From the
The proline utilization ( put) operon from
Salmonella typhimurium consists of the putP gene,
encoding a proline transporter, and the putA gene, encoding an
enzyme with both proline dehydrogenase and 1-pyrroline-5-carboxylate
dehydrogenase activities. In addition to these two enzymatic
activities, the PutA protein is a transcriptional repressor that
regulates the expression of putP and putA in response
to the availability of proline. We report the isolation of
super-repressor mutants of PutA that decrease expression from the
putA promoter in the presence or absence of proline. None of
the mutants exhibited increased affinity for the DNA in the put regulatory region in vitro. Although DNA binding by
wild-type PutA was prevented by the addition of proline and an
artificial electron acceptor, DNA binding by the two strongest
super-repressors was not prevented under identical conditions. The
proline dehydrogenase activity of the purified mutant proteins showed
altered kinetic properties (increased
K
Escherichia coli and Salmonella typhimurium can use proline as a sole carbon and nitrogen source. In these
bacteria, proline utilization requires the presence of two gene
products. The putP gene encodes a membrane protein that is the
major proline permease. The putA gene encodes a bifunctional
dehydrogenase with both proline dehydrogenase (EC 1.5.99.8) and
1-pyrroline-5-carboxylate (P5C)
In addition
to its catalytic functions, PutA is a transcriptional regulator that
represses the expression of both put genes in the absence of
proline. PutA has been shown to bind specifically to operator sites in
the put regulatory region
(2, 6) . Although
proline induces the expression of the put operon in
vivo, incubation of PutA with proline does not prevent the
formation of a PutA-DNA complex in vitro (2, 7) unless an electron acceptor is also available
(7) ,
suggesting that prevention of the formation of a PutA-DNA complex
depends on redox events that follow the binding of proline by PutA. The
following model has been proposed to explain how PutA autoregulates its
own expression. In the absence of proline, PutA remains in the
cytoplasm, where it binds to the put operators, preventing
put gene expression. When a sufficient concentration of
proline is available, PutA binds proline and functionally associates
with the electron transport chain in the cytoplasmic membrane, where it
is enzymatically active
(8) . The resulting decrease in
cytoplasmic PutA levels releases the repression of the operators,
allowing expression of the put genes
(5, 9) .
We report the isolation of mutants of PutA with a super-repressor
phenotype. The purified mutant PutA proteins were defective in proline
dehydrogenase activity, but were not altered in P5C dehydrogenase
activity or DNA binding affinity. Analysis of the mutants indicated
that prevention of the formation of a PutA-DNA complex requires proline
dehydrogenase activity.
About 8000 Tc
DNA
sequencing of the mutant putA
shows
the proline dehydrogenase and P5C dehydrogenase activities of the
purified proteins. Proline dehydrogenase activity (the first of the two
enzymatic steps carried out by PutA) was decreased to a different
extent in all three PutA
To determine if the
decreased proline dehydrogenase activity in the mutant PutA
Although the super-repressor mutations did not directly increase DNA
binding, they could indirectly affect DNA binding by preventing
induction by proline. The formation of a PutA-DNA complex can be
prevented in vitro by preincubating PutA with proline and INT
prior to the DNA addition
(7) . Although neither proline nor INT
alone affects DNA binding, when added together, proline and INT alter
the conformation of the PutA protein so that it assumes a low mobility
form that no longer enters native polyacrylamide gels
(7) .
These observations suggest that the conformational change caused by
proline and INT prevents DNA binding. Therefore, gel retardation assays
in the presence of proline and INT were carried out to determine
whether the PutA
Since these results indicate that proline
dehydrogenase activity is required to prevent DNA binding, we tested
the possibility that a diffusible product of the reaction might be
responsible for this effect. Combinations of the wild-type and mutant
PutA proteins were preincubated with proline and INT before DNA binding
assays were performed (Fig. 7). As expected, DNA binding by
PutA1223 and PutA1224 was not prevented by the addition of proline and
INT (Fig. 7, lanes 2 and 4). However,
in samples that contained wild-type PutA in addition to PutA1223 or
PutA1224, the binding of DNA was prevented, and both the wild-type and
mutant proteins assumed a low mobility form (Fig. 7, lanes 3 and 5) that did not enter the gel
(Fig. 7 B). Thus, when the proteins were mixed before
binding to DNA, the mixture of wild-type and mutant proteins behaved as
the wild-type protein, i.e. the presence of the wild-type
protein abolished the mutant phenotype of PutA1223 and PutA1224 in
vitro. However, if the proteins were mixed after they were bound
to DNA, wild-type PutA did not release PutA1223 or PutA1224 from the
DNA (data not shown).
The PutA protein is unusual because it functions both as a
membrane-associated enzyme and as a DNA-binding protein that represses
its own synthesis. Previous genetic and biochemical evidence indicates
that the choice between its enzymatic or regulatory role is determined
by its cellular distribution: when bound to the membrane, the PutA
protein functions as an enzyme, but when it accumulates in the
cytoplasm, the PutA protein functions as a DNA-binding protein
(5) . Ultimately, this distribution is determined by the
availability of proline, which is both the inducer of the put operon and the substrate for the enzymatic activity of the PutA
protein. But how does proline mediate these two opposing roles of the
PutA protein? One approach to answer this question is to isolate
mutants that affect the regulation of the put operon.
A
large number of loss-of-function mutants have been previously isolated
in the putA gene. Most of the characterized mutants are due to
null mutations that eliminate all three activities of PutA: proline
dehydrogenase, P5C dehydrogenase, and DNA binding
(5) . Such
mutants indicate that the PutA protein is required for transcriptional
regulation, but do not indicate the mechanism of regulation. To
identify the functions of PutA involved in the regulation of the
put operon, we isolated mutants with a super-repressor
phenotype. The scheme we used to isolate such rare mutations had four
important features. (i) The mutants were required to retain the ability
to bind DNA, precluding the isolation of null mutants, which are much
more frequent. (ii) A chromosomal duplication was used, so the ratio of
the gene for the regulator (one copy of putA) to the reporter
gene (one copy of putA::MudJ) was equivalent (in contrast to
plasmid-based schemes, in which one of the genes is present in excess),
eliminating the isolation of mutants that affect the copy number of one
gene or titrate a gene product. (iii) Two copies of the putP gene were included to reduce the probability of isolating mutants
defective in the transport of the inducer proline. (iv) Localized
mutagenesis with P22 transducing particles treated with hydroxylamine
in vitro allowed the isolation of rare mutants while avoiding
the isolation of nonspecific mutants that map outside of the put region.
This approach allowed us to identify several
super-repressor mutants with decreased expression from the putA promoter relative to the wild type. One of the super-repressor
mutants (PutA1224) has a single amino acid substitution in the
predicted proline dehydrogenase domain
(27) . The amino acid
substitutions in the other two super-repressor mutants are located in
another region of PutA. In each of these mutants, the strength of
repression correlates with defects in the proline dehydrogenase
activity of the PutA protein, but the P5C dehydrogenase activity and
DNA binding affinity are not altered. The observation that induction by
proline and proline dehydrogenase activity are simultaneously altered
in all three mutants suggests that proline binds to a single site on
the protein and that proline binding at the active site is responsible
for both enzymatic activity and induction.
It has been previously
shown that reduction by proline induces a conformational change in PutA
(28) . However, reduction by proline does not prevent binding to
the put control region
(7, 28) . Analysis of
the mutants isolated here indicates that proline dehydrogenase activity
itself mediates regulation by proline. PutA1222, which is defective in
the recognition of proline, is a slightly better repressor than the
wild-type protein. Although this mutant requires higher concentrations
of proline to achieve wild-type proline dehydrogenase activity, the
rate of enzymatic conversion of proline to glutamate is not altered.
PutA1223 and PutA1224 are much stronger repressors than the wild-type
protein. These two mutants are severely defective in the proline
dehydrogenase reaction.
The super-repressor phenotype observed
in vivo for the mutants isolated correlates with their DNA
binding properties in vitro. The addition of proline plus INT
to the wild-type PutA protein prevented the formation of a PutA-DNA
complex and caused the wild-type PutA protein to assume a low mobility
form that did not enter the gel (Fig. 6)
(7) . Under
identical conditions, PutA1223 and PutA1224 showed an altered response
to proline in DNA binding assays. In the case of PutA1224, which is
defective in both proline recognition and proline dehydrogenase
activity, the addition of proline plus INT neither induced the
formation of a low mobility form nor prevented DNA binding
(Fig. 6, lanes 14 and 16). In
contrast, with PutA1223, which recognizes proline but is defective in
proline dehydrogenase activity, the addition of proline plus INT
induced the formation of a low mobility form, but did not prevent DNA
binding. This result indicates that the two effects of proline plus INT
on the PutA protein (producing a low mobility form of PutA and
preventing DNA binding) are separate events: binding of proline in the
presence of INT induces a conformational change in PutA that results in
a low mobility form, but prevention of DNA binding requires the proline
dehydrogenase activity of PutA.
Previous studies have shown that a
stable association of PutA with bacterial membranes in vitro requires proline dehydrogenase activity, i.e. simply
reducing PutA by proline is not sufficient to promote stable
PutA-membrane association
(29) . Furthermore, full induction of
the put genes in vivo requires both proline and a
terminal electron acceptor
(30) , suggesting that a functional
coupling of the proline dehydrogenase activity of PutA with the
electron transport chain in the membrane is required for induction of
the put operon. The results presented here provide direct
genetic evidence that proline dehydrogenase activity is required for
induction of the put operon in vivo and prevention of
DNA binding in vitro.
A trivial reason why proline
dehydrogenase activity is required for induction by proline might be
because a product of the reaction, not proline itself, is the actual
inducer. However, three lines of evidence indicate that this is not the
case. (i) The super-repressor mutations are dominant to the wild-type
gene in vivo. (ii) The product of proline dehydrogenase
activity did not prevent DNA binding by wild-type PutA in vitro (Fig. 8). (iii) The addition of P5C to DNA binding assays
did not prevent the formation of a PutA-DNA complex in vitro (data not shown).
In summary, these results indicate that the
redox events associated with the proline dehydrogenase activity of PutA
are required for induction of the put operon by proline. Full
induction of putA expression requires that the PutA protein
synthesized is enzymatically active, thus avoiding wasteful synthesis
of PutA if either membrane sites or proline (which are both required
for enzymatic activity) is not available. Autoregulation of the
putA gene may play an important physiological role because,
like many membrane-associated proteins, overproduction of the PutA
protein is lethal. Hence, by turning off its own synthesis when
membrane sites are saturated, the PutA protein may avoid lethal
overexpression. Some preliminary evidence suggests that certain other
membrane-associated enzymes may also be regulated by a similar
mechanism.
Bacterial strains and plasmids
ccc
Strain/plasmid & Description & Source or Ref.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, reduced
V
, or a completely null phenotype). The
observation that these mutations simultaneously affect induction by
proline and proline dehydrogenase activity suggests that a single
proline-binding site is involved in both proline dehydrogenase activity
and induction of the expression of the put operon.
Furthermore, the results indicate that the proline dehydrogenase
activity of PutA is essential for induction of the put operon
by proline.
(
)
dehydrogenase
(EC 1.5.1.12) activities that catalyzes the oxidation of proline to
glutamate
(1, 2) . The proline dehydrogenase reaction
couples proline oxidation with the reduction of an FAD cofactor tightly
associated with the PutA protein
(2, 3) . The electrons
are transferred to the electron transport chain in the cytoplasmic
membrane in vivo (4) or can be transferred to an
artificial electron acceptor ( p-iodonitrotetrazolium violet
(INT)) in vitro. The reduction of INT can be used to monitor
proline dehydrogenase activity. P5C dehydrogenase activity couples P5C
oxidation with the reduction of NAD. Most previously isolated putA mutants lack both enzymatic activities
(5) .
Bacterial Strains, Media, and Genetic
Techniques
The strains used are listed in . P22
phage lysates and transductional crosses were performed as described
(15) . Strains with partial duplications of the chromosome were
constructed following a modification of a previously described method
(16) . To construct strain MST3324, a P22 lysate from strain
TT1788 was used to transduce strain MST179, selecting KmTc
colonies. The resulting strain, MST3028 (see
Fig. 1A), was then transduced with a P22 lysate from
strain TT1794, selecting PSN
Km
colonies.
One of the resulting Tc
colonies was chosen for further
studies. Chromosomal duplications are unstable in the absence of
selection, so the structure of the duplication in strain MST3324 was
confirmed by segregation of the two copies of the putA gene
after growth in the absence of tetracycline (the selection for the
Tn 10 element at the duplication join point). Cells were grown
on rich medium in the absence of selection and plated onto the same
medium. The colonies were then replica-plated onto rich medium
supplemented with the corresponding antibiotics plus the indicator
X-Gal and to PSN (proline as sole nitrogen source) medium. Homologous
recombination resulted in two types of Tc
segregants
retaining either copy I (PSN
Km
X-Gal-blue) or copy II (PSN
Km
X-Gal-white).
Figure 1:
Strategy for the isolation of
super-repressor mutants. A, construction of strain MST3324
bearing a partial chromosomal duplication that includes the put operon; B, localized mutagenesis of the putA gene in the right side of the duplication (copy II). The different
genetic elements in the diagram are not drawn to scale. Antibiotic
resistance genes and the lacZ gene in the MudJ element are
indicated. The location of the pyrD gene is shown for
orientation relative to the chromosome map. putA* denotes a
potential putA mutant; x y z denote
arbitrary neighboring genes.
Difco nutrient broth (0.8%) containing NaCl at a
final concentration of 0.5% (w/v) was used as rich medium. The minimal
medium used was E medium
(17) supplemented with 0.6% succinate.
When specified, 0.2% proline was added to E minimal medium. The
nitrogen-free medium used to score growth on proline as sole nitrogen
source was NCN medium
(18) supplemented with 0.6% succinate,
0.2% proline, and 1 mM MgCl. PSN plates also
contained the indicator tetrazolium red (2,3,5-triphenyltetrazolium
chloride) at a concentration of 0.0025% (w/v). Solid medium contained
1.5% Difco Bacto-agar (rich or E minimal medium) or Difco Noble agar
(PSN medium). Except where indicated, the final concentrations of
antibiotics in rich medium were as follows: tetracycline, 20 µg/ml;
kanamycin, 50 µg/ml; chloramphenicol, 20 µg/ml; and ampicillin,
30 µg/ml. Concentrations in minimal medium were as follows:
tetracycline, 10 µg/ml; kanamycin, 125 µg/ml; chloramphenicol,
5 µg/ml; and ampicillin, 15 µg/ml. Other supplements used were
X-Gal (20 µg/ml) and sucrose (50 mg/ml). Cells were grown at 37
°C.
Chemical Mutagenesis and Isolation of Super-repressor
Mutants
A concentrated P22 lysate (2 10
plaque-forming units/ml) grown on strain MST1697 was mutagenized
in vitro with hydroxylamine
(15) to 0.1% survival.
Mutagenized phage were centrifuged and resuspended, and then
10
plaque-forming units were used to transduce 100 ml
of an overnight culture of strain MST3324. Cells were concentrated and
plated onto rich medium supplemented with tetracycline (15 µg/ml)
and chloramphenicol, and the colonies obtained were replica-plated onto
different media after 3 days of growth at 37 °C.
Construction of Expression Plasmids by in Vivo
Recombination
The plasmids used are listed in .
Plasmid pPC34 is an Apexpression vector in which the
putA gene is fused to the P
promoter
(19) . pPC34 also contains the lacI gene; thus, the
expression of putA can be induced by IPTG. To construct
pPC101, plasmid pPC34 was partially digested with SalI to
remove the 1932-base pair SalI fragment internal to the
putA gene. This fragment was replaced with a SalI
fragment of
4 kilobase pairs from pRL250 containing the sacB gene from Bacillus subtilis and a Km
gene
( npt)
(14) . In plasmid pPC101a, the transcription of
the sacB gene is in the opposite orientation to that of the
bla gene in the vector. The strategy followed to select for
in vivo recombination of mutant putA
alleles into pPC101 is described in Fig. 2. This strategy
was based on two observations: (i) plasmids derived from pBR322 do not
replicate in polA mutants
(21) , and (ii) cells
containing the sacB gene from B. subtilis do not grow
in the presence of sucrose
(22) .
Figure 2:In vivo recombination of the
mutant putAalleles into an expression vector.
First, the mutant putA alleles were transduced into AA3007, a
S. typhimurium polA strain, selecting for the chloramphenicol
resistance of the linked Tn 10dCm. The resulting
PSN
colonies had also incorporated the mutant
putA allele into the chromosome. Then, pPC101, a plasmid
unable to replicate in a polA mutant, was transduced into each
of the polA putA
strains constructed, and Ap
Km
colonies were selected. Since survival of the
plasmid requires integration into the bacterial chromosome by
homologous recombination, the resulting strains contained a small
duplication bearing two copies of the putA gene ( putA disrupted by the sacB-npt cassette and the mutant
putA
allele). P22 lysates grown in the strains
obtained were then used to transduce TT962, a PolA
strain, and the cells were plated onto rich medium containing
ampicillin and sucrose. Homologous recombination events ( a or
b) would allow the recircularization of the transducing
fragment in two different ways (rendering either pPC101 or the plasmids
containing the putA
alleles). Selection of
Ap
sucrose
and further screening of Km
colonies allowed the identification of cells bearing the plasmids
of interest (pPC102, pPC103, and pPC104). Due to the sucrose
sensitivity of cells containing the sacB gene, colonies in
which pPC101 had been regenerated did not grow in the presence of
sucrose. This method is described in more detail in Ref.
20.
Molecular Biology Techniques
Sequencing of both
strands in double-stranded plasmids was carried out by the dideoxy
chain termination method
(31) using a Sequenase sequencing kit
(U. S. Biochemical Corp.). DNA isolation, restriction, and ligation
followed standard procedures
(23) . Plasmid construction was
done in E. coli DH5. Plasmids that were to be introduced
into S. typhimurium r
m
strains were first propagated in MS1882 (r
m
).
Purification of PutA Protein
The purification of
the wild-type and mutant PutA proteins was carried out by a
modification of the method previously described
(7) . A 1-liter
culture of strain MST2614 carrying the expression plasmid (pPC34,
pPC102, pPC103, or pPC104) was grown to early exponential phase in
plasmid broth
(15) containing ampicillin at 100 µg/ml. The
expression of the putA gene in the plasmid was induced by the
addition of IPTG to a final concentration of 0.1 mM and
incubation of the culture for 1 h. Cells were harvested by
centrifugation, washed once with 1 volume of 0.85% NaCl, resuspended in
35 ml of G buffer (70 mM Tris-HCl, pH 8.2, 20% (v/v)
glycerol), and ruptured in a French pressure cell. The membranes were
removed by centrifugation at 110,000 g for 1 h, and
the resulting crude extract was subjected to precipitation with
(NH
)
SO
at a final concentration of
40%. The pellet was resuspended in 1.5 ml of G buffer and dialyzed
against the same buffer. The samples were brought to 100 mM
KCl, and several 0.5-ml aliquots were subjected to anion-exchange
chromatography on a MonoQ HR 5/5 column using a fast protein liquid
chromatography system (Pharmacia Biotech Inc.). After a 16.5-ml wash
with G buffer containing 100 mM KCl, elution of the proteins
was carried out in G buffer using a linear gradient of KCl
(100-185 mM) followed by isocratic elution at 185
mM KCl in G buffer. The PutA protein eluted from the column
during the isocratic step. Elution of the peak corresponding to PutA
was monitored by the absorbance at 280 nm and confirmed by proline
dehydrogenase assays. For the mutants (PutA1223 and PutA1224) deficient
in proline dehydrogenase activity, the identity of the peak was also
confirmed by SDS-polyacrylamide gel electrophoresis. This modified
purification scheme renders pure PutA protein (as judged by
electrophoresis on SDS-polyacrylamide gels) without the gel filtration
step previously used
(7) . Fractions containing PutA were
pooled, precipitated with (NH
)
SO
at
a final concentration of 50%, resuspended in G buffer, and dialyzed
against the same buffer. The final purified samples (
2 ml)
contained between 1.6 and 4 mg of PutA protein/ml. Purified samples
were stored frozen at
80 °C until use. Protein concentration
was determined using the Bradford protein assay
(24) .
Electrophoresis on SDS-polyacrylamide gels was carried out as described
(15) using discontinuous gels with a 4% stacking gel and a 7%
separating gel. Proteins on SDS or native gels were stained with
FastStain (Zoion Research Inc., Allston, MA).
Gel Retardation Assays
Assays contained 2-20
pmol of PutA protein in G buffer. (The final concentrations of G buffer
were 0.5 in the experiment described for Fig. 5,
0.13
in the experiments described for Fig. 6and 7, and
0.11
in the experiment described for Fig. 8.) 50 ng of the
DNA fragment corresponding to the 420-base pair put control
region (
0.2 pmol) were present in the assays. This fragment was
synthesized by polymerase chain reaction using plasmid pPC6
(12) as a template as described
(23) . The fragment was
used in gel retardation assays without further purification. The 1
binding buffer contained 12 mM Tris-HCl, pH 8.0, 5
mM NaCl, 1 mM MgCl
, 1 mM
CaCl
, 0.1 mM dithiothreitol, and 60 µg/ml
bovine serum albumin. The nonspecific competitor DNA used in the assays
was an HaeIII digest of phage
X174 (500 ng/assay). Where
indicated, PutA was preincubated for 2 min in the presence of proline
and INT (see concentrations and volumes in the legends of
Fig. 6
-8) before mixing with the DNA and binding buffer.
The protein and DNA mixture was then incubated at room temperature for
15 min before adding loading solution (1
loading solution
= 5% (v/v) glycerol, 50 mM EDTA, pH 8.0) and loading in
a gel constantly running at 300 V (the gel was previously prerun at 200
V for 2 h). The DNA fragments were separated by electrophoresis on a 7%
polyacrylamide gel with a 30:0.8
acrylamide/ N, N-methylenebisacrylamide weight ratio.
Electrophoresis was carried out at 4 °C in 1
TBE buffer (89
mM Tris, 89 mM borate, 2.5 mM EDTA, pH 9.5)
at 200 V. The bands on the gel were then visualized with ethidium
bromide (10 µg/ml in 1
TBE buffer), a proline dehydrogenase
activity stain (see below), and/or a protein stain in that order.
Figure 5:
DNA binding affinity of the mutant
PutA proteins. Gel retardation assays containing purified
wild-type or mutant PutA were performed as described under
``Materials and Methods.'' As a control for nonspecific DNA
binding, the assays contained several fragments of DNA not bound by
PutA. Assays contained no protein (
) or increasing amounts (0.3,
0.6, 1.5, and 3 µg) of purified PutA in a 10-µl volume. DNA and
binding buffer were added in a 10-µl volume, and the samples were
incubated for 15 min. Loading solution was then added, and the samples
were loaded on a constantly running 7% polyacrylamide gel.
Electrophoresis was carried out at 4 °C in TBE buffer. The gel was
subsequently subjected to ethidium bromide staining. The positions of
put DNA control region free and bound DNAs are indicated by
arrowheads.
Figure 6:
Effect of proline on the DNA binding
properties of the mutant PutA proteins. Gel retardation
assays were carried out as described under ``Materials and
Methods'' with 3 µg of wild-type or mutant PutA. The protein
was preincubated for 2 min at room temperature in the presence of
proline (0.3 or 0.6 M) and in the presence or absence of 3
mM INT in a 5-µl volume. (It should be noted that at the
concentrations of proline used, the activities of both wild-type PutA
and PutA1222 were able to reduce all the electron acceptor supplied;
thus, INT becomes limiting.) DNA and binding buffer were added in a
10-µl volume, and incubation was continued for 15 min before
subjecting the samples to electrophoresis as described in the legend of
Fig. 5. The gel was subsequently subjected to ethidium bromide staining
( A), a proline dehydrogenase activity stain ( B), and
a protein stain ( C). Only the upper portion of the gel (where
PutA is located) is shown in B and C. The positions
of free and bound DNAs are indicated by
arrowheads.
Figure 8:
Effect of the product of the proline
dehydrogenase reaction on DNA binding by the wild-type PutA protein. A
400-µl sample containing proline (468 mM), INT (2.2
mM), and wild-type PutA (187 µg) and a control sample
without PutA were incubated for 5 min at room temperature. Samples were
filtered through Centricon filters (membrane cutoff of 30,000 Da), and
the treated and control filtrates were collected. 3 µg of wild-type
PutA were preincubated with 6.5 µl of treated ( + lane) or control ( lane) filtrate for 2 min at
room temperature in a 8.5-µl volume. In the sample containing the
control filtrate, the final concentrations in this preincubation were
350 mM proline and 1.68 mM INT. DNA and binding
buffer were added in a 9.25-µl volume, and incubation was continued
for 15 min before subjecting the samples to electrophoresis as
described in the legend of Fig. 5. The gel was subjected to ethidium
bromide staining. The positions of free and bound DNAs are indicated by
arrowheads.
Enzymatic Assays
-Galactosidase activity was
assayed as described
(15) using CHCl
/SDS to
permeabilize whole cells. Proline dehydrogenase activity was assayed by
following the proline-dependent reduction of the electron acceptor INT
as described previously
(1) using crude extracts or purified
protein. Crude extracts used for proline dehydrogenase assays were
prepared from cells grown in E minimal medium supplemented with
succinate and proline. After washing with 1 volume of 0.1 M
sodium cacodylate, pH 6.8, cells were ruptured in a French pressure
cell, and the extract was centrifuged at 5000 rpm to remove intact
cells. The supernatant was used as crude extract. To calculate the
K
and V
values of the proline dehydrogenase reaction, the proline
concentration in the assays was varied from 22 to 454 mM
(wild-type PutA), from 45 to 909 mM (PutA1222), or from 2.3 to
181 mM (PutA1223). Initial rates were measured in all the
assays performed to determine the kinetic parameters of the proline
dehydrogenase reaction, with substrate depletion kept to <5% of the
initial concentration. The proline dehydrogenase bands on native gels
were identified by an activity stain. The gel was immersed in the same
reaction buffer used for purified samples and gently agitated for
5-10 min
(7) . DL-P5C was prepared by hydrolysis
of DL-P5C dinitrophenylhydrazone (Sigma) as described
(25) . The concentration of P5C was determined using
o-aminobenzaldehyde as described
(26) . Due to its
instability at neutral pH, P5C was stored at a concentration of
100 mM in 1 M HCl and vacuum-dried just before
use in enzymatic assays. P5C dehydrogenase activity was assayed by
following the P5C-dependent, NAD-dependent reduction of INT as
described
(1) using purified protein. All proline dehydrogenase
and P5C dehydrogenase assays were corrected for a no-enzyme blank. When
using crude extracts, proline dehydrogenase assays were also corrected
for a no-proline blank.
Isolation of Super-repressor Mutants
To isolate
super-repressor mutants of the PutA protein, a strain was constructed
with a partial chromosomal duplication containing two reporter genes
for the regulation of the transcription from the putA promoter: (i) the putA gene itself and (ii) a
putA::MudJ fusion that places the expression of lacZ under the control of the putA promoter
(Fig. 1 A). Both copies contain wild-type putP genes. The duplication strain used, MST3324, is able to grow on
PSN medium because it contains a wild-type putA allele and
exhibits a blue phenotype in rich medium supplemented with the
indicator X-Gal because the putA::MudJ fusion allows
expression of the lacZ gene from the putA promoter. A
hydroxylamine-treated P22 lysate grown on strain MST1697 was used to
locally mutagenize copy II of the put operon in strain MST3324
(Fig. 1 B). The resulting TcCm
recombinants were replica-plated onto PSN medium (to test for
ability to grow on proline as sole nitrogen source as an indicator of
the expression of the putA gene in copy II) and rich medium
supplemented with X-Gal (to monitor the expression of
-galactosidase as an indicator of the expression of the
putA:: lacZ fusion in copy I). Colonies were also
replica-plated onto two control plates: E minimal medium supplemented
with succinate + proline (to detect mutants in which the
PSN
phenotype was due to auxotrophic mutations) and
rich medium with kanamycin (to detect colonies in which the X-Gal-white
phenotype was due to excision of the MudJ element by recombination
between the mutagenized phage and copy II). In the most common class of
putA mutants, which inactivate the PutA protein, the
expression of
-galactosidase is increased because the mutant PutA
protein is unable to repress the expression of the putA::MudJ
fusion. These putA mutants exhibit a PSN
X-Gal-dark blue phenotype. In contrast, super-repressor mutants
were expected to show reduced expression from both putA promoters ( i.e. low levels of
-galactosidase and
proline dehydrogenase activities), resulting in a PSN
X-Gal-white phenotype.
Cm
colonies were screened, yielding several colonies with the
expected phenotype for super-repressor mutants (PSN
X-Gal-white). Three of these putA
mutants
were characterized in detail: putA1222, putA1223, and
putA1224. putA1225, a null putA
allele used as a control in further studies, was also isolated in
the same screening as a PSN
X-Gal-dark blue colony.
shows the
-galactosidase and proline dehydrogenase
activities of the corresponding strains. In duplication strains with a
null putA allele, the putA:: lacZ fusion was
fully derepressed regardless of the presence or absence of proline. In
duplication strains with a wild-type or putA
allele, the putA:: lacZ fusion was repressed to
different extents. Proline derepressed the putA:: lacZ fusion in duplications with the wild-type putA gene or,
to a lesser extent, in duplications with the putA1222 allele.
In contrast, the putA:: lacZ fusion was fully
repressed in duplications with the putA1223 or putA1224 allele regardless of the presence or absence of proline. Proline
dehydrogenase activity in crude extracts was reduced in all three
putA
mutants, although it was more dramatically
reduced in mutants putA1223 and putA1224. Thus, based
on the level of repression and induction of the putA promoter
by proline, these results indicate that at least two different classes
of super-repressor mutants were isolated. The super-repressor phenotype
of mutant putA1222 seems to be weaker than that of
putA1223 or putA1224.
Dominance of putA
To test whether the putAMutations
mutations were dominant over the wild-type putA allele,
duplication strains containing each of the three putA
alleles on one copy and wild-type putA
on the other copy were constructed. In all cases, the resulting
diploids were unable to grow on proline as sole nitrogen source,
indicating that the super-repressor mutations are dominant over
putA
in trans, resulting in a very
low level of expression of the wild-type protein. The presence of both
the wild-type and mutant putA
genes in these
duplications was confirmed by transduction with a lysate from strain
MST179 ( putA::MudJ) followed by selection of Km
colonies. Recombination of the donor DNA fragment resulted in
PSN
X-Gal-white colonies (yielding a
putA::MudJ/ putA
duplication) or
PSN
X-Gal-blue colonies (yielding a
putA/ putA::MudJ duplication) depending on whether the
recombination event removed the wild-type or mutant putA
allele, respectively. The results of the dominance tests also
confirmed that, in all three cases, the putA
mutations were responsible for the reduced expression of the
putA:: lacZ fusion, excluding the possibility that
this phenotype was due to a second, spontaneous mutation affecting the
expression of the fusion.
Cloning and Sequencing of putA
Because the putA gene is
autoregulated, the expression of the PutA protein is severely reduced
in strains with a putAAlleles
mutation (),
making it difficult to accurately measure the enzymatic activities. In
addition, the putA
mutations also severely reduce
the expression of the proline transporter encoded by the putP gene. The decreased level of proline transported into these cells
would prevent induction of the putA gene by intracellular
proline. Therefore, to overexpress and to purify the mutant proteins,
we cloned the mutant putA
alleles in an expression
vector by an in vivo recombination strategy (Fig. 2). In
the resulting plasmids, pPC102, pPC103, and pPC104, the expression of
the mutant proteins is no longer autoregulated, but is instead driven
by the P
promoter. To confirm that the
putA
mutations had been recombined into the
expression vector, plasmids pPC102, pPC103, and pPC104 were tested for
their ability to regulate the expression of a putA:: lacZ fusion (I). In all three cases, the presence of a
putA
allele in the plasmid resulted in the reduced
expression of the putA:: lacZ fusion compared with the
level of expression in the presence of the putA
allele. Cells containing the putA
plasmids
and a putA::Tn 10 mutation on the chromosome were also
tested for their ability to grow on proline as sole nitrogen source.
None of the plasmids containing putA
alleles were
able to confer a PSN
phenotype, confirming that the
super-repressor mutations had recombined into these plasmids.
alleles indicated
that each of the three mutants contains a single base substitution that
changes the amino acid sequence. Gly-288 is changed to Asn in PutA1222,
Asp-285 is changed to Asn in PutA1223, and Arg-556 is changed to His in
PutA1224. PutA1223 also contains a second mutation at codon 761 (TTC to
TTT) that does not affect the amino acid sequence of the corresponding
PutA protein.
Overexpression and Purification of Mutant
Proteins
The wild-type and mutant PutA proteins were purified
after overexpression from plasmids pPC34, pPC102, pPC103, and pPC104,
respectively. The purification protocol was identical in all four cases
since none of the mutations affected the relevant physical properties
of the mutant proteins with respect to the wild-type protein. After
induction of cells containing pPC34 or any of the plasmids bearing
putAalleles with IPTG, a band corresponding to
PutA (predicted molecular mass of 144 kDa) was evident.
Fig. 3
shows the induction and purification steps for the
wild-type protein and the final purified samples of wild-type and
mutant PutA proteins.
Figure 3:
Overexpression and purification of PutA.
Shown is the SDS-polyacrylamide gel electrophoresis of the wild-type
and mutant PutA proteins. A, induction of wild-type PutA.
Samples contained 100 µl of saturated cultures of strain MST2614
containing the vector pCKR101 ( lanes 1 and
2) or the putA expression plasmid pPC34 ( lanes 3 and 4). Cultures in lanes 2 and 4 were induced with IPTG. Cultures in lanes 1 and 3 were noninduced controls. The position
of the PutA band is indicated by the arrowhead. B,
purification of the wild-type and mutant PutA proteins. Lanes
5-7 correspond to the purification steps described under
``Materials and Methods'': crude extract,
(NH)
SO
pellet, and 3 µg of
purified wild-type PutA, respectively. Lanes 8-10 contained 3 µg of purified PutA1222, PutA1223, and PutA1224,
respectively. The positions of the following prestained high molecular
mass standards (Sigma) are indicated:
-macroglobulin
(190 kDa),
-galactosidase (125 kDa), fructose-6-phosphate kinase
(88 kDa), and pyruvate kinase (65 kDa).
Enzymatic Properties of Mutant PutA
The PutA protein can either associate with the
cytoplasmic membrane, where it is enzymatically active, or bind to the
put regulatory region, where it represses the expression of
both put genes
(5) . Thus, super-repressor mutants
could result from mutations that either increase the affinity of PutA
for DNA or decrease its enzymatic activity. Therefore, we analyzed the
DNA binding and enzymatic properties of the purified mutant PutA
proteins compared with the wild-type protein.
Proteins
proteins. In the case of PutA1222
(a weak super-repressor), the activity observed was
40% of the
wild-type level, whereas in mutants PutA1223 and PutA1224 (the two
strong super-repressors), the activity was reduced to 6 and 0.25% of
the wild-type level, respectively. Thus, there is a direct correlation
between the strength of super-repression (Tables II and III) and the
level of proline dehydrogenase activity (). In contrast to
proline dehydrogenase activity, P5C dehydrogenase activity (the second
enzymatic step carried out by PutA) was not decreased in any of the
PutA
mutants (). The calculated
K
was
8 mM
for the wild-type and the three mutant proteins.
proteins was due to decreased affinity for proline or a decreased
rate of catalysis, the kinetic parameters of proline dehydrogenase
activity were determined (Fig. 4). The
K
and V
values of the proline dehydrogenase reaction were calculated
using Lineweaver-Burk inverse plots for wild-type PutA
( K
= 43
mM; V
= 6900 nmol of INT reduced
min
mg of protein
), PutA1222
( K
= 285
mM; V
= 6670 nmol of INT reduced
min
mg of protein
), and PutA1223
( K
= 7
mM; V
= 434 nmol of INT reduced
min
mg of protein
). Finally, the
activity observed in PutA1224 was extremely low at all the substrate
concentrations tested, preventing the accurate calculation of
K
and V
for this mutant. The values calculated for the wild-type protein
are in good agreement with those previously reported for purified PutA
from S. typhimurium ( K
= 83 mM; V
= 5000
nmol of INT reduced min
mg of
protein
)
(3) , although in Ref. 3, PutA was
purified by a different protocol. Kinetic analysis of the mutant
proteins indicates that PutA1222 is defective in the recognition of
proline ( K
), but not in
the rate of conversion of proline to P5C, whereas PutA1223 is defective
in the conversion of proline to P5C, but not in the recognition of
proline. Furthermore, like wild-type PutA
(3) , PutA1223 was
inhibited by high concentrations of substrate (Fig. 4 B),
also indicating that this mutant protein is not altered in the
recognition of proline.
Figure 4:
Proline dehydrogenase activity of the
mutant PutA proteins. A, proline dehydrogenase
activity of the purified wild-type and mutant PutA proteins as a
function of substrate concentration; B, Lineweaver-Burk plot
of the data in A for the wild-type protein and PutA1223
showing inhibition by the substrate. The amounts of protein used in
these assays were those used in Table IV for proline dehydrogenase
determinations.
DNA Binding Properties of Mutant PutA
To determine whether any of the
super-repressor proteins exhibited enhanced DNA binding affinity, a
series of gel retardation assays were carried out using a DNA fragment
corresponding to the put control region
(6) and
variable amounts of the purified wild-type or mutant protein. As a
control for nonspecific binding, all the assays contained an excess of
DNA fragments that were not bound by PutA. None of the mutant proteins
was significantly enhanced in its affinity for DNA in vitro (Fig. 5). Thus, the super-repressor phenotype observed
in vivo was not the result of increased DNA affinity.
Proteins
proteins, which showed reduced induction
by proline in vivo, responded to the presence of proline
in vitro. The results indicate that proline alone has no
effect on DNA binding by the wild-type and mutant PutA proteins
(Fig. 6). In contrast, when the proteins were preincubated with
proline and INT, several differences between the wild-type and mutant
PutA proteins were evident. At the concentrations of proline used,
there was no difference in the observed DNA binding by wild-type PutA
(Fig. 6, lanes 1-4) and the weak super-repressor,
PutA1222 ( lanes 5-8). In both cases, the formation of a
PutA-DNA complex was prevented by preincubation of PutA with proline
and INT, and in both cases, PutA assumed a low mobility form that did
not enter the gel. In contrast, preincubation of PutA1223 and PutA1224
with proline and INT did not prevent the formation of a PutA-DNA
complex. However, PutA1223 and PutA1224 were different with respect to
the conformational changes induced by proline: whereas PutA1223 assumed
a low mobility form (Fig. 6 C, lanes 10 and 12), PutA1224 did not ( lanes 14 and
16).
Figure 7:
Effect of wild-type PutA on DNA binding by
mutant PutA proteins. Gel retardation assays were carried
out as described under ``Materials and Methods'' with a total
of 3 µg of wild-type and/or mutant PutA. PutA was preincubated for
2 min at room temperature in the presence of proline (0.46 M)
and INT (2.2 mM) in a 6.5-µl volume. Samples containing
the wild-type and mutant proteins were then mixed and incubated
together for 5 min. DNA and binding buffer were added in a 8.75-µl
volume, and incubation was continued for 15 min before subjecting the
samples to electrophoresis as described in the legend of Fig. 5. The
gel was subsequently subjected to ethidium bromide staining
( A) and a protein stain ( B). Only the upper portion
of the gel is shown in B. The positions of free and bound DNAs
are indicated by arrowheads.
There are two potential explanations for the
dominance of the wild-type protein in vitro: (i) the product
of the wild-type reaction (P5C in equilibrium with -glutamyl
semialdehyde) could be the actual effector that prevents DNA binding
(not proline or INT), or (ii) the wild-type and mutant proteins could
form heterodimers that no longer bind DNA in the presence of proline
and INT. Considering the observation that there is no effect on DNA
binding by the mutant proteins when the wild-type and mutant proteins
are mixed after binding to DNA, the second possibility seems more
likely. To directly test whether a diffusible intermediate such as P5C
was the actual effector that prevented DNA binding, proline and INT
were preincubated with wild-type PutA, and the protein was then removed
by filtration. A control sample without PutA was also prepared in
parallel. In the sample preincubated with wild-type PutA, the amount of
enzyme used would exhaust all the oxidized INT present. Both the
treated and untreated filtrates were added to gel retardation assays
with wild-type PutA (Fig. 8). The control filtrate not
preincubated with wild-type PutA (containing proline and INT) prevented
DNA binding as efficiently as when PutA was directly preincubated with
proline and INT (Fig. 8,
lane). However, the
filtrate preincubated with wild-type PutA (containing P5C, the product
of the proline dehydrogenase reaction, and an excess of proline, but no
oxidized INT) did not prevent DNA binding by PutA (Fig. 8,
+ lane). These data suggest that the redox changes
induced in PutA upon the binding of proline in the presence of INT (not
a diffusible intermediate such as P5C) prevent DNA binding.
S. typhimurium
&
AA3007 & ara-9 polA2(Am) & 10
MS1882 & leuA414(Am) hsdL(rm
) endA & M. Susskind
TT962 & putA826::Tn 10 & J. Roth
TT1788 & zcc-4::Tn 10 (20% linked to
put on pyrD side) & J. Roth
TT1794 & zcc-6::Tn 10 (50% linked to
put on pyrC side) & J. Roth
MST179 & putA1020::MudJ
& Laboratory collection
MST1697 & zcc-2473::Tn 10dCm
(80% linked to put on pyrD side) & Laboratory collection
MST2614 & putA1020::MudJ recA1
srl-203::Tn 10dCm