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, gcvT::lacZ, GcvA, GcvR, repression
Abbreviations: CRP, cyclic AMP receptor protein; GCV, glycine cleavage; RNAP, RNA polymerase; WT, wild-type
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INTRODUCTION |
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Regulation of the gcvTHP operon is complex, involving both global-acting and gcv-specific regulatory proteins (Fig. 1). Lrp, a global regulator involved in regulation of amino acid metabolic pathways (Calvo & Matthews, 1994
), is required for expression of a gcvT::lacZ fusion (Lin et al., 1992
; Stauffer & Stauffer, 1994
). Lrp binds to and protects from DNase I digestion a large region of DNA upstream of the gcv promoter from about base -92 to -229 (Stauffer & Stauffer, 1994
). In addition, Lrp binding to this region results in a sharp bend in the gcv DNA (Stauffer & Stauffer, 1998a
). Its primary role may be structural, facilitating formation of stereospecific nucleoprotein complexes required to either activate or repress the gcv operon (Stauffer & Stauffer, 1999
).
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The cAMP receptor protein (CRP) has recently been shown to play a positive role in controlling gcvT::lacZ expression (Wonderling & Stauffer, 1999 ). CRP binds to a site from about base -324 to -303, and although its specific mechanism for regulating gcvT::lacZ expression has not yet been identified, CRP likely interferes with repression by the GcvA protein.
The GcvA protein is a member of the LysR family of activators that share amino acid homology and domain structure (Schell, 1993 ). GcvA binds to three sites in the gcv control region, from base -34 to -69 (site 1), from base -214 to -241 (site 2) and from base -242 to -271 (site 3) (Fig. 1
) (Wilson et al., 1995
). GcvA acts as an activator of gcv expression when glycine is present in the growth medium, causing a six- to sevenfold induction (Wilson et al., 1993b
). GcvA also represses the gcv operon over a fivefold range when the purine inosine is added to the growth medium (Wilson et al., 1993a
). In the presence of both glycine and purines, the activator function of GcvA is dominant (Stauffer & Stauffer, 1994
). Activation and repression by GcvA requires Lrp, presumably to bend the DNA appropriately (Stauffer & Stauffer, 1998a
, b
, 1999
). Mutations in GcvA have been isolated that affect either the activation or repression function without altering its DNA-binding capabilities (Jourdan & Stauffer, 1998
), suggesting that these two activities lie in separate functional regions.
The GcvR protein is required for repression by GcvA (Ghrist & Stauffer, 1995 ). A Tn10 insertion in the gcvR gene causes high constitutive levels of gcvT::lacZ expression due to the loss of GcvA-mediated repression, and overexpression of gcvR leads to superrepression of the fusion that also requires the GcvA protein (Ghrist & Stauffer, 1995
). Preliminary evidence suggests that GcvR interacts directly with GcvA rather than binding gcv DNA (A. Ghrist, unpublished results), but the mechanism by which GcvR causes repression is unknown.
Previous studies suggested that all three GcvA binding sites are required for repression by GcvA, but only sites 2 and 3 are necessary for activation (Wilson et al., 1995 ). However, interpretation of the role of site 1 was complicated because the single site 1 allele examined only reduced GcvA binding to site 1 about twofold, and also resulted in a twofold promoter-down phenotype. A prediction based on these early studies is that mutations in site 1 that prevent GcvA binding should affect GcvA-mediated repression without interfering with GcvA-mediated activation. Such mutations would be useful in understanding the mechanism of the GcvA/GcvR-mediated repression system. In this study, mutations were created in GcvA binding site 1 to determine which nucleotides are necessary for GcvA binding and the effects of these mutations on GcvA-mediated repression and activation. The results are consistent with the previous hypothesis that GcvA site 1 is primarily involved in GcvA-mediated repression. The results also suggest that GcvA has different requirements for binding at sites 2 and 3 compared to site 1.
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METHODS |
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Media.
The glucose minimal (GM) medium used was the minimal salts medium of Vogel & Bonner (1956 ) supplemented with 0·4% glucose. Supplements were added at the following concentrations in µg ml-1: thiamin, 1; phenylalanine, 50; glycine, 300; inosine, 50; ampicillin, 100; tetracycline, 10. GM medium was always supplemented with phenylalanine and thiamin since all strains used carry the pheA905 and thi mutations.
Enzyme assays.
Cells for enzyme assays were harvested from mid-exponential phase cultures (OD600 0·6). ß-Galactosidase assays were performed as described by Miller (1992
), using the chloroform/SDS lysis procedure. All results are the means of two or more assays with each reaction performed in triplicate.
Site-directed mutagenesis and construction of lysogens.
Using plasmid pGS239 as template, the PCR megaprimer mutagenesis procedure (Sarkar & Sommer, 1990 ) was used to create nucleotide changes in GcvA binding site 1 in the gcv control region. The specific base changes were verified by DNA sequence analysis. The approximately 5400 bp EcoRIMfeI fragments carrying mutant gcvT::lacZ fusions along with the lacY and lacA genes were isolated from each plasmid and ligated into the EcoRI site of phage
gt2 (Panasenko et al., 1977
). The phages generated were purified as single plaques and designated
gcvT::lacZ(-55T),
gcvT::lacZ(-60G),
gcvT::lacZ(-61G),
gcvT::lacZ(-67A) and
gcvT::lacZ(-60G,-61G,-67A). The designations in parentheses after the fusions indicate the nucleotide changes and positions relative to the +1 transcription start site. The gcvT::lacZ(1-A) and gcvT::lacZ(1-B) fusions have four and six base changes, respectively, in site 1 and are described in Fig. 4
. Appropriate strains were lysogenized with the above phages and the lysogens verified to carry a single copy of
by infection with phage
cI90c17 (Shimada et al., 1972
).
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RESULTS |
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A three-base mutation in the conserved 5'-CTAAT-3'sequence of site 1 causes a phenotype similar to the single-base changes
Despite the loss of GcvA-mediated repression caused by the point mutations when the GS852 lysogens were grown in GM+inosine, the levels of gcvT::lacZ expression are still significantly lower than the levels seen in the gcvR strain GS1053 (Table 1). If binding site 1 is required for repression by GcvA and GcvR, why are the levels of expression lower in the site 1 mutants compared to a gcvR mutant? One possibility is the inability of single-base changes to eliminate binding of GcvA to site 1. To test this possibility, a triple mutant was constructed, combining the changes at bases -60, -61 and -67 in a single gcvT::lacZ fusion. This fusion was cloned into the
gt2 vector and the resulting phage, designated
gcvT::lacZ(-60G,-61G,-67A) was used to lysogenize strains GS852 and GS986. Cells were grown in GM, GM+glycine or GM+inosine and ß-galactosidase levels were measured (Table 2
). Expression of the gcvT::lacZ(-60G,-61G,-67A) fusion was two- to threefold higher in GS852 compared to the WT gcvT::lacZ fusion when cells were grown in either GM or GM+inosine. The mutations in site 1 had no significant effect on ß-galactosidase expression when the lysogen was grown in GM+glycine. In the gcvA purR strain GS986, the site 1 triple mutant no longer caused a two- to threefold increase in gcvT::lacZ expression compared to WT, although there was a slight but reproducible increase (
1·4-fold). Since GS986 is a gcvA strain, these results indicate that the site 1 mutations cause a small GcvA-independent effect, probably an increase in promoter strength. Despite this small GcvA-independent effect, the majority of the increase seen in GS852 can be attributed to a loss of GcvA-mediated repression. However, the site 1 triple mutant did not completely relieve GcvA-mediated repression of the gcvT::lacZ fusion as seen in a gcvR strain, but exhibited a phenotype similar to the single-base mutants.
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The mutations in the conserved sequence of site 1 are involved in DNA conformation
Recent experiments demonstrated that GcvA bends DNA when it binds to any of the three binding sites in the gcv control region (G. Stauffer, unpublished results). Since the mutations in the conserved 5'-CTAAT-3' sequence of site 1 alter repression to varying degrees but reduce GcvAs binding affinity less than twofold, it is possible that some other function, such as DNA bending, is affected by mutations in the region that leads to altered regulation. To test this possibility, the triple site 1 mutant -60G,-61G,-67A, the four-base mutant site 1 described previously (Wilson et al., 1995 ) (Fig. 1
) and the WT site 1 were cloned into the vector pBEND2. The plasmids were digested with restriction enzymes to generate equal length DNA fragments with site 1 located at varying distances from the centre of the fragments (Fig. 3
). These fragments were then used as templates in gel mobility-shift assays. Since protein-induced DNA bending alters the end-to-end distance of a fragment, a determinant of mobility through a polyacrylamide gel, DNA bending was detected by a change in mobility when site 1 was located at different distances from the centre of the fragment. As expected, when the WT site 1 was located at the right end or the left end of the fragment, it showed a faster mobility than when site 1 was located in the centre of the fragment (Fig. 3
, lanes 1, 4 and 7). The mobilities of the two mutant fragments were faster than the WT fragment when the target site was located at the right end of the fragment (Fig. 3
, lanes 13), but slower when the target sites were located at the left end of the fragment (lanes 79). When the target sites were located near the middle of the DNA fragment, the three-base mutant fragment exhibited a similar mobility to the WT and the four-base mutant fragment exhibited a faster mobility than the WT fragment (lanes 46). The results of the DNA bending assays show that the mutations cause some disruption of the bending at site 1.
Isolation of a GcvA site 1 that relieves repression
Since previous mutations (Wilson et al., 1995 ) and the mutations described above did not completely relieve GcvA/GcvR-mediated repression of gcvT::lacZ, the role of site 1 in regulation is difficult to ascertain. To further characterize site 1, a partially random mutagenesis was performed that could potentially alter site 1 at six different nucleotides in the region surrounding the conserved 5'-CTAAT-3' sequence, or at seven positions promoter proximal to the conserved sequence (Fig. 4
). This was accomplished by the use of two oligonucleotides, referred to as oligo site 1-A and oligo site 1-B, that have any of the four nucleotides incorporated at bases -58, -59, -64, -65, -66, -68 or at bases -50, -51, -52, -53, -54, -56 and -57 relative to the transcription initiation site at +1. Using a second WT primer, a pool of PCR products was generated, the fragments were cloned into pMC1403 to reconstruct gcvT::lacZ fusions and used to transform GS852. One transformant from each selection was sequenced to determine the nucleotide changes. The transformants isolated using oligo 1-A and oligo 1-B had four and five base changes, respectively (Fig. 4
).
To test for their abilities to relieve repression by GcvA, the 1-A and 1-B fragments were cloned as gcvT::lacZ fusions and used to lysogenize strains GS852 and GS986. The resulting lysogens were grown in GM, GM+glycine and GM+inosine, and assayed for ß-galactosidase activity. The 1-A mutations had little effect when compared to the WT gcvT::lacZ fusion in purR strain GS852 (Table 3
, compare rows 1 and 2). However, in the gcvA purR strain GS986, the 1-A mutations caused a two- to threefold decrease in expression compared to the WT fusion, indicating that one or more of the altered nucleotides in the 1-A mutant are important for general promoter strength (compare rows 4 and 5). In addition, these nucleotides might be important for normal gcvT::lacZ expression since any effect on GcvA-mediated regulation may be masked by the promoter-down effect. The 1-B fusion had about twofold higher levels of gcvT::lacZ expression than the WT fusion when cells were grown in GM and over a sixfold increase in GM+inosine. There was no significant effect of the mutations in GM+glycine. These results suggest that the mutations present in the 1-B fusion interfere with negative regulation, especially in the presence of inosine (Table 3
, compare rows 1 and 3). It should be noted that the 1-B mutations also caused a two- to threefold decrease in gcvT::lacZ expression when compared to the WT gcvT::lacZ fusion in GS986, indicating that in the absence of GcvA these mutations decrease the basal levels of expression (compare rows 4 and 6). This GcvA-independent decrease may indicate that the 1-B mutations cause an even larger relief of repression than reflected by the ß-galactosidase levels in Table 4
, that is concealed by the GcvA-independent promoter decrease. However, it is possible that the promoter-down effect exhibited by the 1-B mutations only occurs in the absence of GcvA, perhaps by interfering with RNAP binding to the promoter. This possibility is difficult to test since attempts at DNase I protection assays with RNAP and gcv DNA have proven unsuccessful.
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Binding of GcvA to the site 1-A and site 1-B mutants
The 464 bp BamHISspI fragments, identical to those described in the mobility-shift assays above except for the base changes, were isolated from the 1-A and 1-B mutants, 32P-labelled at the BamHI ends and used in gel mobility-shift assays. The 1-A mutant site 1 fragment was able to bind GcvA with approximately a twofold decreased affinity compared to WT site 1, suggesting that the nucleotides at positions -57, -58, -64 and -65 have some role in GcvA binding (Fig. 5, compare lanes 14 with 58). The 1-B mutant fragment showed at least a fourfold decrease in affinity for GcvA, indicating that one or more of the nucleotides altered are important for binding of GcvA (Fig. 5
, compare lanes 14 with 912). Although the 1-B mutations did not completely abolish binding by GcvA, the 1-B mutant is the first site 1 mutant to exhibit more than a twofold decrease in binding affinity for GcvA, suggesting that the central region of site 1 is important for GcvA binding, rather than the conserved 5'-CTAAT-3' sequence important for recognition and binding of GcvA at sites 2 and 3 (Wilson et al., 1995
).
To separate the GcvA-dependent and -independent effects of the 1-B mutations, site-directed mutagenesis was used to create single-base changes at positions -50, -52, -53, -56 and -57. We expected that one or more of these nucleotides would be required for GcvA binding and repression, and for general promoter strength independent of GcvA. The fragments containing the single-base changes were verified by DNA sequencing and tested for their effects on these various functions. Surprisingly, none of the single-base changes had any significant effect on regulation by GcvA, general promoter strength or the ability to bind GcvA (data not shown).
Superrepression by multi-copy gcvR in the presence of the 1-B mutations in site 1
Our current model proposes that GcvA binding to site 1 is required for GcvA-mediated repression in the presence of inosine. However, we do not know if the repression mediated by the GcvR protein requires the GcvA/site 1 interaction or if GcvR represses through a separate mechanism. To determine if repression by GcvR is affected by the mutations at site 1, the multi-copy plasmid pGS334 that overexpresses gcvR and causes superrepression of a WT fusion was transformed into GS852 gcvT::lacZ, GS852
gcvT::lacZ(1-B), GS986
gcvT::lacZ and GS986
gcvT::lacZ(1-B). If the 1-B mutations at site 1 interfere with repression by GcvR, then superrepression should be antagonized by the nucleotide changes. However, if site 1 is involved in a secondary mechanism of repression that does not involve GcvR, then superrepression should occur in the presence of multi-copy gcvR. The transformants were grown in GM medium and assayed for ß-galactosidase activity. Plasmid pGS334 repressed the WT gcvT::lacZ fusion nearly 2·8-fold (Table 5
, rows 1 and 2) and the gcvT::lacZ (1-B) fusion about 1·6-fold (rows 3 and 4). GS986 served as the control strain to demonstrate that multi-copy gcvR cannot cause repression in the absence of GcvA. Thus, the 1-B mutations eliminate most of the GcvR-mediated superrepression, suggesting that repression by GcvR requires an intact site 1.
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DISCUSSION |
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The most severe allele examined that alters both GcvA binding and repression is the 1-B mutant, with five base changes (Fig. 4). These changes result in about a fourfold decrease in GcvA binding affinity in vitro when compared to WT site 1 (Fig. 5
), and in vivo caused a sixfold loss of GcvA-mediated repression when cells were grown in GM+inosine (Table 3
). The mutations also caused a two- to threefold decrease in promoter strength (Table 3
). However, when the five bases were changed individually, none of the changes had a significant effect on gcvT::lacZ expression or on the binding affinity of GcvA, again suggesting that several bases must be altered to interfere with binding and repression by GcvA.
The 1-A and 1-B mutants isolated in this study, as well as the four-base mutant isolated previously (Wilson et al., 1995 ), showed GcvA-independent promoter-down effects (Table 3
). It should be noted that the promoter-down effects are only observed in the absence of GcvA. Thus, it is possible that RNAP interacts with this region of gcv in the absence of GcvA to maintain basal promoter activity, and the mutations interfere with this interaction. It is known that the C-terminal domain of the
subunit of RNAP interacts with up-elements located upstream of the promoter -35 sequence element (Estrem et al., 1999
). Although a sequence is present in gcv from base -44 to -54 that shows a 7 out of 11 base match with the proposed up-element consensus distal subsite (Fig. 1
), this degree of homology is considered insignificant as a subsite (Estrem et al., 1999
). Furthermore, the 1-A mutant and the four-base mutant (Wilson et al., 1995
) lie upstream of this sequence. We have been unsuccessful at footprinting RNAP at the gcv promoter to verify an RNAPDNA interaction in this region.
Previous studies showed that overexpressing gcvR favours repression of a gcvT::lacZ fusion even under activating conditions (glycine supplementation), and overexpressing gcvA favours activation even under repressing conditions (purine supplementation) (Ghrist & Stauffer, 1998 ). Since GcvA activates a gcvT::lacZ fusion in the absence of GcvR, and GcvR-mediated repression is dependent on a functional GcvA protein, a model was proposed that GcvA homo-oligomers activate the gcv operon, whereas GcvA/GcvR hetero-oligomers repress the gcv operon (Ghrist & Stauffer, 1995
). The small co-regulators glycine and purines possibly determine whether the activator or the repressor form of GcvA is favoured in the cell. We believe our results are consistent with this model. Since GcvA/GcvR, in the presence of inosine, represses a gcvT::lacZ fusion four- to fivefold in a site 1-dependent manner, GcvA binding at site 1 in the presence of GcvR appears to be necessary to repress transcription. Furthermore, as none of the changes have a significant effect on activation, site 1 appears necessary only for repression. However, the role(s) of GcvR in the repression mechanism is still unclear. Also unclear are the different sequence requirements for site 1 compared to sites 2 and 3. It is possible that the difference is related to the mechanism GcvA employs to switch between activation and repression.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 July 2000;
accepted 1 August 2000.