©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proline Dehydrogenase Activity of the Transcriptional Repressor PutA Is Required for Induction of the put Operon by Proline (*)

Alicia M. Muro-Pastor (§) , Stanley Maloy (¶)

From the (1) Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

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)() 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) .

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.


MATERIALS AND METHODS

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 KmTccolonies. The resulting strain, MST3028 (see Fig. 1A), was then transduced with a P22 lysate from strain TT1794, selecting PSNKmcolonies. One of the resulting Tccolonies 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 Tcsegregants retaining either copy I (PSNKmX-Gal-blue) or copy II (PSNKmX-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 10plaque-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 10plaque-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 Ppromoter (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 Kmgene ( 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 putAalleles 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 PSNcolonies 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 putAstrains constructed, and ApKmcolonies 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 putAallele). P22 lysates grown in the strains obtained were then used to transduce TT962, a PolAstrain, 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 putAalleles). Selection of Apsucroseand further screening of Kmcolonies 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 rmstrains were first propagated in MS1882 (rm).

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)SOat 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)SOat 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 Kand Vvalues 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.


RESULTS

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 TcCmrecombinants 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 PSNphenotype 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 PSNX-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 PSNX-Gal-white phenotype.

About 8000 TcCmcolonies were screened, yielding several colonies with the expected phenotype for super-repressor mutants (PSNX-Gal-white). Three of these putAmutants were characterized in detail: putA1222, putA1223, and putA1224. putA1225, a null putAallele used as a control in further studies, was also isolated in the same screening as a PSNX-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 putAallele, 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 putAmutants, 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 putAMutations

To test whether the putAmutations were dominant over the wild-type putA allele, duplication strains containing each of the three putAalleles on one copy and wild-type putAon 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 putAin trans, resulting in a very low level of expression of the wild-type protein. The presence of both the wild-type and mutant putAgenes in these duplications was confirmed by transduction with a lysate from strain MST179 ( putA::MudJ) followed by selection of Kmcolonies. Recombination of the donor DNA fragment resulted in PSNX-Gal-white colonies (yielding a putA::MudJ/ putAduplication) or PSNX-Gal-blue colonies (yielding a putA/ putA::MudJ duplication) depending on whether the recombination event removed the wild-type or mutant putAallele, respectively. The results of the dominance tests also confirmed that, in all three cases, the putAmutations 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 putAAlleles

Because the putA gene is autoregulated, the expression of the PutA protein is severely reduced in strains with a putAmutation (), making it difficult to accurately measure the enzymatic activities. In addition, the putAmutations 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 putAalleles 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 Ppromoter. To confirm that the putAmutations 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 putAallele in the plasmid resulted in the reduced expression of the putA:: lacZ fusion compared with the level of expression in the presence of the putAallele. Cells containing the putAplasmids 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 putAalleles were able to confer a PSNphenotype, confirming that the super-repressor mutations had recombined into these plasmids.

DNA sequencing of the mutant putAalleles 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)SOpellet, 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 PutAProteins

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.

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 PutAproteins. 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 PutAmutants (). The calculated Kwas 8 mM for the wild-type and the three mutant proteins.

To determine if the decreased proline dehydrogenase activity in the mutant PutAproteins 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 Kand Vvalues 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 minmg of protein), PutA1222 ( K= 285 mM; V= 6670 nmol of INT reduced minmg of protein), and PutA1223 ( K= 7 mM; V= 434 nmol of INT reduced minmg of protein). Finally, the activity observed in PutA1224 was extremely low at all the substrate concentrations tested, preventing the accurate calculation of Kand Vfor 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 minmg 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 PutAProteins

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.

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 PutAproteins, 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).

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).


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.


DISCUSSION

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.
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