulator ppGpp increase under conditions of starvation. Thus the intracellular concentrations of mazEF products strongly depend on the growth conditions.
The regulation of the systems responsible for programmed cell death
must be very carefully controlled; otherwise, fluctuations in the
concentrations of the system's products may result in death. The
duplication of the regulatory elements in the operator of the
mazEF operon provides such safety to the "addiction
system." We propose that limited space on the E. coli
chromosome is the reason for the metamorphosis of two palindromes for
autoregulation into the alternating structure. Thus, the
mazEF promoter is elegantly engineered to respond to any
possible changes in the nutritional environment of the bacterium.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Rahel Warshaw-Dadon for critical reading of the manuscript. We are grateful to G. Muskhelishvili for providing pure FIS protein.
![]() |
FOOTNOTES |
---|
* This work was supported by Israel Science Foundation Grant 467/99-2 (to G. G.).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.
¶ To whom correspondence should be addressed. Tel.: 972-2-675-8168; Fax: 972-2-641-5848; E-mail: glaser@cc.huji.ac.il.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M008832200
2 I. Marianovsky and G. Glaser, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
bp, base pair(s);
FIS, factor for inversion stimulation;
PCR, polymerase
chain reaction;
IPTG, isopropyl-1-thio-D-galactopyranoside.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Couturier, M., Bahassi, el-M., and Van Melderen, L. (1998) Trends Microbiol. 6, 269-275[CrossRef][Medline] [Order article via Infotrieve] |
2. | Engelberg-Kulka, H., and Glaser, G. (1999) Annu. Rev. MicroBiol. 53, 43-70[CrossRef][Medline] [Order article via Infotrieve] |
3. | Jensen, R. B., and Gerdes, K. (1995) Mol. Microbiol. 17, 205-210[Medline] [Order article via Infotrieve] |
4. | Yarmolinsky, M. B. (1995) Science 267, 836-837[Medline] [Order article via Infotrieve] |
5. | de Feyter, R., Wallace, C., and Lane, D. (1989) Mol. Gen. Genet. 218, 481-486[Medline] [Order article via Infotrieve] |
6. | Tam, J. E., and Kline, B. C. (1989) J. Bacteriol. 171, 2353-2360[Medline] [Order article via Infotrieve] |
7. | Tam, J. E., and Kline, B. C. (1989) Mol. Gen. Genet. 219, 26-32[Medline] [Order article via Infotrieve] |
8. | Ruiz-Echevarria, M. J., Berzal-Herranz, A., Gerdes, K., and Diaz-Orejas, R. (1991) Mol. Microbiol. 5, 2685-2693[Medline] [Order article via Infotrieve] |
9. | Tsuchimoto, S., and Ohtsubo, E. (1993) Mol. Gen. Genet. 237, 81-88[Medline] [Order article via Infotrieve] |
10. |
Magnuson, R.,
Lehnherr, H.,
Mukhopadhyay, G.,
and Yarmolinsky, M. B.
(1996)
J. Biol. Chem.
271,
18705-18710 |
11. |
Magnuson, R.,
and Yarmolinsky, M. B.
(1998)
J. Bacteriol.
180,
6342-6351 |
12. |
Aizenman, E.,
Engelberg-Kulka, H.,
and Glaser, G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6059-6063 |
13. | Gotfredsen, M., and Gerdes, K. (1998) Mol. Microbiol. 29, 1065-1076[CrossRef][Medline] [Order article via Infotrieve] |
14. | Masuda, Y., Miyakawa, K., Nishimura, Y., and Ohtsubo, E. (1993) J. Bacteriol. 175, 6850-6856[Abstract] |
15. | Masuda, Y., and Ohtsubo, E. (1994) J. Bacteriol. 176, 5861-5863[Abstract] |
16. |
Metzger, S.,
Dror, I. B.,
Aizenman, E.,
Schreiber, G.,
Toone, M.,
Friesen, J. D.,
Cashel, M.,
and Glaser, G.
(1988)
J. Biol. Chem.
263,
15699-15704 |
17. | Cashel, M., Gentry, D. R., Hernandez, V. Z., and Vinella, D. (1996) Escherichia coli and Salmonella: Cellular and Molecular Biology , 2nd Ed , pp. 1458-1496, ASM Press, Washington, D. C. |
18. |
Engelberg-Kulka, H.,
Reches, M.,
Narasimhan, S.,
Schoulaker-Schwarz, R.,
Klemes, Y.,
Aizenman, E.,
and Glaser, G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15481-15486 |
19. | Aviv, M., Giladi, H., Oppenheim, A. B., and Glaser, G. (1996) FEMS Microbiol. Lett. 140, 71-76[CrossRef][Medline] [Order article via Infotrieve] |
20. | Diederich, L., Roth, A., and Messer, W. (1994) BioTechniques 16, 916-923[Medline] [Order article via Infotrieve] |
21. | Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 80, 7010-7013 |
22. | Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
23. | Giladi, H., Koby, S., Gottesman, M. E., and Oppenheim, A. B. (1992) J. Mol. Biol. 224, 937-948[Medline] [Order article via Infotrieve] |
24. | Miller, J. H. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
25. | Gafny, R., Hyman, H. C., Razin, S., and Glaser, G. (1988) Nucleic Acids Res. 16, 61-76[Abstract] |
26. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Koch, C.,
and Kahmann, R.
(1986)
J. Biol. Chem.
261,
15673-15678 |
28. | Finkel, S. E., and Johnson, R. C. (1992) Mol. Microbiol. 6, 3257-3265[Medline] [Order article via Infotrieve] |
29. | Xu, J., and Johnson, R. C. (1997) J. Mol. Biol. 270, 346-359[CrossRef][Medline] [Order article via Infotrieve] |
30. | Ball, C. A. R., Osuna, R., Ferguson, K. C., and Johnson, R. C. (1992) J. Bacteriol. 174, 8043-8056[Abstract] |
31. | Pan, C. Q., Finkel, S. E., Cramton, S. E., Feng, J.-A., Sigman, D., and Johnson, R. C. (1996) J. Mol. Biol. 264, 675-695[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Roberts, R. C.,
Spangler, C.,
and Helinski, D. R.
(1993)
J. Biol. Chem.
268,
27109-27117 |
33. | Casadaban, M. J., and Cohen, S. N. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4530-4533[Abstract] |
34. | Casadaban, M. J., Chou, J., and Cohen, S. N. (1980) J. Bacteriol. 143, 971-980[Medline] [Order article via Infotrieve] |