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. 2001 Feb;183(3):921-7.
doi: 10.1128/JB.183.3.921-927.2001.

Lowering S-adenosylmethionine levels in Escherichia coli modulates C-to-T transition mutations

G Macintyre  1 , C V Atwood, C G Cupples
Affiliations

Lowering S-adenosylmethionine levels in Escherichia coli modulates C-to-T transition mutations

G Macintyre et al. J Bacteriol. 2001 Feb.

Abstract

Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro either directly by enzyme-induced deamination of cytosine to uracil in the absence of the methyl donor, S-adenosylmethionine (SAM), or indirectly through spontaneous deamination of [5-methyl]cytosine to thymine. Using a Lac reversion assay, we investigated the contribution of the first mechanism to Dcm mutagenesis in vivo by lowering the levels of SAM. Escherichia coli SAM levels were lowered by reducing SAM synthetase activity via the introduction of a metK84 allele or by hydrolyzing SAM using the bacteriophage T3 SAM hydrolase. The metK84 strains exhibited increased C-to-T mutagenesis. Expression of the T3 SAM hydrolase gene, under the control of the arabinose-inducible P(BAD) promoter, effectively reduced Dcm-mediated genomic DNA methylation. However, increased mutagenesis was not observed until extremely high arabinose concentrations were used, and genome methylation at Dcm sites was negligible.

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Figures

FIG. 1
FIG. 1
Pathways to mutation at C · G.
FIG. 2
FIG. 2
Number of Lac revertants per 108 cells (plus standard error of the mean) resulting from C-to-T transition mutations at a Dcm recognition site, CCAGG, in an episomally located lac gene, in various genetic backgrounds. CC500 (wild type), CC502 (metK84), CC503 (vsr), CC505 (vsr metK84), CC504 (ung), CC507 (ung metK84), CC506 (ung vsr), CC508 (ung vsr metK84). Six replicate cultures for each strain were analyzed in triplicate.
FIG. 3
FIG. 3
Effect of the dcm-6 allele on VSP repair. Five-microliter aliquots of saturated bacterial cultures were spotted onto papillation medium. Columns: A, CC112; B, CC112 Δdcm vsr; C, CC112 dcm-6. Strains containing plasmids: row 1, pACYC184; row 2, pDV101; row 3, pDV102.
FIG. 4
FIG. 4
Dcm methylation of genomic DNA is reduced upon induction of T3 SAM hydrolase. (A) MvaI, a methylation-insensitive enzyme, cleaves at Dcm sites in genomic DNA from CC509 containing pBAD24 (lanes 1 to 6) or pBADT3SH (lanes 7 to 12). (B) EcoRII, a methylation-sensitive enzyme, does not cut DNA from CC509 containing pBAD24 (lanes 1 to 6). EcoRII cleaves at unmethylated Dcm sites in CC509 overexpressing T3SH (lanes 7 to 12). Lane U, typical sample of uncut DNA. (A and B) Arabinose concentrations: Lanes 1 and 7, 0 mM (plus 0.2% glucose); lanes 2 and 8, 1.3 μM; lanes 3 and 9, 13 μM; lanes 4 and 10, 130 μM; lanes 5 and 11, 1.3 mM; lanes 6 and 12, 13.3 mM.
FIG. 5
FIG. 5
The effect of reduced SAM levels on C-to-T transition mutations. A T3SH-containing plasmid was compared to a pBAD24 control plasmid under various induction conditions. Lac revertants were measured per 108 cells (± standard error of the mean) due to C-to-T transition mutations at a Dcm recognition site, CCAGG, in an episomally located lac gene in two strains, CC509 vsr Δara and CC510 vsr ung Δara. Four replicate cultures were assayed in triplicate. CC509/pBAD24 ⧫; CC509/pBAD-T3SH ◊; CC510/pBAD24 ■; CC510/pBAD-T3SH □.
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