Regulation of
            <i>S</i>
            -Adenosylmethionine Synthetase in
            <i>Escherichia coli</i>

Holloway, Caroline T.; Greene, Ronald C.; Su, Ching-Hsiang

doi:10.1128/jb.104.2.734-747.1970

Cited by 83 publications

(46 citation statements)

References 32 publications

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“…It is likely that overexpression of SAM synthetase depleted the methionine pool, either by draining it to make SAM or by repression of methionine biosynthesis. A methionine requirement due to SAM overexpression has also been reported by several investigators in E. coli (Holloway et al , 1970; Posnick and Samson, 1999) and in Bacillus subtilis (Yocum et al. , 1996).…”

Section: Resultssupporting

confidence: 65%

Studies on the role of the metK gene product of Escherichia coli K‐12

Wei

Newman

2002

Molecular Microbiology

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Studies on the role of the metK gene product of Escherichia coli K-12be expected to be an essential metabolite if any one of them were essential. As permeability problems prevent its being supplied exogenously, a functional metK gene should be essential for growth. We show here that in fact the metK gene cannot be deleted in the absence of a second functional copy of the gene. We also show that metK is transcribed from a start site at -140, and that the metK 84 allele has an A(r)G transition in its -10 sequence. Results Construction of a viable DmetK strain by gene replacementWe created a metK deletion by inverse polymerase chain reaction (PCR) of pZAP-K31 and subcloned it onto the gene replacement vector pKO3, making pKO3DmetK. We transformed pKO3DmetK into our parent E. coli K-12, MEW308, and into the same strain carrying a rescue plasmid, pBADmetK, inserted pKO3 into the chromosome and selected for recombinants. We then improved this construct by replacing pBADmetK with lower-copy, betterregulated p15ApLtet metK, adding the gene coding for the tet repressor to the chromosome, and making the strain recA -to avoid recombination of functional metK into the chromosome.Without metK in trans, we were unable to find strains with chromosomal deletions as judged by colony PCR of randomly selected colonies. However, with metK on the relatively high-copy pBR322-based pBADmetK, we found colony PCR produced two kinds of bands, some of the size of full-length metK (3.2 kb total) and some with metK 683 bp shorter (2.5 kb). It is thus possible to make a deletion of metK only in the presence of a functional metK, and we named the strain carrying such a deletion MEW308DmetK pBADmetK. These strains grew well with arabinose. They were not totally growth deficient in the absence of inducer, but grew slowly, resulting in small colonies, presumably because the pBAD promoter is quite leaky (Guzman et al., 1995).Using a p15A-based lower-copy plasmid carrying metK under the control of the pLtet promoter (Lutz and Bujard, 1997), and the tet repressor supplied from the chromosome, we could show a strong dependence of growth on inducer concentration (see below). However, because we had made only a partial deletion of metK, albeit a long one, the plasmid gene could recombine into the chromo- SummaryWe show here that the metK gene is essential to the growth of Escherichia coli K-12 and can be deleted only in the presence of a rescue plasmid carrying a functional metK gene. When metK expression was limited, genomic DNA methylation decreased and cell division was hampered. Through primer extension, the transcription start site of metK was located at 140 bp upstream of the translation start site. The frequently used metK 84 mutant has been shown to carry an A(r)G transition in the -10 region of the metK promoter. This accounts for its low level of S-adenosylmethionine (SAM) synthetase and SAM deficiency.

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Section: Resultssupporting

confidence: 65%

Studies on the role of the metK gene product of Escherichia coli K‐12

Wei

Newman

2002

Molecular Microbiology

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“…In analogy, ethionine in eukaryotic cells forms the homolog S-adenosylethionine (SAE) (54). Interestingly, S-adenosylmethionine synthases from E. coli and Salmonella do not accept ethionine as a substrate (55,56). Therefore, ethionine does not interfere with transmethylation reactions in those bacteria, and they are protected from cytotoxic ethionine effects.…”

Section: Discussionmentioning

confidence: 99%

Two‐carbon folate cycle of commensalLactobacillus reuteri6475 gives rise to immunomodulatory ethionine, a source for histone ethylation

Röth

Chiang

et al. 2018

FASEB j.

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Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1‐carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2‐carbon‐transporting folate in the form of 5, 10‐ethenyl‐tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2‐carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP‐1 cells with ethionine, their transcription of TNF‐α was inhibited and cell proliferation reduced. Mass spectrometry of THP‐1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone‐methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2‐carbon transporting variant of the folate cycle and modify human chromatin via ethylation.—Roth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two‐carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation. FASEB J. 33,3536‐3548 (2019). http://www.fasebj.org

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“…Strain E-40 (RG-109 metK-) was characterized as an S-adenosylmethionine synthetase mutant with low levels of SAM and high levels of methionine (6). Strain D-8 (RG 69, metJ-) was constitutive for SAM synthetase (9).…”

Section: Meedei and Pizermentioning

confidence: 99%

“…Unfortunately, the effect of SAM on these enzymes is not directly ascertainable, since it is relatively impermeable to the cells. Recently, however, Greene and co-workers (7,9) and Ahmed (1) isolated mutants of Escherichia coli K-12 defective in SAM synthetase with lowered levels of SAM and increased levels of methionine. With these mutants, one may examine the possibility that SAM levels regulate one-carbon formation and distribution.…”

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confidence: 99%

Regulation of One-Carbon Biosynthesis and Utilization in Escherichia coli

Meedel

Pizer

1974

J Bacteriol

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The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Escherichia coli K-12 defective in S-adenosylmethionine synthetase. The mutant that was reported to have a low endogenous concentration of S-adenosylmethionine had elevated levels of N-5, 10-methylene tetrahydrofolate reductase and serine transhydroxymethylase, but the level of N-5, 10methylene tetrahydrofolate dehydrogenase was not altered. These results suggest that S-adenosylmethionine plays a role in the regulation of one-carbon production and utilization. An enzyme system that cleaved glycine to one-carbon units was demonstrated. The enzymes responsible for the cleavage of glycine were induced by exogenous glycine but were independent of S-adenosylmethionine or purine levels in the cell.The regulation by methionine of several enzymes involved in one-carbon metabolism has been demonstrated by several authors (1,5,10,11,16,26). Generally, their approach was to use methionine-cyanocobalamin (B 12) auxotrophs, whose requirement for methionine was furnished by cyanocobalamin. By increasing the concentration of methionine in the medium, they were able to demonstrate a corresponding decrease in levels of certain enzymes involved in one-carbon metabolism, especially methionine biosynthesis. However, these methods do not distinguish between the effect of methionine and S-adenosylmethionine (SAM) levels, which presumably were increased with increasing methionine concentrations. Unfortunately, the effect of SAM on these enzymes is not directly ascertainable, since it is relatively impermeable to the cells. Recently, however, Greene and co-workers (7,9) and Ahmed (1) isolated mutants of Escherichia coli K-12 defective in SAM synthetase with lowered levels of SAM and increased levels of methionine. With these mutants, one may examine the possibility that SAM levels regulate one-carbon formation and distribution. They may also provide the means to resolve the apparent contradictions between the results of Mansouri et al. (16) and Taylor et al. (26) as to whether the supply of methionine controls the levels of serine transhydroxymethylase (EC 2.1.2.1) and obtain information on the interrelationship between the metabolism of one-carbon units and methionine. Our studies with these mutants showed a pronounced effect of SAM levels on N-5, 10-methylene tetrahydrofolate reductase (EC 1.1.1.68), a much smaller effect on serine transhydroxymethylase, and no effect on N-5, 10-methylene tetrahydrofolate dehydrogenase (EC 1.5.1.5). These results indicate that SAM rather than methionine plays a role in controlling serine transhydroxymethylase levels. They also confirm the results of Kung et al. that N-5, 10-methylene tetrahydrofolate reductase levels are regulated by SAM rather than by methionine (12).As suggested by earlier workers, an alternative means of generating one-carbon units is by cleavage of glycine (23). Serine auxotrophs which cannot synthesize serine via the normal phosphorylated pathway may have their requirement for serine su...

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Regulation of S -Adenosylmethionine Synthetase in Escherichia coli

Cited by 83 publications

References 32 publications

Studies on the role of the metK gene product of Escherichia coli K‐12

Studies on the role of the metK gene product of Escherichia coli K‐12

Two‐carbon folate cycle of commensalLactobacillus reuteri6475 gives rise to immunomodulatory ethionine, a source for histone ethylation

Regulation of One-Carbon Biosynthesis and Utilization in Escherichia coli

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