GcvR interacts with GcvA to inhibit activation of the Escherichia coli glycine cleavage operon

Angela C. Ghrist1, Gary Heil1 and George V. Stauffer1

Department of Microbiology, The University of Iowa, Iowa City, IA 52242, USA1

Author for correspondence: George V. Stauffer. Tel: +1 319 335 7791. Fax: +1 319 335 9006. e-mail: george-stauffer{at}uiowa.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Escherichia coli glycine cleavage enzyme system, encoded by the gcvTHP operon, catalyses the oxidative cleavage of glycine to CO2, NH3 and a one-carbon methylene group. Transcription of the gcv operon is positively regulated by GcvA and negatively regulated by GcvA and GcvR. Using a LexA-based system for analysing protein heterodimerization, it is shown that GcvR interacts directly with GcvA in vivo to repress gcvTHP expression. Several mutations in either gcvA or gcvR that result in a loss of gcv repression also result in a loss of GcvA/GcvR heterodimerization. Finally, it is shown that the C-terminal half of GcvA is involved in its interaction with GcvR, whilst the entire GcvR protein appears to be necessary for heterodimerization.

Keywords: gcvTHP, repressor, activator, repression, antiactivation

Abbreviations: AP, ampicillin; DBD, DNA-binding domain; gcv, gcvTHP; TC, tetracycline; TPEG, phenylethyl-ß-D-thiogalactoside


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Escherichia coli glycine cleavage enzyme system, encoded by the gcvTHP (gcv) operon, catalyses the oxidative cleavage of glycine to CO2, NH3 and a one-carbon methylene group (Kikuchi, 1973 ; Meedel & Pizer, 1974 ). This one-carbon group is transferred to the acceptor tetrahydrofolate to form 5,10-methylenetetrahydrofolate, a methyl donor used in the biosynthesis of purines, thymine, methionine and numerous methylated products (Mudd & Cantoni, 1964 ). Transcription of the gcv operon is activated by the GcvA protein in the presence of excess glycine. When glycine is limiting, GcvA actively represses expression of the operon; this repression is more severe in the presence of excess purines (Wilson et al., 1993a , b ). GcvA, a member of the LysR family of transcriptional regulators, binds by way of a helix–turn–helix motif to three sites upstream of the gcv promoter (Wilson et al., 1995 ; Jourdan & Stauffer, 1998 ) (Fig. 1). Binding sites 2 and 3 are required for activation of the operon in response to glycine, whereas all three sites are required for repression in response to purines (Wilson et al., 1995 ; Wonderling et al., 2000 ).



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Fig. 1. gcv control region. The GcvA upstream (sites 3 and 2) and downstream (site 1) binding sites (Wilson et al., 1995 ) and the Lrp-binding region (Stauffer & Stauffer, 1994 ) are shown relative to the gcv promoter -35, -10 and +1 sequences (Stauffer et al., 1993 ). Not to scale.

 
GcvA-mediated regulation of gcv expression requires two additional proteins, Lrp and GcvR. Lrp, a global transcriptional regulator of genes involved in amino acid metabolism (Lin et al., 1992 ; Calvo & Matthews, 1994 ), is required for both activation and repression of gcv expression (Stauffer & Stauffer, 1994 ). It has been proposed that Lrp plays a structural role in gcv regulation, binding to and bending the DNA between GcvA-binding sites 1 and 2 to facilitate the formation of appropriate regulatory complexes (Stauffer & Stauffer, 1999 ) (Fig. 1).

GcvR, a negative-acting protein required for GcvA-mediated repression of gcv transcription (Ghrist & Stauffer, 1995 ), does not appear to regulate gcv expression directly, as it fails to bind to the gcv control region in gel mobility shift assays (A. Ghrist, unpublished results). In addition, GcvR does not regulate gcv expression indirectly by regulating the expression of gcvA or gcvR (Ghrist & Stauffer, 1998 ). Rather, repression of the gcv operon depends on the relative levels of expression of gcvA and gcvR; overexpression of gcvA results in gcv activation, whereas overexpression of gcvR results in super-repression (Ghrist & Stauffer, 1995 ). In light of these observations, we hypothesized that GcvR interacts directly with GcvA to control activation and repression of the gcv operon (Ghrist & Stauffer, 1998 ). In this study, we present evidence that supports this hypothesis. Using a LexA-based system for analysing protein heterodimerization, we show that GcvA and GcvR interact in vivo. In addition, we show that gcvA and gcvR mutations which decrease gcv repression also decrease heterodimerization.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
All strains used in this study were constructed in our laboratory and are derivatives of the E. coli K-12 strain GS162 [pheA905 thi {Delta}(argF–lac)U169 araD129 rpsL150 relA1 deoC1 flbB5301 ptsF25 rbsR]. Additional strain designations and mutations are as follows: GS1111, {Delta}gcvR bcp{Sigma}neo; GS1118, {Delta}gcvA orf2{Sigma}aadA; GS1131, {Delta}gcvR bcp{Sigma}neo, {Delta}gcvA orf2{Sigma}aadA. All lysogens denoted with {lambda}gcvT::lacZ carry a single chromosomal copy of a gcvT::lacZ reporter fusion in phage {lambda}gt2 (Stauffer et al., 1993 ). The lysogen GS1131{lambda}sulA::lacZ carries a single chromosomal copy of the sulA::lacZ reporter fusion from strain SU202 (Dmitrova et al., 1998 ).

The multi-copy plasmid used in the gcvR mutant screen was pBR322 (Bolivar et al., 1977 ). The low-copy-number plasmid used in this study was pSC101 (Cohen & Chang, 1977 ) and the single-copy plasmid used was pGS311 (Ghrist & Stauffer, 1995 ).

Media and growth conditions.
The rich medium used was Luria–Bertani broth (LB) (Miller, 1992 ). The minimal media used were minimal salts (Vogel & Bonner, 1956 ) supplemented with phenylalanine (50 µg ml-1), thiamin (1 µg ml-1), and either 0·4% glucose (GM) or 0·4% lactose (LM). Agar was added at 1·5% to make solid media. Additional supplements were added, where indicated, at the following concentrations: inosine, 50 µg ml-1; glycine, 300 µg ml-1; phenylethyl-ß-D-thiogalactoside (TPEG), 2 mM; ampicillin (AP), 150 µg ml-1 for multi-copy plasmids and 30 µg ml-1 for single-copy plasmids; tetracycline (TC), 10 µg ml-1. {lambda}gcvT::lacZ lysogens carry the cI857 mutation resulting in a temperature-sensitive repressor and were grown at 30 °C. All other strains were grown at 37 °C.

Construction of LexA fusion plasmids.
Wild-type and mutant alleles of gcvA were cloned into pMS604 (Dmitrova et al., 1998 ) on PCR-generated BstEII–XhoI fragments, creating in-frame fusions with the LexA DNA-binding domain (DBD). Wild-type and mutant gcvR alleles were cloned into pDP804 (Dmitrova et al., 1998 ) on PCR-generated BssHII–BglII fragments, creating in-frame fusions with the LexA408 DBD variant. The nucleotide sequence of each PCR-generated fragment was verified by DNA sequence analysis.

ß-Galactosidase assays.
ß-Galactosidase assays were performed as described by Miller (1992) . Each experiment was repeated at least twice, with each sample assayed in triplicate.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
GcvR interacts with GcvA in vivo
To determine if GcvR interacts with GcvA in vivo, we used a LexA-based system for analysing protein heterodimerization developed for use in E. coli (Dmitrova et al., 1998 ). In this system, the reporter strain carries a {lambda}sulA::lacZ fusion under the control of a hybrid LexA operator containing a wild-type half-site and a mutant half-site (Fig. 2). The wild-type half-site is recognized by the wild-type LexA DBD and the mutant half-site is recognized by the LexA408 DBD variant. Interaction between protein domains fused to these LexA DBDs will create LexA/LexA408 heterodimers capable of repressing the {lambda}sulA::lacZ reporter (Fig. 2). For this experiment, gcvA was fused downstream of the wild-type LexA DBD sequence of pMS604 and gcvR was fused downstream of the LexA408 DBD sequence of pDP804. The expression of both fusion proteins is under the control of a lacUV5 promoter with a down mutation in the ribosome-binding site. Since the reporter strain GS1131{lambda}sulA::lacZ carries a lacI deletion, both fusion proteins are constitutively expressed in this strain. The reporter strain transformed with one or both of these fusion plasmids was grown in LB (plus appropriate antibiotics) and ß-galactosidase activity was determined. Expression of either fusion protein alone resulted in a small decrease in ß-galactosidase activity (less than 30%) (Table 1). However, co-expression of both fusion proteins resulted in a significant decrease in ß-galactosidase activity (Table 1, row 4). Because plasmids pMS604 and pDP804 are high-copy number and since each fusion protein alone had a small effect on sulA::lacZ expression, we tested whether the effects of the GcvA and GcvR fusion proteins alone were additive and partly responsible for some of the reduced expression of the sulA::lacZ fusion in the co-expression experiment. The wild-type gcvA gene fused to the LexA DBD in pMS604 was cloned into the low-copy-number plasmid pSC101. Expression of the fusion protein from the low-copy-number plasmid did not cause a decrease in ß-galactosidase activity (Table 1, compare rows 1 and 10). When co-expressed with the GcvR fusion protein, there was still a significant reduction in ß-galactosidase levels (Table 1, compare rows 1 and 11). These results indicate that GcvA and GcvR interact to form heterodimers in vivo.



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Fig. 2. Transcriptional repression by LexA heterodimer formation of GcvA and GcvR. The sulA -35 and -10 promoter elements are indicated by a line between the sequences and the mutant 408 and wild-type (wt) operator half-sites are in lower case (Dmitrova et al., 1998 ).

 

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Table 1. Heterodimerization of GcvA and GcvR and their mutants

 
Isolation of gcvR mutants unable to properly repress the gcv operon
To determine if gcvR mutants unable to properly repress gcv expression might also be defective in their ability to interact with GcvA, the following strategy was used to isolate gcvR mutants impaired in their ability to negatively regulate the gcv operon. The wild-type lysogen GS162{lambda}gcvT::lacZ, which carries a chromosomal gcvT::lacZ reporter fusion, was transformed with a multi-copy plasmid pool carrying PCR-induced random base pair changes in gcvR. Transformants were selected on LB+AP plates and then transferred to LM+AP plates supplemented with the purine nucleoside inosine and TPEG (an inhibitor of ß-galactosidase); this medium results in maximal repression of the gcvT::lacZ fusion and the inability to grow with lactose as the carbon source. Assuming that GcvR acts as a dimer (G. Heil, unpublished results), we reasoned that transformants expressing mutant GcvR proteins that could not interact with GcvA but that could still dimerize with wild-type GcvR and interfere with its ability to repress gcv expression would be able to grow on the LM plates due to increased ß-galactosidase activity. Five transformants with this phenotype were isolated. Plasmid DNA was purified from each of these transformants and used to transform the {Delta}gcvR strain GS1111{lambda}gcvT::lacZ. The resulting transformants were grown in GM supplemented with inosine and assayed for ß-galactosidase activity. All five were found to have increased ß-galactosidase activity as compared to GS1111{lambda}gcvT::lacZ transformed with a multi-copy plasmid carrying the wild-type gcvR allele, indicating that each encoded a GcvR protein defective in negative regulation of the gcv operon (data not shown).

Each of the mutant gcvR alleles was sequenced and the following mutations were identified: gcvR{Delta}99–189 and gcvR{Delta}131–189 contain stop codons at positions 99 and 131, respectively; gcvR{Delta}106 contains a single nucleotide deletion (C) at codon 106, resulting in a reading frameshift and a stop codon at position 108, producing a protein that is 107 amino acids in size with two missense amino acids at its C-terminus; gcvRR129H contains an arginine to histidine change at position 129; and gcvRQ144P contains a glutamine to proline change at position 144.

To determine the effects of these mutant alleles on gcv expression when they are expressed at wild-type levels, each mutant gcvR allele was cloned into the single-copy plasmid pGS311. The resulting single-copy constructs were used to transform the {Delta}gcvR strain GS1111{lambda}gcvT::lacZ. Transformants were grown in GM, GM + glycine and GM+inosine media and assayed for ß-galactosidase activity (Table 2). The three deletion alleles as well as gcvRQ144P led to a complete loss of gcv repression in all three media, exhibiting ß-galactosidase activities nearly identical to the levels observed in the {Delta}gcvR control. The gcvRR129H allele also led to a loss of repression, but the phenotype was less severe. It should be noted that the 1·5–2-fold repression observed in GM+inosine medium is due to the PurR repressor, which has been shown to repress transcription of the gcv operon independently of GcvA/GcvR-mediated repression (Wilson et al., 1993a ).


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Table 2. gcvA and gcvR mutations and their effects on gcvT::lacZ expression

 
GcvR mutants unable to repress gcv expression are impaired in their ability to interact with GcvA
If GcvA interacts directly with GcvR to regulate the gcv operon, one possible explanation for the loss of repression displayed by the gcvR mutants described above is that they are unable to interact with GcvA. Therefore, the gcvRQ144P and gcvRR129H alleles were cloned into pDP804 and tested for their ability to interact with GcvA using the protein heterodimerization system. Both mutant alleles were impaired in their ability to interact with GcvA, displaying an approximately 4·5–5·5-fold increase in expression of the {lambda}sulA::lacZ reporter fusion as compared to the wild-type gcvR allele (Table 1, compare row 4 with rows 5 and 6).

Deletion analysis of gcvR
To define the region(s) of GcvR involved in its interaction with GcvA, a 3' deletion (gcvR{Delta}143–189) and a 5' deletion (gcvR{Delta}1–35) of gcvR (Fig. 3a) were cloned into pDP804 and tested for their ability to interact with GcvA using the LexA heterodimerization assay. Since all of the gcvR mutations described above were located in the 3' half of gcvR, it was not surprising that deletion of the C-terminal 47 residues of GcvR, which includes the glutamine at position 144, resulted in a loss of interaction with GcvA (Table 3, row 4). It was somewhat surprising, however, that deletion of the N-terminal 35 residues also resulted in a loss of interaction with GcvA (Table 3, row 5).



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Fig. 3. (a) The 189 amino acid GcvR protein and the GcvR fragments fused to the LexA87 408 headpiece in plasmid pDP804 used to define regions of GcvR involved in interacting with GcvA. The R129 and Q144 amino acids involved in negative regulation are indicated relative to the deletion end points. (b) The 305 amino acid GcvA protein and the GcvA fragments fused to the LexA87 wild-type headpiece in plasmid pMS604 used to define the region of GcvA involved in interacting with GcvR. The C169 and R197 amino acids and the C-terminal 14 amino acids involved in negative regulation are indicated. H-T-H, helix–turn–helix domain. Not to scale.

 

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Table 3. Deletions of gcvA and gcvR and their effects on GcvA/GcvR interaction

 
Isolation of GcvA mutants refractory to inhibition by GcvR
In a previous study, two gcvA mutants that are able to activate but not repress gcv expression were isolated but not characterized (Jourdan & Stauffer, 1998 ). In that same study, a deletion of the sequence encoding the C-terminal 14 amino acids of GcvA ({Delta}gcvA944; renamed gcvA{Delta}292–305 in this study) (Fig. 3b) was constructed and shown to have the same phenotype. During this study, the uncharacterized gcvA mutant alleles were sequenced and the mutations were identified as a cysteine to arginine change at position 169 (gcvAC169R) and an arginine to glycine change at position 197 (gcvAR197G) (Fig. 3b). To measure the effects of these mutations on gcv expression, they were cloned into the single-copy plasmid pGS311 and the resulting constructs were used to transform the {Delta}gcvA reporter strain GS1118{lambda}gcvT::lacZ. Transformants were grown in GM, GM+glycine and GM+inosine media and assayed for ß-galactosidase activity. As expected, the {Delta}gcvA control lysogen showed no significant change in ß-galactosidase activity under any growth condition, except for the twofold PurR effect (Table 2). The gcvAR197G and gcvA{Delta}292–305 mutations resulted in a complete loss of gcv repression, producing levels of ß-galactosidase activity similar to that observed in the {Delta}gcvR control (Table 2, rows 10 and 11). The gcvAC169R mutation also led to a loss of gcv repression, although the phenotype was not as severe (Table 2, row 9).

GcvA mutants unable to repress gcv expression are impaired in their ability to interact with GcvR
To test whether the gcvA mutants described above are defective in their ability to interact with GcvR, each mutant allele was cloned into pMS604 and its interaction with GcvR measured using the LexA heterodimerization system. The gcvAC169R mutation resulted in a small loss of interaction with GcvR, whilst the gcvAR197G and gcvA{Delta}292–305 mutations resulted in more severe losses of interaction with GcvR (Table 1, compare row 4 with rows 7, 8 and 9). These results are consistent with the effects of the mutations on repression of the gcv operon.

The C-terminal half of GcvA is able to interact with GcvR
To define the region(s) of GcvA that is involved in its interaction with GcvR, several 5' and 3' deletions of gcvA (gcvA{Delta}1–151, gcvA{Delta}1–230 and gcvA{Delta}1–151·{Delta}231–305) (Fig. 3b) were cloned into pMS604 and tested for their ability to interact with GcvR using the protein heterodimerization system. Deletion of the N-terminal 151 amino acids of GcvA had no effect on its ability to interact with GcvR (Table 3, row 6). However, deletion of the N-terminal 230 amino acids, which deletes the cysteine at position 169 and the arginine at position 197, or deletion of amino acids 1–151 and 231–305, resulted in significant loss of interaction with GcvR (Table 3, rows 7 and 8). These results suggest that the GcvA/GcvR interaction requires the C-terminal half of GcvA.

The C-terminal half of GcvA interferes with inhibition of wild-type GcvA by GcvR
Sequence and mutational analysis indicate that the N-terminal half of GcvA is involved in DNA binding and activation (Wilson & Stauffer, 1994 ; Jourdan & Stauffer, 1998 ) and results from this study suggest that the C-terminal half of GcvA is involved in an interaction with GcvR. To determine if overexpression of the C-terminal half of GcvA would lead to activation of the gcv operon by interfering with the ability of GcvR to inhibit wild-type GcvA, the wild-type strain GS162{lambda}gcvT::lacZ and the {Delta}gcvA strain GS1118{lambda}gcvT::lacZ were transformed with plasmids carrying the lexA–gcvA{Delta}1–151 and lexA–gcvA{Delta}1–230 alleles used in the heterodimerization experiment. Transformants were grown in GM, GM+glycine and GM+inosine media and assayed for ß-galactosidase activity. Transformation of the {Delta}gcvA strain GS1118{lambda}gcvT::lacZ with either plasmid resulted in low non-inducible gcv expression, identical to that observed in the untransformed control (data not shown). Thus, neither LexA–{Delta}GcvA protein fusion is able to activate the gcvT::lacZ fusion. Transformation of wild-type strain GS162{lambda}gcvT::lacZ resulted in normal regulation of gcv expression in the case of the lexA–gcvA{Delta}1230 allele, whereas transformation with the lexA–gcvA{Delta}1151 allele led to constitutive activation in all three media (Table 4).


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Table 4. LexA–GcvA{Delta}1–151 interferes with inhibition of wild-type GcvA by GcvR

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this report, we present evidence to support a model in which GcvR interacts with GcvA to prevent activation of the gcv operon. Using a LexA-based system for measuring protein heterodimerization, we were able to show that GcvA and GcvR interact in vivo. In addition, mutations in both gcvA and gcvR which result in a loss of gcv repression also result in a loss of GcvA/GcvR interaction. Thus, there is a strong correlation between the ability of these proteins to form heterodimers and their ability to repress expression of the gcv operon.

Three lines of evidence suggest that the C-terminal half of GcvA is involved in heterodimerization with GcvR. First, substitution mutants of GcvA which result in a loss of interaction with GcvR lie in the C-terminal half of GcvA. Second, deletion of the N-terminal half of GcvA has no effect on its ability to interact with GcvR, whereas deletion of the C-terminal 14 amino acids results in a complete loss of heterodimerization. Finally, although unable to activate gcv transcription, the C-terminal half of GcvA is able to interfere with GcvR-mediated repression of wild-type GcvA, probably by titrating the available antiactivator, leading to constitutive activation of the operon (Table 4). Together, these results suggest that the C-terminal half of GcvA is involved in an interaction with GcvR.

All of the gcvR mutations identified in this study that result in a decrease in repression of the gcv operon as well as a decrease in heterodimerization lie in the C-terminal half of the protein, suggesting that this region is involved in an interaction with GcvA. Consistent with these results, a deletion of the C-terminal 47 amino acids resulted in a loss of interaction with GcvA (Table 3). However, deletion of the N-terminal 35 amino acids also resulted in a loss of heterodimerization (Table 3). Since our screen relied on the ability of mutant GcvR to interact with wild-type GcvR, it is possible that mutations in the N-terminal region that would have resulted in a loss of GcvR interaction with GcvA were excluded from the screen as the mutations might have also resulted in a loss of GcvR homodimerization. It is also possible that deletion of this region of gcvR results in a decrease in expression or protein stability.

While it is clear that GcvR interacts with GcvA to repress the gcv operon, it is not clear how this interaction is modified by glycine and purines and, in turn, how this modification leads to the correct regulation of gcv expression. In previous studies, we have shown that neither glycine nor purines have an effect on the expression of gcvA or gcvR (Wilson & Stauffer, 1994 ; Ghrist & Stauffer, 1998 ). These results led us to propose a model in which GcvA homocomplexes function as activators, whilst GcvA/GcvR heterocomplexes function as repressors. In this model, the coeffectors influence the type of complex formed; glycine promotes the formation of activation complexes, whilst purines promote the formation of repression complexes. Increasing the expression of GcvA or GcvR would force the formation of activation or repression complexes, respectively. We used the LexA-based heterodimerization system to measure the effects of glycine and inosine on the ability of GcvA and GcvR to interact by growing transformants in GM, GM+glycine and GM+inosine media prior to assaying for ß-galactosidase activity. However, glycine and inosine had no significant effect on the GcvA/GcvR interaction (data not shown). This may be due to the high level of expression of both fusion proteins in this assay and in vitro studies with purified GcvA and GcvR will be needed to provide further data to prove or disprove this model.

Regulation of the gcv operon by GcvA and GcvR is part of a growing number of prokaryotic regulatory systems involving an activator/antiactivator complex, including TraR/TraM regulation of conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens (Hwang et al., 1999 ), NifA/NifL regulation of nitrogen fixation in Klebsiella pneumoniae (Narberhaus et al., 1995 ), ComK/MecA/ClpC regulation of competence in Bacillus subtilis (Turgay et al., 1998 ) and CRP/CytR regulation of operons encoding the nucleoside transport and biosynthesis enzymes (Valentin-Hansen et al., 1996 ). In the case of TraR/TraM, NifA/NifL and ComK/MecA/ClpC, the activator is sequestered by the antiactivator(s), thus preventing binding and activation. In the case of CRP/CytR, the antiactivator binds DNA between activator-binding sites, forming a nucleoprotein complex which prevents activation (Valentin-Hansen et al., 1996 ). Evidence suggests that GcvR could act in both ways to negatively regulate gcv expression. As discussed above, the C-terminal half of GcvA, deleted for the DNA-binding region (Jourdan & Stauffer, 1998 ), prevents GcvR-mediated repression. This suggests that GcvR may function by sequestering GcvA in an inactive form. On the other hand, overexpressing GcvR in a gcvA+ strain results in a lower level of gcv expression than that observed in a gcvA mutant (Ghrist & Stauffer, 1995 ). In addition, GcvA/GcvR, in the presence of inosine, repress a gcvT::lacZ fusion four- to fivefold in a GcvA-binding site 1-dependent manner (Wonderling et al., 2000 ). These results suggest that GcvA and GcvR act together at the gcv control region to directly repress gcv expression.


   ACKNOWLEDGEMENTS
 
We thank Michele Granger for generously providing pMS604, pDP804 and SU202.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 29 December 2000; revised 23 March 2001; accepted 23 April 2001.