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.

Characterization of the gcv control region from Escherichia coli.

We constructed a set of deletions upstream of the gcv promoter and analyzed the effects of the deletions on expression of a gcvT-lacZ gene fusion. A deletion that ends at position -313 upstream of the transcription initiation site (+1) results in reduced levels of gcvT-lacZ expression, but the fusion is still inducible by glycine and repressible by purines. A deletion that ends at position -169 results in loss of both GcvA- and Lrp-mediated activation of the gcvT-lacZ fusion. The endpoints of delta -313 and delta -169 also define a site that down-regulates gcvT-lacZ expression two- to threefold. A deletion that ends at position -89 upstream from the transcription initiation site still shows PurR-mediated repression, suggesting that PurR-mediated repression is not by direct interference with the GcvA- and Lrp-mediated regulatory mechanism(s). Gel mobility shift assays and DNase I footprinting showed that Lrp protein binds to multiple sites upstream of the gcv promoter, from about bp -92 to bp -229. The results suggest that the gcv regulatory region is complex, with numerous cis-acting sites that are required for normal gcv expression.

Characterization of the Escherichia coli gcv operon.

The nucleotide (nt) sequence of the Escherichia coli gcvP gene was determined. The polypeptide deduced from the DNA sequence has an M(r) of 104,375 (957 amino acids). In a minicell system, gcvP encodes a polypeptide that migrates at 93.3 kDa on sodium dodecyl sulfate-polyacrylamide gels. After the coding region, there is a 39-nt sequence followed by a T-rich sequence within which transcription appears to terminate. This region is preceded by a G/C-rich sequence that could form a stable stem-loop structure once transcribed, and is characteristic of Rho-independent transcription terminators. A Northern analysis identified an approx. 4700-nt RNA molecule, large enough to encode the T-, H-and P-proteins of the glycine cleavage enzyme complex. Analyses of gcvP::lacZ fusions with and without stop codons in gcvT, the first gene in the operon, confirmed gcvT, gcvH and gcvP lie in an operon. RNA slot blot analyses indicated that induction of gcv by glycine, and PurR-mediated repression of gcv occur at the level of transcription.

Roles of the GcvA and PurR proteins in negative regulation of the Escherichia coli glycine cleavage enzyme system.

When Escherichia coli was grown in medium containing both inosine and glycine, the PurR repressor protein was shown to be responsible for a twofold reduction from the fully induced glycine cleavage enzyme levels. This twofold repression was also seen by measuring beta-galactosidase levels in cells carrying a lambda gcvT-lacZ gene fusion. In this fusion, the synthesis of beta-galactosidase is under the control of the gcv regulatory region. A DNA fragment carrying the gcv control region was shown by gel mobility shift assay and DNase I footprinting to bind purified PurR protein, suggesting a direct involvement of the repressor in gcv regulation. A separate mechanism of purine-mediated regulation of gcv was shown to be independent of the purR gene product and resulted in an approximately 10-fold reduction of beta-galactosidase levels when cells were grown in medium containing inosine but lacking the inducer glycine. This additional repression was dependent upon a functional gcvA gene, a positive activator for the glycine cleavage enzyme system. A dual role for the GcvA protein as both an activator in the presence of glycine and a repressor in the presence of inosine is suggested.

The Escherichia coli gcvT gene encoding the T-protein of the glycine cleavage enzyme system.

Plasmid pGS146 carries the Escherichia coli gcv system on a 7.12 kb SalI-BamHI DNA insert fragment. The DNA sequence of a gene which presumably encodes the T-protein of the glycine cleavage (GCV) enzyme complex was determined. The gene, designated gcvT, encodes a polypeptide of 364 amino acids with a calculated molecular weight of 40,146 daltons. In a minicell system, the SalI-BamHI fragment directs the synthesis of three polypeptides with Mr values of about 93,300, 43,300 and 17,400 daltons. When gcvT was inactivated by insertion of a translation terminator sequence, the Mr 43,300 dalton polypeptide was not observed. The deduced amino acid sequence of the E. coli T-protein was compared with the sequence of the T-protein from bovine liver. 190 of 364 amino acid residues are identical or chemically similar between the two proteins. An S1 nuclease mapping experiment located the transcription start point for gcvT. Single basepair changes were made in the promoter -10 and -35 sequences. These mutations significantly reduced expression from a gcvT-lacZ gene fusion. The gcvT gene is transcribed and translated in the same direction as the gcvH gene.

An Escherichia coli protein with homology to the H-protein of the glycine cleavage enzyme complex from pea and chicken liver.

The nucleotide sequence of an Escherichia coli gene which presumably encodes the H-protein of the glycine cleavage (GCV) enzyme complex is presented. The gene, designated gcvH, encodes a polypeptide of 128 amino acids with a calculated molecular weight of 13,665 daltons. The translation start site was determined by N-terminal amino acid sequence analysis of a gcvH-lacZ encoded fusion protein. The E. coli H-protein shows extensive homology with the H-proteins from the pea (Pisum sativum) and the chicken liver GCV enzyme complexes. 85 of 128 amino acid residues are identical or chemically similar between the E. coli and the pea H-proteins, and 74 of 128 amino acid residues are identical or chemically similar between the E. coli and the chicken liver H-proteins. All three proteins have identical amino acid sequences from residues 61-65. This sequence contains the lysyl residue involved in lipoic acid attachment in the chicken liver H-protein.

The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system.

The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene.

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.

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.

Salmonella typhimurium LT2 metF operator mutations.

Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (lambda Flac), in which beta-galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring beta-galactosidase levels in E. coli strains lysogenized with the wild-type lambda Flac phage and mutant lambda Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B12 control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B12 is dependent on a functional metJ gene product.

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 metF gene and its homology with the corresponding sequence of Escherichia coli.

The Salmonella typhimurium LT2 metF gene, encoding 5,10-methylenetetrahydrofolate reductase, has been cloned. Strains with multicopy plasmids carrying the metF gene overproduce the enzyme 44-fold. The nucleotide sequence of the metF gene was determined, and an open reading frame of 888 nucleotides was identified. The polypeptide deduced from the DNA sequence contains 296 amino acids and has a molecular weight of 33,135 daltons. Mung bean nuclease mapping experiments located the transcription start point and possible transcription termination region for the gene. There is a 25 bp nucleotide sequence between the translation termination site and the possible transcription termination region. This region possesses a GC-rich sequence that could form a stable stem and loop structure once transcribed (delta G = -9 kcal/mol), followed by an AT-rich sequence, both of which are characteristic of rho-independent transcription terminators. The nucleotide and deduced amino acid sequences of the S. typhimurium metF gene are compared with the corresponding sequences of the Escherichia coli metF gene. The nucleotide sequences show 85% homology. Most of the nucleotide differences found do not alter the amino acid sequences, which show 95% homology. The results also show that a change has occurred in the metF region of the S. typhimurium chromosome as compared to the E. coli chromosome.

A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium.

We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF gene product. Transduction analysis indicates that the mutant carries normal metE, metH, and metF genes; the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmid-borne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitamin B12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitamin B12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whose product acts in trans to activate the metE and metH genes.

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.

Cloning and characterization of the Salmonella typhimurium metE gene.

The metE gene of Salmonella typhimurium was cloned into plasmid pACYC184 by using a lambda gt7 -metE transducing phage as a source of metE DNA. The recombinant plasmid, designated pGS41 , carries a 12.8-kilobase-pair EcoRI insert fragment. The metE gene was subcloned from pGS41 into plasmid pBR322 on a 4.2-kilobase-pair EcoRI-HindIII fragment (plasmid pGS47 ) and a 4.5-kilobase-pair PstI fragment (plasmid pGS69 ). The location of metE in these plasmids was determined by transposon Tn5 insertional inactivation experiments. A metE-encoded polypeptide of 92,500 Mr was detected in a minicell system by using metE+ plasmids as templates. Truncated polypeptides replaced the 92,500-Mr polypeptide when plasmid derivatives containing Tn5 insertions that inactivate metE were used in the system. A comparison of the site of each Tn5 insertion and the size of the polypeptide made in the minicell system allowed us to determine the direction of transcription and translation. The location of the metE promoter was determined by using an in vitro transcription system and by RNA polymerase protection of restriction endonuclease sites.

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.

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.