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
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ABSTRACT |
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Keywords: gcvTHP, repressor, activator, repression, antiactivation
Abbreviations: AP, ampicillin; DBD, DNA-binding domain; gcv, gcvTHP; TC, tetracycline; TPEG, phenylethyl-ß-D-thiogalactoside
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INTRODUCTION |
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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gcv expression directly, as it fails to bind to the gcv control region in gel mobility shift assays (A. Ghrist, unpublished results).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.
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METHODS |
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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 LuriaBertani 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.
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 BstEIIXhoI 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 BssHIIBglII 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.
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RESULTS |
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Each of the mutant gcvR alleles was sequenced and the following mutations were identified: gcvR99189 and gcvR
131189 contain stop codons at positions 99 and 131, respectively; gcvR
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 gcvR strain GS1111
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
gcvR control. The gcvRR129H allele also led to a loss of repression, but the phenotype was less severe. It should be noted that the 1·52-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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Deletion analysis of gcvR
To define the region(s) of GcvR involved in its interaction with GcvA, a 3' deletion (gcvR143189) and a 5' deletion (gcvR
135) 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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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 gcvA292305 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 (gcvA1151, gcvA
1230 and gcvA
1151·
231305) (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 1151 and 231305, 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
gcvT::lacZ and the
gcvA strain GS1118
gcvT::lacZ were transformed with plasmids carrying the lexAgcvA
1151 and lexAgcvA
1230 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
gcvA strain GS1118
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
GcvA protein fusion is able to activate the gcvT::lacZ fusion. Transformation of wild-type strain GS162
gcvT::lacZ resulted in normal regulation of gcv expression in the case of the lexAgcvA
1230 allele, whereas transformation with the lexAgcvA
1151 allele led to constitutive activation in all three media (Table 4
).
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 December 2000;
revised 23 March 2001;
accepted 23 April 2001.