metJ Gene Research Articles

Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli

Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids (l-cysteine (Cys), l-histidine, and l-methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using Escherichia coli cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in E. coli cells carrying the egtBCDE genes from Mycobacterium smegmatis was effective for ERG production. Furthermore, coexpression of the egtA gene, which encodes γ-glutamylcysteine synthetase that synthesizes the γ-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though E. coli intrinsically has γ-glutamylcysteine synthetase. Additionally, disruption of the metJ gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.

A Metabolic Regulator Modulates Virulence and Quorum Sensing Signal Production in Pectobacterium atrosepticum

Plant cell wall-degrading enzymes (PCWDE) are key virulence determinants in the pathogenesis of the potato pathogen Pectobacterium atrosepticum. In this study, we report the impact on virulence of a transposon insertion mutation in the metJ gene that codes for the repressor of the methionine biosynthesis regulon. In a mutant strain defective for the small regulatory RNA rsmB, PCWDE are not produced and virulence in potato tubers is almost totally abolished. However, when the metJ gene is disrupted in this background, the rsmB(-) phenotype is suppressed and virulence and PCWDE production are restored. Additionally, when metJ is disrupted, production of the quorum-sensing signal, N-(3-oxohexanoyl)-homoserine lactone, is increased. The metJ mutant strains showed pleiotropic transcriptional impacts affecting approximately a quarter of the genome. Genes involved in methionine biosynthesis were most highly upregulated but many virulence-associated transcripts were also upregulated. This is the first report of the impact of the MetJ repressor on virulence in bacteria.

Mechanism of L-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of L-methionine biosynthetic enzymes.

We derived L-methionine-analogue-resistant mutants from Escherichia coli JM109 strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and selected the potent L-methionine-overproducing strains by microbioassay using lactic acid bacteria. One of the mutants, strain TN1, produced approximately 910 mg L-methionine/l following the addition of 0.1% yeast extract to fundamental medium containing glucose and ammonium sulfate. The L-methionine biosynthetic enzymes, cystathionine gamma-synthase and cystathionine beta-lyase, of the L-methionine-overproducing mutants were little repressed by L-methionine. To analyse the mechanism of L-methionine overproduction in the mutant strains, the metJ gene coding for the E. coli met repressor, MetJ protein, was cloned and sequenced by the polymerase chain reaction. The same single-amino-acid subsitution (wild-type Ser-->Asn) at position 54 was observed in four independent L-methionine-producing mutants. When the wild-type metJ gene was then introduced into strain TN1 having the mutant metJ gene, the level of enzyme synthesis and the L-methionine productivity in the transformants were found to revert to those of the wild-type. It was therefore considered that only one point mutation in the metJ gene occurred in the L-methionine-producing mutants. These results demonstrate the important role of residue 54 of the MetJ protein in L-methionine overproduction, probably because of the derepression of L-methionine biosynthetic enzymes.

Role of the metF and metJ genes on the vitamin B 12 regulation of methionine gene expression: Involvement of N 5-methyltetrahydrofolic acid

The repression of MetE synthesis in Escherichia coli by vitamin B 12 is known to require the MetH holoenzyme (B 12-dependent methyltransferase) and the metF gene product. Experiments using trimethoprim, an inhibitor of dihydrofolate reductase, show that the MetF protein is not directly involved in the repression, but that N 5-methyltetrahydrofolic acid (N 5-methyl-H 4-folate), the product of the MetF enzymatic reaction is required. Since the methyl group from N 5-methyl-H 4-folate is normally transferred to the MetH holoenzyme to form a methyl-B 12 enzyme, the present results suggest that a methyl-B 12 enzyme is involved in the vitamin B 12 repression of metE expression. Other results argue against the possibility that a methyl-B 12 enzyme functions in this repression solely by decreasing the cellular level of homocysteine, which is required for MetR activation of metE expression. Experiments with metJ mutants show that the MetJ protein mediates about 50% of the repression of metE expression by B 12 but is totally responsible for the regulation of metF expression by vitamin B 12.

Control of methionine biosynthesis in Escherichia coli K12: a closer study with analogue-resistant mutants

Control of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue-resistant mutants. Norleucine (NL) and alpha-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization. Adenosylation of Et to generate S-adenosylethionine (AdoEt) by cell-free enzyme from E. coli K12 was demonstrated. Tolerance of increasing concentrations of NL by E. coli K12 mutants is expressed serially as phenotypes NLR, NLREtR, NLRMMR and finally NLREtRMMR. All spontaneous NLR mutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metK and metJ genes. The kinetics of methionine adenosylation by the E. coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture. NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet.

Nucleotide sequence of the metH gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2

The Escherichia coli K-12 metH gene, encoding the vitamin B12-dependent homocysteine transmethylase, is located between iclR and lysC in the 91-min region of the chromosome. The metH gene has been sequenced and reveals an open reading frame of 3600 bp encoding a polypeptide of 1200 amino acids (aa) with a calculated Mr of 132628. The first 414 aa of the deduced polypeptide sequence are 92% identical to the 414 aa deduced from the partially sequenced Salmonella typhimurium LT2 metH gene. In-frame fusions of metH to lacZ were used to confirm the reading frame of the metH gene and to study its regulation, metH was repressed tenfold, presumably indirectly, by l-methionine and the metJ gene product, while vitamin B12 did not induce de novo synthesis of MetH.

Salmonella typhimurium metC operator-constitutive mutations

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (lambda Clac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying lambda Clac mutant phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.

Salmonella typhimurium metCoperator-constitutive mutations

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (λClac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying λClac mutant phage exhibit high β-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.

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.

Regulation of the metR gene of Salmonella typhimurium

Regulation of the Salmonella typhimurium metR gene was studied by measuring beta-galactosidase levels in Escherichia coli strains lysogenic for a lambda bacteriophage carrying a metR-lacZ fusion. The results indicate that the metR gene is negatively regulated by its own gene product and that this autoregulation involves homocysteine as a corepressor. In addition, the results indicate that the metR gene is negatively regulated by the metJ gene product over a 70- to 80-fold range.

Operator-constitutive mutations of the Escherichia coli metF gene.

The Escherichia coli metF gene codes for 5,10-methylene-tetrahydrofolate reductase, the enzyme that leads to the formation of N-methyltetrahydrofolate, supplying the methyl group of methionine. Transcription of metF, as well as most of the methionine genes, is repressed by the metJ gene product complexed with S-adenosylmethionine. A metF'-'lacZ gene fusion was used to isolate mutants that have altered expression from the metF promoter. The nucleotide sequences of the metF regulatory region from five such mutants were determined. The mutations were located in the region previously defined as the potential target of the methionine repressor by its similarity to other binding sites. The mutationally defined metF operator thus consists of a 40-base-pair-long region, with five 8-base-pair imperfect palindromes spanning the metF transcription start. The altered operators do not recognize the purified repressor in an in vitro transcription-translation system, although the repressor binds efficiently to the metF wild-type operator.

Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine.

The metJ gene encoding the methionine aporepressor was placed under the control of a strong and inducible promoter, ptac. Bacterial strains carrying the recombinant plasmid pIP35 overproduced the regulatory protein by a factor of 200 over the wild type strain as determined by the immunoblot technique. The purified metJ gene product negatively controls the expression of the metF gene, in a cell-free system as shown by repression of beta-galactosidase synthesis under the control of the metF promoter. The metJ protein binds to a DNA fragment containing the potential operator of the metF gene with an affinity which is 10 times greater in the presence of S-adenosylmethionine than in its absence. Equilibrium dialysis experiments showed that the met aporepressor binds 2 mol of S-adenosylmethionine per mol of dimer with a dissociation constant of 200 microM.

Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene.

Regulation of the Salmonella typhimurium metJ gene was examined by measuring beta-galactosidase activity in Escherichia coli strains lysogenic for a phage carrying a metJ-lacZ gene fusion. The results indicated that the metJ gene is regulated by its own gene product and by methionine supplementation to the growth medium. This autoregulatory mechanism involved two tandem promoters, pJ1 and pJ2, separated by approximately 65 base pairs. Deletion analysis permitted the assessment of the activity of promoters pJ1 and pJ2 individually. Promoter Pj1 was negatively regulated by the metJ gene product and by methionine. Although Pj2 regulation remained unclear, evidence is presented which suggests that it is not negatively regulated like pJ1.

Regulation of in vivo transcription of the Escherichia coli K-12 metJBLF gene cluster

We subcloned DNA of the intercistronic region between the divergently transcribed metJ and metB genes of Escherichia coli into the transcription-fusion vector pK01 and localized the metJ promoters by deletion analysis. The plasmid-borne promoters of both genes were repressed by chromosomal metJ. In addition, S1 nuclease mapping of chromosomally derived mRNA from a derepressed strain revealed the start sites of transcription for metBL, metF, and metJ. The metBL and metF genes each had a single transcript which was repressed by metJ, while the metJ gene had three transcripts, of which the first was strongly repressed by metJ, the second was less strongly repressed, and the third was not repressed.

Regulation of methionine synthesis in Escherichia coli: Effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes

The regulation of the expression of three Escherichia coli met genes, metB, which codes for cystathionine γ-synthetase (EC 4.2.99.9), metL, which codes for aspartokinase II-homoserine dehydrogenase II (EC 2.7.2.4-EC 1.1.1.3) and metJ, which codes for the methionine regulon aporepressor, has been studied using highly purified DNA-directed in vitro protein synthesis systems. In a system where the entire gene product is synthesized, the expression of the metB and metL genes is specifically inhibited by MetJ protein (repressor protein) and S-adenosylmethionine (AdoMet). In a simplified system that measures the formation of the first dipeptide of the gene product (fMet-Ala for the metJ gene), MetJ protein and AdoMet partially repress (∼40–60%) metJ gene expression. Thus, the metJ gene can be partially autoregulated by its gene product.

Regulation of the methionine regulon in Escherichia coli.

AbstractThe genes involved in methionine biosynthesis are scattered throughout the Escherichia coli chromosome and are controlled in a similar but not coordinated manner. The product of the metJ gene and S‐adenosylmethionine are involved in the repression of this ‘regulon’.

Isolation and characterization of the product of the methionine-regulatory gene metJ of Escherichia coli K-12.

We have modified a previously isolated metJ plasmid by removing a segment of DNA including the rop gene. Bacterial strains carrying this plasmid produce elevated levels of the metJ gene product, presumably because of the high number of gene copies in the cell. We have isolated the metJ gene product in nearly homogeneous form from such a strain. The subunit size and the amino acid composition are the same as those predicted from the DNA sequence of the metJ gene. Sedimentation equilibrium measurements show that the native metJ gene product is a dimer. The purified dimer protects a short segment of DNA in the regulatory region of the metB and metJ genes from hydrolysis by DNase I.

Cloning and initial characterization of the metJ and metB genes from Salmonella typhimurium LT2

The metJ and metB genes of Salmonella typhimurium have been cloned into Escherichia coli K-12 on a 19-kb EcoRI fragment in the plasmid vector pACYC184. The presence of a functional metB+ gene on this plasmid, designated pGS89, was demonstrated by its ability to complement a metB− E. coli mutant. The presence of a functional metJ+ gene on this plasmid was demonstrated by its ability to repress metC+ gene expression in a metJ− mutant transformed with this plasmid. The metJ gene product was identified in a minicell system as a polypeptide of Mr 12000. This polypeptide was not produced when the metJ gene was inactivated by insertion of a TnJ element. Transformation of an E. coli metB− mutant with plasmid pGS89 (metB+, metJ+) results in transformants that grow slowly on glucose-minimal medium or glucose-minimal medium supplemented with homocysteine. Methionine addition, however, restores normal growth. This phenotype requires the relA− mutation in the host strain and at least two other plasmid loci, one of which is the metJ+ gene. Transformation of an E. coli metJ− mutant with metJ− derivatives of plasmid pGS89 results in transformants that are unable to grow on either glucose-minimal medium or glucose-minimal medium supplemented with methionine. This phenotype requires the presence of a functional metB+ gene on the plasmid, and is unrelated to the status of the relA gene.

Nucleotide sequence and biochemical characterization of the metJ gene from Salmonella typhimurium LT2.

The nucleotide sequence of the Salmonella typhimurium metJ gene is presented along with the sequence of the promoter region for the closely linked metB gene. The two genes are transcribed in opposite directions, with transcription initiating from a single promoter for metB, and from two apparent promoters for metJ. RNA polymerase binding sites for metJ and metB, determined by in vitro protection studies, lie adjacent to each other and may overlap. The two metJ promoters, PJ1 and PJ2, are separated by approximately 65 base pairs. Binding of RNA polymerase in vitro could only be observed for PJ1, even though transcripts are initiated from both promoters in vivo. The metJ gene codes for a polypeptide of 105 amino acids with a calculated Mr of 12,110. The translation start site was determined by N-terminal amino acid sequence analysis of a metJ-lacZ fusion protein.

Structure and autoregulation of the metJ regulatory gene in Escherichia coli.

The nucleotide sequence of the Escherichia coli metJ regulatory gene (312 nucleotides) has been determined as well as that of two mutations located within the gene. Analysis of the sequence downstream from the metJ gene has revealed inverted repeats homologous to several intercistronic regions, also reported to occur between operons. A hybrid protein that contains the 55 first amino acid residues of the metJ protein substituting for the 8 amino acid residues at the NH2 terminus of beta-galactosidase was produced by gene fusion. The hybrid protein retaining beta-galactosidase activity was purified. Its amino-terminal sequence was determined and this allowed us to locate the translational start codon of the metJ gene. Evidence was provided for autoregulation by repression of the metJ gene. By sequencing upstream from metJ, the region situated between the metJ and metB genes was found to contain putative operator structures that we propose to call "Met boxes."