glyA Gene Research Articles

Site-Directed Mutagenesis Techniques in the Study ofEscherichia coliSerine Hydroxymethyltransferase

The 3340-bp fragment containing theEscherichia coli glyA gene coding for serine hydroxymethyltransferase was reduced in size by PCR, and the 1600-bp fragment obtained was cloned into the vector pBR322 in both orientations (5′–3′ and 3′–5′). This DNA manipulation allowed us to perform site-directed mutagenesis by PCR on theglyA gene. To overcome the problem of the presence of wild-type protein in the various mutant enzyme preparations, theE. colistrain GS245 used to express recombinant serine hydroxymethyltransferase was maderecA deficient through generalized transduction mediated by phage P1. The new strain was used for the production of a mutant form of the enzyme, in which the pyridoxal 5′-phosphate-binding lysine was substituted by a glutamine. The preparation of this mutant form was completely devoid of wild-type enzyme contamination and measurements of its catalytic activity in the transamination reactions ofl- andd-alanine confirmed the suggestion that the active site lysine is not the base that removes the α-proton from the substrate (Schirchet al.,1993,J. Biol. Chem.268, 23132–23138).

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.

Characterization of Campylobacter upsaliensis fur and its localization in a highly conserved region of the Campylobacter genome

Despite increasing recognition of the importance of Campylobacter upsaliensis in human disease little is known about either the virulence properties or genetics of this enteric pathogen. The complete coding sequence of a C. upsaliensis gene has yet to be published. We have cloned and sequenced the complete iron-uptake regulatory ( fur) gene from the type strain of this species. The C. upsaliensis fur homolog was isolated from a genomic library of C. upsaliensis ATCC 43954 constructed in phage lambdaGEM-11. The open reading frame identified encodes a polypeptide consisting of 156 amino acids. The 5′-flanking region of the C. upsaliensis fur gene contains 3 putative Fur-binding sequences and two catabolite activator-binding sequences indicating the potential for autogenous and cAMP-mediated regulation, respectively. Primer extension analysis identified a single transcription start site 262 nt upstream from the AUG initiation codon. Sequence analysis indicates that the Fur protein of C. upsaliensis is highly homologous (87% amino acid identity) to Campylobacter jejuni Fur. Furthermore, the arrangement of the lysS and glyA genes downstream of fur is precisely conserved in both C. upsaliensis ATCC 43954 and C. jejuni TGH9011. Using the polymerase chain reaction close linkage of fur with lysS and glyA was also observed among multiple isolates of C. upsaliensis, C. jejuni and C. coli suggesting a possible functional relevance for this conserved genetic arrangement in campylobacteria.

Characterization of the MetR binding sites for the glyA gene of Escherichia coli.

Sequence analysis of the glyA control region of Escherichia coli identified two regions with homology to the consensus binding sequence for MetR, a lysR family regulatory protein. Gel shift assays and DNase I protection assays verified that both sites bind MetR. Homocysteine, a coregulator for MetR, increased MetR binding to the glyA control region. The MetR binding sites were cloned into the pBend2 vector. Although the DNA did not show any significant intrinsic bend, MetR binding resulted in a bending angle of about 33 degrees. MetR-induced bending was independent of homocysteine. To verify that the MetR binding sites play a functional role in glyA expression, site-directed mutagenesis was used to alter the two binding sites in a lambda glyA-lacZ gene fusion phage. Changing the binding sites toward the consensus MetR binding sequence caused an increase in glyA-lacZ expression. Changing either binding site away from the consensus sequence caused a decrease in expression, suggesting that both sites are required for normal glyA regulation.

Modulation of the heat shock response by one-carbon metabolism in Escherichia coli.

A genetic screen designed to isolate mutants of Escherichia coli W3110 altered in the ability to induce the heat shock response identified a strain unable to induce the heat shock proteins in a rich, defined medium lacking methionine after exposure to 2,4-dinitrophenol. This strain also grew slowly at 28 degrees C and linearly at 42 degrees C in this medium. The abnormal induction of the heat shock proteins and abnormal growth at both high and low temperatures were reversed when methionine was included in the growth medium. The mutation responsible for these phenotypes mapped to the glyA gene, a biosynthetic gene encoding the enzyme that converts serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. This reaction is the major source of glycine and one-carbon units in the cell. Because fixed one-carbon units, in the form of methionine, allowed mutant cells to induce the heat shock response after exposure to 2,4-dinitrophenol, a one-carbon restriction may be responsible for the phenotypes described above.

Lysyl-tRNA synthetase gene of Campylobacter jejuni

We report the cloning and complete nucleotide sequence of the Campylobacter jejuni lysyl-tRNA synthetase gene (lysS). The C. jejuni lysS gene sequence shows high homology to the two Escherichia coli lysyl-tRNA synthetase genes, lysS and lysU. The Campylobacter lysyl-tRNA synthetase protein (LysRS) shows 47.9 and 46.6% sequence identity to the E. coli enzymes encoded by the lysS and lysU genes, respectively. The LysRS encoded by the C. jejuni gene is a polypeptide of 501 amino acids with a deduced molecular weight of 57,867. The enzyme is active in E. coli. The gene is expressed from its own promoter, and the transcription start site has been mapped. The carboxyl-terminal codon of the C. jejuni lysS gene overlaps by 1 bp with the Met initiation codon of the glyA gene, which has been shown to have a promoter which is functional in E. coli (V.L. Chan and H.L. Bingham, Gene 101:51-58, 1991). C. jejuni, unlike E. coli, has only one lysyl-tRNA synthetase gene.

Complete sequence of the Campylobacter jejuni glyA gene encoding serine hydroxymethyltransferase

The complete nucleotide sequence of the Campylobacter jejuni glyA gene was determined and the amino acid (aa) sequence of its product, serine hydroxymethyltransferase (SHMT), was deduced. The deduced polypeptide has 414 aa residues ( M r 45758). The aa sequences of C. jejuni and Escherichia coli show 55.6% identity. Comparative analysis of the aa sequences of the SHMTs of E. coli and C. jejuni identified two new putative functional domains. The translational product of the C. jejuni glyA gene was identified using both minicell and maxicell systems and the transcription start point was mapped. The deduced transcription-regulatory signals, −10 and −35 sequences, show high homology to the corresponding consensus sequences for σ 70 promoters in E. coli. The C. jejuni glyA promoter may be useful in the construction of shuttle vectors between E. coli and C. jejuni.

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of Mr 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.

Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum

A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.

Regulation of the Escherichia coli glyA gene by the purR gene product

The purine regulon repressor protein, PurR, was shown to be a purine component involved in glyA regulation in Escherichia coli. Expression of glyA, encoding serine hydroxymethyltransferase activity, was elevated in a purR mutant compared with a wild-type strain. When the purR mutant was transformed with a plasmid carrying the purR gene, the serine hydroxymethyltransferase levels returned to the wild-type level. The PurR protein bound specifically to a DNA fragment carrying the glyA control region, as determined by gel retardation. In a DNase I protection assay, a 24-base-pair region was protected from DNase I digestion by PurR. The glyA operator sequence for PurR binding is similar to that reported for several pur regulon genes.

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.

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 expression of the Campylobacter jejuni glyA gene in Escherichia coli

Genetic studies of Campylobacter jejuni are greatly hampered by the lack of genetic markers and an established classical gene transfer mechanism between strains of this species. To facilitate future genetic studies and to provide a recombinant DNA approach for analyzing genes of C. jejuni, we constructed an extensive genomic library of a pathogenic C. jejuni strain TGH9011 (serotype 0:3) using pBR322. We report the isolation of a number of recombinant plasmids containing the complete structural gene of glyA, that encodes serine hydroxymethyltransferase (SHMT) of C. jejuni. Escherichia coli cells containing this multicopy recombinant plasmid with the glyA gene produce high levels of SHMT. The SHMT-encoding fragment was identified by subcloning and functional complementation. The expression of the C. jejuni glyA gene was probably via transcription initiated from its own promoter.

Serine hydroxymethyltransferase from Escherichia coli: purification and properties.

Serine hydroxymethyltransferase from Escherichia coli was purified to homogeneity. The enzyme was a homodimer of identical subunits with a molecular weight of 95,000. The amino acid sequence of the amino and carboxy-terminal ends and the amino acid composition of cysteine-containing tryptic peptides were in agreement with the primary structure proposed for this enzyme from the structure of the glyA gene (M. Plamann, L. Stauffer, M. Urbanowski, and G. Stauffer, Nucleic Acids Res. 11:2065-2074, 1983). The enzyme contained no disulfide bonds but had one sulfhydryl group on the surface of the protein. Several sulfhydryl reagents reacted with this exposed group and inactivated the enzyme. Spectra of the enzyme in the presence of substrates and substrate analogs showed that the enzyme formed the same complexes and in similar relative concentrations as previously observed with the cytosolic and mitochondrial rabbit liver isoenzymes. Kinetic studies with substrates showed that the affinity and synergistic binding of the amino acid and folate substrates were similar to those obtained with the rabbit liver isoenzymes. The enzyme catalyzed the cleavage of threonine, allothreonine, and 3-phenylserine to glycine and the corresponding aldehyde in the absence of tetrahydrofolate. The enzyme was also inactivated by D-alanine caused by the transamination of the active site pyridoxal phosphate to pyridoxamine phosphate. This substrate specificity was also observed with the rabbit liver isoenzymes. We conclude that the reaction mechanism and the active site structure of E. coli serine hydroxymethyltransferase are very similar to the mechanism and structure of the rabbit liver isoenzymes.

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 λgt7- 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 M r 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.

Sequence homology between prokaryotic and eukaryotic forms of serine hydroxymethyltransferase

The sequence of tryptic and chymotryptic peptides from cytosolic and mitochondrial rabbit liver serine hydroxymethyltransferase are compared to the proposed sequence of a protein coded for by the glyA gene of Escherichia coli. The E. coli glyA gene is believed to code for serine hydroxymethyltransferase. Extensive sequence homology between these peptides were found for the proposed E. coli enzyme in the aminoterminal two-thirds of the molecule. All three proteins have identical sequences from residue 222-231. This sequence is known to contain the lysyl residue which forms a Schiff's base with pyridoxal-P in the two rabbit liver enzymes. These results support the interpretation that the proposed sequence of E. coli serine hydroxymethyltransferase is correct. The data also show that cytosolic and mitochondrial serine hydroxymethyltransferase are homologous proteins.

Characterization of the Escherichia coli gene for serine hydroxymethyltransferase

Plasmid pGSl 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 M r values of about 46500 and 45500. When the glyA gene is inactivated with the transposable element Tn5, the M r 46 500 polypeptide is not observed, identifying it as the glyA gene product. The M r 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.

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 gene

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 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 SalI-BclI fragment carries the glyA gene. A physical map of this fragment is presented.