Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium

The Salmonella typhimurium metE and metR genes share a common control region, with overlapping, divergently transcribed promoters. A double gene fusion was constructed in which the metE promoter directs expression of the Escherichia coli lacZ gene and the metR promoter directs expression of the E. coli galK gene. By using an E. coli strain lysogenized with a lambda bacteriophage carrying the metE-lacZ metR-galK double fusion (lambda Elac.Rgal), two classes of cis-acting mutations were isolated that increase metR-galK expression. The first class of mutations causes a simultaneous decrease in metE-lacZ expression by disrupting the normal MetR-mediated activation of the metE promoter. The mutations are located within a region extending from 17 to 34 base pairs upstream of the -35 region of the metE promoter. Gel mobility shift assays and DNaseI protection experiments demonstrated that the MetR protein specifically binds to a 24-base-pair region encompassing these mutations. The second class of mutations increases metR-galK expression by directly altering the promoter consensus sequences of the metE and metR promoters.     

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.     

MetJ-mediated regulation of the Salmonella typhimurium metE and metR genes occurs through a common operator region

Abstract In Salmonella typhimurium the metE and metR promoters overlap and are divergently transcribed. Three tandem repeats of an 8 bp sequence defined previously as the metE operator site for MetJ-mediated repression also overlap the −35 region of the metR promoter. Starting with a metE-lacZ · metR-galK double gene fusion, site-directed mutagenesis was used to change nucleotides in each of the repeat units from the consensus sequence. Each mutation, along with the wild-type metE-lacZ · metR-galK gene fusion, was cloned into phage λgt2. Regulation of the metE and metR genes was examined by measuring β-galactosidase and galactokinase levels in Escherichia coli strains lysogenized with phage carrying the wild-type and mutant fusions. Mutations in each of the 8 bp repeat units disrupt MetJ-mediated repression for both the metE-lacZ and metR-galK gene fusions, suggesting that the metE and metR genes share a common operator site for the MetJ repressor.     

The methionine biosynthetic pathway from homoserine in Pseudomonas putida involves the metW, metX, metZ, metH and metE gene products

Biosynthesis of methionine from homoserine in Pseudomonas putida takes place in three steps. The first step is the acylation of homoserine to yield an acyl-L-homoserine. This reaction is catalyzed by the products of the metXW genes and is equivalent to the first step in enterobacteria, gram-positive bacteria and fungi, except that in these microorganisms the reaction is catalyzed by a single polypeptide (the product of the metA gene in Escherichia coli and the met5 gene product in Neurospora crassa). In Pseudomonas putida, as in gram-positive bacteria and certain fungi, the second and third steps are a direct sulfhydrylation that converts the O-acyl-L-homoserine into homocysteine and further methylation to yield methionine. The latter reaction can be mediated by either of the two methionine synthetases present in the cells.     

Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene.

The vitamin B12 (B12)-mediated repression of the metE gene in Escherichia coli and Salmonella typhimurium requires the B12-dependent transmethylase, the metH gene product. It has been proposed that the MetH-B12 holoenzyme complex is involved directly in the repression mechanism. Using Escherichia coli strains lysogenized with a lambda phage carrying a metE-lacZ gene fusion, we examined B12-mediated repression of the metE-lacZ gene fusion. Although B12 supplementation results in a 10-fold repression of metE-lacZ expression, homocysteine addition to the growth medium overrides the B12-mediated repression. In addition, B12-mediated repression of the metE-lacZ fusion is dependent on a functional MetR protein. When a metB mutant was transformed with a high-copy-number plasmid carrying the metE gene, which would be expected to reduce intracellular levels of homocysteine, metE-lacZ expression was reduced and B12 supplementation had no further effect. In a metJ mutant, B12 represses metE-lacZ expression less than twofold. When the metJ mutant was transformed with a high-copy-number plasmid carrying the metH gene, which would be expected to reduce intracellular levels of homocysteine, B12 repression of the metE-lacZ fusion was partially restored. The results indicate that B12-mediated repression of the metE gene is primarily a loss of MetR-mediated activation due to depletion of the coactivator homocysteine, rather than a direct repression by the MetH-B12 holoenzyme.     

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 metE gene in Escherichia coli

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.     

The involvement of methionine regulatory mutants in the suppresion of b12-dependent homocysteine transmethylase (metH) mutants of Salmonella typhimurium

Summary Certain metH mutants (which lack the B₁₂-dependent homocysteine transmethylase) give rise to revertants resistant to the methionine analogue, ethionine. The revertants retain the original metH mutation and its suppression is due to two mutations, supI and supII. The supI mutation, which confers ethionine resistance, appears to be a mutation in the methionine regulatory gene, metJ, but the location and nature of supII have not been determined. It is possible that suppression results from a direct association between the metH and metJ gene products or by the introduction of an alternative pathway of homocysteine methylation.     

Liver mitochondrial aldehyde dehydrogenase: in vitro expression, in vitro import, and effect of alcohols on import

An in vitro expression plasmid (pGRAP) that contained the cDNA coding for the rat mitochondrial aldehyde dehydrogenase precursor was constructed, mRNA was synthesized then translated, and the in vitro synthesized precursor of aldehyde dehydrogenase was used in an in vitro import assay. As expected the 19 amino acid signal peptide of the precursor allowed import of the precursor into rat liver mitochondria. This in vitro system was used to examine the effect of alcohols on import. It was found that the alcohols (ethyl, butyl, hexyl, and octyl) tested inhibited the import of the aldehyde dehydrogenase precursor. Pretreatment of the mitochondria with alcohol was responsible for the inhibition. The inhibition appeared to be relatively specific for pre-aldehyde dehydrogenase as the precursor of ornithine transcarbamylase was still imported in the presence of alcohols. Of potential physiological significance was finding that ethanol inhibited import in a dose-response fashion; 50% inhibition occurred at 75 mM, a concentration achievable during the ingestion of alcohol. In addition, the concentrations of alcohols required to produce an inhibitory effect on import decreased as the hydrocarbon chain length of alcohols increased. The inhibitory effect of alcohols appeared to be specific as other solvents examined did not inhibit import. We postulate that alcohols may perturb the mitochondrial membrane and affect the receptor-translocator necessary for the import of the aldehyde dehydrogenase precursor.     

The effect of MAR elements on variation in spatial and temporal regulation of transgene expression

The level of transgene expression often differs among independent transformants. This is generally ascribed to different integration sites of the transgene into the plant genome in each independently obtained transformant (position effect). It has been shown that in tobacco transformants expressing, for example, a cauliflower mosaic virus (CaMV) 35S promoter-driven -glucuronidase (GUS) reporter gene, these position-induced quantitative differences among individual transformants were reduced by the introduction of matrix-associated regions (MAR elements) on the T-DNA. We have previously shown by imaging of in planta firefly luciferase (luc) reporter gene activity that quantitative differences in transgene activity can be the result of either a variation in (1) level, (2) spatial distribution and/or (3) temporal regulation of transgene expression between independent transformants. It is not known which of these three different aspects of transgene expression is affected when the transgene is flanked by MAR elements. Here we have used the firefly luciferase reporter system to analyse the influence of MAR elements on the activity of a CaMV 35S-luc transgene in a population of independently transformed tobacco plants. Imaging of in planta LUC activity in these tobacco plant populations showed that the presence of MAR elements does not result in less variation in the average level of transgene expression between individual transformants. This result is different from that obtained previously with a 35S-GUS reporter gene flanked by MAR elements and reflects the differences in the stability of the LUC and GUS reporter proteins. Also the variation in spatial patterns of in vivo LUC activity is not reduced between independent transformants when the transgene is flanked by MAR elements. However, MAR elements do seem to affect the variation in temporal regulation of transgene expression between individual transformants. The potential effects of MAR elements on the variability of transgene expression and the relation to the stability of the (trans)gene product are discussed.     

In vitro effect of homocysteine on nucleotide hydrolysis by blood serum from adult rats

During the past few years, elevated blood levels of homocysteine (Hcy) have been linked to increased risk of premature coronary artery disease, stroke and thromboembolism. These processes can be also related to the ratio adenine nucleotide/adenosine, since extracellularly these nucleotides are associated with modulation of processes such as platelet aggregation, vasodilatation and coronary flow. Furthermore, there are some studies that suggest a relationship between Hcy and plasma adenosine concentrations. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes one of the systems for rapid inactivation of circulating adenine nucleotides. Thus, the main objective of this study was to evaluate if Hcy can participate in the modulation of the extracellular adenine nucleotide hydrolysis by rat blood serum. Our results showed that Hcy, at final concentrations of 5.0 mM, inhibits in vitro ATP, ADP and AMP hydrolysis by 26, 21 and 16%, respectively. Also Hcy, at final concentrations of 8.0mM, inhibited the in vitro hydrolysis of ATP, ADP and AMP by 46, 44 and 44%, respectively. Kinetic analysis showed that the inhibitions of the three adenine nucleotide hydrolyses in the presence of Hcy, by serum of adult rats, is of the uncompetitive type. The IC50 calculated from the results obtained were 6.52+/-1.75 mM (n = 4), 5.18 +/- 0.64 mM (n = 3) and 5.16 +/- 1.22 mM (n = 3) for ATP, ADP and AMP hydrolysis, respectively.