Escherichia coli cis- and trans-acting mutations that increase glyA gene expression.

We used an Escherichia coli strain blocked in serine biosynthesis and carrying a partial glyA deletion to isolate strains with altered regulation of the glyA gene. The glyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increased glyA expression were isolated on glycine supplemented plates. One class of mutations increased glyA expression 10-fold by directly altering the -35 consensus sequence of the glyA promoter. The two other classes increased glyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of the metE gene of the folate branch of the methionine pathway, but not metA in the nonfolate branch of the methionine pathway, or the gcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on the E. coli chromosome.

Nucleotide sequence of the Salmonella typhimurium glyA gene.

The DNA sequence of the Salmonella typhimurium glyA gene has been determined. The polypeptide deduced from the DNA sequence contains 417 amino acids and has a calculated molecular weight of 45428 daltons. S1 nuclease mapping experiments located the transcription start point and possible transcription termination region. The nucleotide and amino acid sequences for the S. typhimurium and Escherichia coli glyA genes were compared. The nucleotide sequences show 89% identity, and the amino acid sequences show 93% identity. In S. typhimurium there is an absence of REP sequences between the translation termination site and the proposed transcription termination site that are present in the E. coli sequence. A conserved sequence is found in both organisms extending from 79 to 117 bp upstream of the consensus -35 sequences of the glyA promoters. This conserved sequence shows homology to a sequence preceding the S. typhimurium metE gene determined to bind the MetR regulatory protein.

Escherichia coli glyA mRNA decay: the role of 3' secondary structure and the effects of the pnp and rnb mutations.

The Escherichia coli glyA structural gene is followed by two REP sequences and a rho-independent transcription terminator. These sequences are essential for maintaining glyA mRNA stability and gene expression by blocking the 3' to 5' exonucleolytic activities of polynucleotide phosphorylase and ribonuclease II. The results support the model of cooperative endonucleolytic and 3' to 5' exonucleolytic activities in mRNA decay.

The Salmonella typhimurium glycine cleavage enzyme system.

A glycine cleavage enzyme system, inducible by glycine, has been demonstrated in Salmonella typhimurium. The induced enzyme levels, however, are only about 20% of the induced levels found in Escherichia coli. Starting with a serine auxotroph, mutants were isolated that grow with a serine supplement, but not with a glycine supplement. Three independently isolated mutants have reduced or nondetectable glycine cleavage enzyme levels. The new mutations, designated gcv, were mapped between the serA and lys genes at 62.5 min on the S. typhimurium chromosome.

Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine.

The methionine component of glyA gene regulation in Escherichia coli K-12 was investigated. The results indicate that the glyA gene is positively controlled by the metR gene product. Activation of glyA by the MetR protein requires homocysteine, an intermediate in methionine biosynthesis. The positive-acting metR regulatory system functions independently of a regulatory system shown previously to control glyA gene expression.

Cloning and characterization of the glycine-cleavage enzyme system of Escherichia coli.

The glycine-cleavage enzyme system of Escherichia coli has been cloned in the cosmid vector pMF7. The recombinant plasmid, designated pGS64, carries two 19.4-kb EcoRI insert fragments. One of these fragments, which carries the gcv system, was subcloned from plasmid pGS64 into the plasmid vectors pACYC184 and pSC101 (creating plasmids pGS96 and pGS97, respectively). Plasmid pGS97, but not pGS96, complements a gcv mutant on glycine-supplemented plates. Enzyme assays, however, verified that both plasmids carry an inducible gcv system. The location of the gcv system in plasmid pGS97 was determined by Tn5 insertional inactivation. Subcloning experiments identified the region on the 19.4-kb fragment that inhibits growth in strains transformed with plasmid pGS96 and a region that is possibly involved in negative regulation of the gcv system.

Characterization of a cis-acting regulatory mutation that maps at the distal end of the Escherichia coli glyA gene.

We have isolated a phage Mu cts-generated glyA mutant with only 30% of the normal level of serine hydroxymethyltransferase activity. Genetic and physical mapping studies show that Mu cts has inserted between the end of the glyA structural gene and its proposed transcription termination site. The mutation is cis acting and shows that sequences distal to the glyA structural gene play an important role in the expression of this gene.

Cloning and characterization of the gene for Salmonella typhimurium serine hydroxymethyltransferase.

A plasmid containing the glyA gene of Salmonella typhimurium LT2 was constructed in vitro using plasmid pACYC184 as the cloning vector and a lambda gt7-glyA transducing phage as the source of glyA DNA. The recombinant plasmid (pGS30) contains a 10-kb EcoRI insert fragment. Genetic and biochemical experiments established that the fragment contains a functional glyA gene. From plasmid pGS30 we subcloned a 4.4-kb SalI-EcoRI fragment containing the glyA gene and its neighboring regions (plasmid pGS38). The location and orientation of the glyA gene within the 4.4-kb insert fragment was determined in four ways: (1) comparison of the physical map of the 4.4-kb SalI-EcoRI fragment with the physical map of a 2.6-kb SalI-PvuII fragment that carries the Escherichia coli glyA gene; (2) deletion analysis; (3) transposon Tn5 insertional inactivation experiments; (4) deoxyribonucleic acid sequencing and comparison of the S. typhimurium DNA sequence with the E. coli DNA sequence. A presumptive glyA-encoded polypeptide of Mr 47000 was detected using plasmid pGS38 as template in a minicell system, but not when the glyA gene was inactivated by insertion of a Tn5 element.

Complete nucleotide sequence of the E. coli glyA gene.

The nucleotide sequence of the Escherichia coli glyA gene has been determined. The amino acid sequence predicted from the DNA sequence consists of 417 residues. After the coding region there is a 185 nucleotide sequence preceding the proposed transcription termination region for the glyA gene. This region is preceded by a G-C rich sequence that could form a stable stem-loop structure once transcribed, followed by an A-T rich sequence within which transcription appears to terminate. There is a long region of dyad symmetry and numerous smaller symmetrical regions between the site of translation termination and the proposed transcription termination region. These stem-loop structures show remarkable homology with intercistronic elements of other prokaryotic operons and may play a role in the regulation of glyA gene expression.

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.

Escherichia coli K12 mutants defective in the glycine cleavage enzyme system.

Two 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 cell's 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.

Construction and expression of hybrid plasmids containing the Escherichia coli glyA genes.

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 EcoRI insert. Genetic and biochemical experiments indicate that the region controlling STHM synthesis is present on the insert. Strains bearing multi-copy 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 the plasmid pBR322 established that a 2.5 kb SalI-BclI fragment carries the glyA gene. A physical map of this fragment is presented.