Antagonistic Roles for GcvA and GcvB in hdeAB Expression in Escherichia coli.

In E. coli, the periplasmic proteins HdeA and HdeB have chaperone-like functions, suppressing aggregation of periplasmic proteins under acidic conditions. A microarray analysis of RNA isolated from an E. coli wild type and a ΔgcvB strain grown to mid-log phase in Luria-Bertani broth indicated the hdeAB operon, encoding the HdeA and HdeB proteins, is regulated by the sRNA GcvB. We wanted to verify that GcvB and its coregulator Hfq play a role in regulation of the hdeAB operon. In this study, we show that GcvB positively regulates hdeA::lacZ and hdeB::lacZ translational fusions in cells grown in Luria-Bertani broth and in glucose minimal media + glycine. Activation also requires the Hfq protein. Although many sRNAs dependent on Hfq regulate by an antisense mechanism, GcvB regulates hdeAB either directly or indirectly at the level of transcription. GcvA, the activator of gcvB, negatively regulates hdeAB at the level of transcription. Although expression of gcvB is dependent on GcvA, activation of hdeAB by GcvB occurs independently of GcvA's ability to repress the operon. Cell survival and growth at low pH are consistent with GcvA negatively regulating and GcvB positively regulating the hdeAB operon.

The Escherichia coli GcvB sRNA Uses Genetic Redundancy to Control cycA Expression.

The Escherichia coli sRNA GcvB regulates several genes involved in transport of amino acids and peptides (sstT, oppA, dppA, and cycA). Two regions of GcvB from nt +124 to +161 and from nt +73 to +82 are complementary with essentially the same region of the cycA mRNA. Transcriptional fusions of cycA to lacZ showed the region of cycA mRNA that can pair with either region of GcvB is necessary for regulation by GcvB. However, mutations in either region of gcvB predicted to disrupt pairing between cycA mRNA and GcvB did not alter expression of a cycA-lacZ translational fusion. A genetic analysis identified nts in GcvB necessary for regulation of the cycA-lacZ fusion. The results show that either region of GcvB complementary to cycA mRNA can basepair with and independently repress cycA-lacZ and both regions need to be changed to cause a significant loss of repression.

Role of the sRNA GcvB in regulation of cycA in Escherichia coli.

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria-Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria-Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

Role of the Escherichia coli Hfq protein in GcvB regulation of oppA and dppA mRNAs.

The gcvB gene encodes a small non-translated RNA (referred to as GcvB) that regulates oppA and dppA, two genes that encode periplasmic binding proteins for the oligopeptide and dipeptide transport systems. Hfq, an RNA chaperone protein, binds many small RNAs and is required for the small RNAs to regulate expression of their respective target genes. We showed that repression by GcvB of dppA : : lacZ and oppA : : phoA translational fusions is dependent upon Hfq. Double mutations in gcvB and hfq yielded similar expression levels of dppA : : lacZ and oppA : : phoA compared with gcvB or hfq single mutations, suggesting that GcvB and Hfq repress by the same mechanism. The effect of Hfq is not through regulation of transcription of gcvB. Hfq is known to increase the stability of some small RNAs and to facilitate the interactions between small RNAs and specific mRNAs. In the absence of Hfq, there is a marked decrease in the half-life of GcvB in cells grown in both Luria-Bertani broth and glucose minimal medium with glycine, suggesting that part of the role of Hfq is to stabilize GcvB. Overproduction of GcvB in wild-type Escherichia coli results in superrepression of a dppA : : lacZ fusion, but overproduction of GcvB in an hfq mutant does not result in significant repression of the dppA : : lacZ fusion. These results suggest that Hfq also is likely required for GcvB-mRNA pairing.

The small RNA GcvB regulates sstT mRNA expression in Escherichia coli.

In Escherichia coli, the gcvB gene encodes a nontranslated RNA (referred to as GcvB) that regulates OppA and DppA, two periplasmic binding proteins for the oligopeptide and dipeptide transport systems. An additional regulatory target of GcvB, sstT, was found by microarray analysis of RNA isolated from a wild-type strain and a gcvB deletion strain grown to mid-log phase in Luria-Bertani broth. The SstT protein functions to transport L-serine and L-threonine by sodium transport into the cell. Reverse transcription-PCR and translational fusions confirmed that GcvB negatively regulates sstT mRNA levels in cells grown in Luria-Bertani broth. A series of transcriptional fusions identified a region of sstT mRNA upstream of the ribosome binding site needed for negative regulation by GcvB. Analysis of the GcvB RNA identified a sequence complementary to this region of the sstT mRNA. The region of GcvB complementary to sstT mRNA is the same region of GcvB identified to regulate the dppA and oppA mRNAs. Mutations predicted to disrupt base pairing between sstT mRNA and GcvB were made in gcvB, which resulted in the identification of a small region of GcvB necessary for negative regulation of sstT-lacZ. Additionally, the RNA chaperone protein Hfq was found to be necessary for GcvB to negatively regulate sstT-lacZ in Luria-Bertani broth and glucose minimal medium supplemented with glycine. The sstT mRNA is the first target found to be regulated by GcvB in glucose minimal medium supplemented with glycine.

The role of the small regulatory RNA GcvB in GcvB/mRNA posttranscriptional regulation of oppA and dppA in Escherichia coli.

The gcvB gene encodes two small, nontranslated RNAs that regulate OppA and DppA, periplasmic binding proteins for the oligopeptide and dipeptide transport systems. Analysis of the gcvB sequence identified a region of complementarity near the ribosome-binding sites of dppA and oppA mRNAs. Several changes in gcvB predicted to reduce complementarity of GcvB with dppA-lacZ and oppA-phoA reduced the ability of GcvB to repress the target RNAs while other changes had no effect or resulted in stronger repression of the target mRNAs. Mutations in dppA-lacZ and oppA-phoA that restored complementarity to GcvB restored the ability of GcvB to repress dppA-lacZ but not oppA-phoA. Additionally, a change that reduced complementarity of GcvB to dppA-lacZ reduced GcvB repression of dppA-lacZ with no effect on oppA-phoA. The results suggest that different regions of GcvB have different roles in regulating dppA and oppA mRNA, and although pairing between GcvB and dppA mRNA is likely part of the regulatory mechanism, the results do not support a simple base pairing interaction between GcvB and its target mRNAs as the complete mechanism of repression.

The Yersinia pestis gcvB gene encodes two small regulatory RNA molecules.

BACKGROUND: In recent years it has become clear that small non-coding RNAs function as regulatory elements in bacterial virulence and bacterial stress responses. We tested for the presence of the small non-coding GcvB RNAs in Y. pestis as possible regulators of gene expression in this organism. RESULTS: In this study, we report that the Yersinia pestis KIM6 gcvB gene encodes two small RNAs. Transcription of gcvB is activated by the GcvA protein and repressed by the GcvR protein. The gcvB-encoded RNAs are required for repression of the Y. pestis dppA gene, encoding the periplasmic-binding protein component of the dipeptide transport system, showing that the GcvB RNAs have regulatory activity. A deletion of the gcvB gene from the Y. pestis KIM6 chromosome results in a decrease in the generation time of the organism as well as a change in colony morphology. CONCLUSION: The results of this study indicate that the Y. pestis gcvB gene encodes two small non-coding regulatory RNAs that repress dppA expression. A gcvB deletion is pleiotropic, suggesting that the sRNAs are likely involved in controlling genes in addition to dppA.

GcvA interacts with both the alpha and sigma subunits of RNA polymerase to activate the Escherichia coli gcvB gene and the gcvTHP operon.

The glycine cleavage enzyme system in Escherichia coli provides one-carbon units for cellular methylation reactions. The gcvB gene encodes two small RNAs that in turn regulate other genes. The GcvA protein is required for expression of both the gcvTHP (P(gcvT)) and gcvB (P(gcvB)) promoters. However, the architectures of the two promoters are different, with the P(gcvT) promoter representing a class III activator-dependent promoter and the P(gcvB) promoter representing a class II activator-dependent promoter. The RNA polymerase holoenzyme was examined for its role in transcription activation of the gcvTHP operon and the gcvB gene by the GcvA protein. The results suggest that GcvA interacts with the RNA polymerase alpha subunit for activation of the gcvTHP operon and interacts with the RNA polymerase sigma subunit for activation of the gcvB gene.

Regulation of Serine, Glycine, and One-Carbon Biosynthesis.

The biosynthesis of serine, glycine, and one-carbon (C1) units constitutes a major metabolic pathway in Escherichia coli and Salmonella enterica serovar Typhimurium. C1 units derived from serine and glycine are used in the synthesis of purines, histidine, thymine, pantothenate, and methionine and in the formylation of the aminoacylated initiator fMet-TRNAfMet used to start translation in E. coli and serovar Typhimurium. The need for serine, glycine, and C1 units in many cellular functions makes it necessary for the genes encoding enzymes for their synthesis to be carefully regulated to meet the changing demands of the cell for these intermediates. This review discusses the regulation of the following genes: serA, serB, and serC; gly gene; gcvTHP operon; lpdA; gcvA and gcvR; and gcvB genes. Threonine utilization (the Tut cycle) constitutes a secondary pathway for serine and glycine biosynthesis. L-Serine inhibits the growth of E. coli cells in GM medium, and isoleucine releases this growth inhibition. The E. coli glycine transport system (Cyc) has been shown to transport glycine, D-alanine, D-serine, and the antibiotic D-cycloserine. Transport systems often play roles in the regulation of gene expression, by transporting effector molecules into the cell, where they are sensed by soluble or membrane-bound regulatory proteins.

Glycine binds the transcriptional accessory protein GcvR to disrupt a GcvA/GcvR interaction and allow GcvA-mediated activation of the Escherichia coli gcvTHP operon.

The Escherichia coli gcvTHP operon is under control of the LysR-type transcriptional regulator GcvA. GcvA activates the operon in the presence of glycine and represses the operon in its absence. Repression by GcvA is dependent on a second regulatory protein, GcvR. Generally, LysR-type transcriptional regulators bind to specific small co-effector molecules which results in either their altered affinity for specific binding sites on the DNA or altered ability to bend the DNA, resulting in either activation or repression of their respective operons. This study shows that glycine, the co-activator for the gcv operon, does not alter either GcvA's ability to bind DNA nor its ability to bend DNA. Rather, glycine binds to GcvR, disrupting a GcvA/GcvR interaction required for repression and allowing GcvA activation of the gcvTHP operon. Amino acid changes in GcvR that reduce glycine binding result in a loss of glycine-mediated activation in vivo.

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

The Escherichia coli glycine cleavage enzyme system, encoded by the gcvTHP operon, catalyses the oxidative cleavage of glycine to CO(2), NH(3) 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.

GcvA binding site 1 in the gcvTHP promoter of Escherichia coli is required for GcvA-mediated repression but not for GcvA-mediated activation.

GcvA binds to three sites in the gcvTHP control region, from base -34 to -69 (site 1), from base -214 to -241 (site 2) and from base -242 to -271 (site 3). Previous results suggested that sites 3 and 2 are required for both GcvA-dependent activation and repression of a gcvT::lacZ fusion. However, the results were less clear as to the role of site 1. To determine the role of site 1 in regulation, single and multiple base changes were made in site 1 and tested for their ability to alter GcvA-mediated activation and GcvA/GcvR-mediated repression. Several of the mutants were also tested for effects on GcvA binding to site 1 and the ability of GcvA to bend DNA at site 1. The results are consistent with site 1 playing primarily a role in negative regulation of the gcvTHP operon.

Genetic analysis of the GcvA binding site in the gcvA control region.

The GcvA protein both activates and represses the gcv operon and negatively regulates its own transcription. GcvA binds to three sites in the gcv control region and to one site in the gcvA control region; each of these binding sites contains the conserved 5 bp DNA sequence 5'-CTAAT-3'. This report describes the role this DNA sequence plays in autoregulation and expression of gcvA. Through single base-pair mutations, the importance of three of these five basepairs in the autoregulation of gcvA expression is shown. Two of the gcvA control region mutations described cause a gcvA::lacZ fusion to be overexpressed at 9-24 times the wild-type level. The increase in expression is due in part to a complete loss of autoregulation and in part to a GcvA-independent mechanism. One of the mutants was shown by Western blot analysis to increase the intracellular concentration of GcvA. This high level of gcvA expression subsequently causes the loss of purine-mediated repression of a gcvT::lacZ fusion. However, overexpression of gcvR re-established purine-mediated repression of the gcvT::lacZ fusion, supporting the model for gcv regulation that suggests the need for a relatively constant GcvA to GcvR ratio for appropriate regulation of gcv expression in response to glycine and purine availability.

Role for the leucine-responsive regulatory protein (Lrp) as a structural protein in regulating the Escherichia coli gcvTHP operon.

The Escherichia coli glycine-cleavage enzyme system (gcvTHP and lpd gene products) provides C1 units for cellular methylation reactions. Both the GcvA and leucine-responsive regulatory (Lrp) proteins are required for regulation of the gcv operon. One model proposed for gcv regulation is that Lrp plays a structural role, bending the DNA to allow GcvA to function as either an activator or a repressor in response to environmental signals. This hypothesis was tested by replacing all but the upstream 22 bp of the Lrp-binding region in a gcvT::lacZ fusion with the I1A site from phage lambda. Integration host factor (IHF) binds the I1A site and bends the DNA about 140 degrees. Shifting the I1A site by increments of 1 base around the DNA helix resulted in IHF-dependent activation and repression of gcvT::lacZ expression that were face-of-the-helix dependent. Activation was also dependent on the GcvA protein, and repression was dependent on both the GcvA and GcvR proteins, demonstrating that the roles for these proteins were not altered. The results are consistent with Lrp playing primarily a structural role in gcv regulation, although they do not completely rule out the possibility that Lrp also interacts with another gcv-regulatory protein or with RNA polymerase.

Roles for GcvA-binding sites 3 and 2 and the Lrp-binding region in gcvT::lacZ expression in Escherichia coli.

GcvA and Lrp are both necessary for activation of the gcv operon. The upstream GcvA-binding sites 3 and 2 were separated from the Lrp-binding region and the rest of the gcv control region. Moving these sites by 1 or 2 helical turns of DNA further from the gcv promoter reduces, but does not eliminate, either GcvA-mediated activation or repression of a gcvT::lacZ gene fusion. However, moving these sites by 1.5 or 2.5 helical turns of DNA results in a GcvA-mediated super-repression of the operon. This repression is dependent on Lrp and is partially dependent on GcvR. Lrp bound to the gcv control region induces a bend in the DNA. Based on these results, a model for gcv regulation is presented in which Lrp plays a primarily structural role, by bending the DNA and GcvA functions as the activator protein.

Spacing and orientation requirements of GcvA-binding sites 3 and 2 and the Lrp-binding region for gcvT::lacZ expression in Escherichia coli.

Both GcvA and Lrp are required for normal regulation of the gcv operon. Moving the GcvA-binding sites 3 and 2 and the Lrp-binding region either closer to, or further away from, the gcv promoter by approximately one helical turn of DNA resulted in a less than twofold decrease in glycine-mediated activation or inosine-mediated repression of a gcvT::IacZ fusion. Moving these sites approximately two helical turns of DNA away from the gcv promoter resulted in a further loss of both activation and repression; moving these sites approximately three helical turns of DNA from the gcv promoter resulted in an essentially complete loss of both glycine-mediated activation and inosine-mediated repression. However, when these sites were moved by approximately 1.5 and 2.5 helical turns of DNA away from the gcv promoter, there was a complete loss of both glycine-mediated activation and inosine-mediated repression of the gcvT::IacZ fusion. The flexibility in the absolute distance of the GcvA- and Lrp-binding sites relative to the gcv promoter, but strict orientation dependence of these sites is consistent with a possible protein-protein interaction of either GcvA, Lrp, or both of these proteins with RNA polymerase. Because of the location of these target sites relative to the gcv promoter, it is also likely that DNA looping is required for this mechanism of regulation.