George V. Stauffer

Homocysteine Antagonism of Nitric Oxide-Related Cytostasis in Salmonella typhimurium

Nitric oxide (NO) is associated with broad-spectrum antimicrobial activity of particular importance in infections caused by intracellular pathogens. An insertion mutation in the metL gene of Salmonella typhimurium conferred specific hypersusceptibility to S-nitrosothiol NO-donor compounds and attenuated virulence of the organism in mice. The metL gene product catalyzes two proximal metabolic steps required for homocysteine biosynthesis. S-Nitrosothiol resistance was restored by exogenous homocysteine or introduction of the metL gene on a plasmid. Measurement of expression of the homocysteine-sensitive metH gene indicated that S-nitrosothiols may directly deplete intracellular homocysteine. Homocysteine may act as an endogenous NO antagonist in diverse processes including infection, atherosclerosis, and neurologic disease.

The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli

The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic‐binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA–phoA and dppA–lacZ gene fusions. Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.

Construction and expression of hybrid plasmids containing the Escherichia coli glyA gene

Abstract The Escherichia coli glyA gene, encoding serine transhydroxymethylase (STHM), has been cloned in the plasmid vector pACYC184. The recombinant plasmid (pGS1) contains a 13 kb Eco RI insert. Genetic and biochemical experiments indicate that the region controlling STHM synthesis is present on the insert. Strains bearing multicopy plasmid vectors carrying the glyA gene overproduce the enzyme from 17- to 26-fold. The glyA gene was identified on the insert by analyzing a set of plasmids derived from pGS1 that carry random insertions of the transposable kanamycin resistance element Tn5. Cloning of segments of the original insert into plasmid pBR322 established that a 2.5 kb Sal I- Bcl I fragment carries the glyA gene. A physical map of this fragment is presented.

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.

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.

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.

Escherichia coli K12 mutants defective in the glycine cleavage enzyme system

SummaryTwo routes of one-carbon biosynthesis have been described in Escherichia coli K12. One is from serine via the serine hydroxymethyltransferase (SHMT) reaction, and the other is from glycine via the glycine cleavage (GCV) enzyme system. To isolate mutants deficient in the GCV pathway, we used a selection procedure that is based on the assumption that loss of this enzyme system in strains blocked in serine biosynthesis results in their inability to use glycine as a serine source. Mutants were accordingly isolated that grow with a serine supplement, but not with a glycine supplement. Enzyme assays demonstrated that three independently isolated mutants have no detectable GCV enzyme activity. The absence of a functional GCV pathway results in the excretion of glycine, but has no affect on the cells primary source of one-carbon units, the SHMT reaction. The new mutations, designated gcv, were mapped between the serA and lysA genes on the E. coli chromosome.

Characterization of the Escherichia coli gene for serine hydroxymethyltransferase

Plasmid pGS1 carries the Escherichia coli glyA gene and its neighboring regions on a 13-kb EcoRI insert. In a cell-free transcription-translation system, the insert directs the synthesis of two polypeptides with Mr values of about 46 500 and 45 500. When the glyA gene is inactivated with the transposable element Tn5, the Mr 46 500 polypeptide is not observed, identifying it as the glyA gene product. The Mr 45 500 polypeptide is the product of an unknown gene designated gene X. When plasmids with random insertions of the Tn5 element in either the glyA gene or gene X are used as templates in the cell-free transcription-translation system, the polypeptides observed are smaller than the glyA or X gene products. A comparison of the site of each Tn5 insertion within the glyA gene or within gene X and the size of the polypeptide observed in the cell-free system enabled us to determine the direction of transcription and translation of both genes. The glyA gene is transcribed and translated in a direction opposite to that of gene X. Nucleotide sequencing confirmed the location and orientation of the two genes in the insert. DNase I footprinting experiments defined the glyA gene and gene X control regions recognized by RNA polymerase, and S1 nuclease mapping experiments located the transcription start point for each gene. The transcription start points for the two genes are 216 bp apart, and the translation start sites are 327 bp apart. Less than 90 bp separate the two RNA polymerase molecules bound to the two promoters.

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.

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 GcvAs 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.