Formation of DNA methylation patterns: nonmethylated GATC sequences in gut and pap operons - PubMed (original) (raw)

Formation of DNA methylation patterns: nonmethylated GATC sequences in gut and pap operons

M van der Woude et al. J Bacteriol. 1998 Nov.

Abstract

Most of the adenine residues in GATC sequences in the Escherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44. 5) centered 44.5 bp upstream of the transcription start site of the gutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44. 5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of the pap GATC-I and GATC-II sites.

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Figures

FIG. 1

FIG. 1

In vitro methylation protection. Squares represent the percentage of DNA in a complex with protein, as determined by EMSA, and circles represent the percentage of methylated DNA, as determined by an in vitro methylation protection assay (see Materials and Methods). (A) Data obtained with pap regulatory region DNA and purified Lrp. (B) Data obtained with gutABD regulatory region DNA and CAP. (C) Data obtained with gutABD regulatory region DNA and GutR.

FIG. 2

FIG. 2

DNA methylation pattern analyses. The DNA methylation states of GATC-44.5 of the gutABD operon in isolate DL1882 (lanes 1 to 6) and isogenic gutR isolate DL2803 (lanes 7 to 12) were examined as described in Materials and Methods. Odd-numbered lanes show DNAs digested with _Bam_HI and _Acc_65I, and even-numbered lanes show DNAs digested with _Bam_HI, _Acc_65I, and _Mbo_I. DNA was isolated from cultures grown with glucose (lanes 1, 2, 7, and 8), glycerol (lanes 3, 4, 9, and 10), or glucitol (lanes 5, 6, 11, and 12) in the logarithmic phase of growth. DNA fragments A and E were from the gutA′-lacZ fusion, and DNA fragments B, C, and D were from the wild-type gutABD operon.

FIG. 3

FIG. 3

DNase I footprint analyses. (A and B) The DNase I footprint of the top strand of gutABD regulatory region DNA with CAP (A) or GutR (B) is shown. Results obtained with nonmethylated gutABD regulatory region DNA (A, lanes 1 to 7; B, lanes 1 and 2) and methylated gutABD regulatory region DNA (A, lanes 8 to 14; B, lanes 3 and 4) are shown. In panel A, the relative levels of CAP added are shown, with 1 being equal to 280 nM. In panel B, a level of GutR sufficient for binding saturation was used. In panel A, lanes 2 and 9, the asterisk indicates that the reactions took place in the presence of 840 nM CAP but in the absence of cAMP. (C) Protection by GutR and CAP of the top DNA strand. Open circles below a base designate DNase I-hypersensitive sites, and thin lines designate protection from DNase I digestion. The asterisk indicates the base (G−46) that was sensitive in methylated DNA but not in nonmethylated DNA. The GATC-44.5 sequence is boxed, and the predicted CAP binding site is underlined with a thick line (30). The numbering of the gut DNA is that of Yamada and Saier (32).

FIG. 3

FIG. 3

DNase I footprint analyses. (A and B) The DNase I footprint of the top strand of gutABD regulatory region DNA with CAP (A) or GutR (B) is shown. Results obtained with nonmethylated gutABD regulatory region DNA (A, lanes 1 to 7; B, lanes 1 and 2) and methylated gutABD regulatory region DNA (A, lanes 8 to 14; B, lanes 3 and 4) are shown. In panel A, the relative levels of CAP added are shown, with 1 being equal to 280 nM. In panel B, a level of GutR sufficient for binding saturation was used. In panel A, lanes 2 and 9, the asterisk indicates that the reactions took place in the presence of 840 nM CAP but in the absence of cAMP. (C) Protection by GutR and CAP of the top DNA strand. Open circles below a base designate DNase I-hypersensitive sites, and thin lines designate protection from DNase I digestion. The asterisk indicates the base (G−46) that was sensitive in methylated DNA but not in nonmethylated DNA. The GATC-44.5 sequence is boxed, and the predicted CAP binding site is underlined with a thick line (30). The numbering of the gut DNA is that of Yamada and Saier (32).

FIG. 3

FIG. 3

DNase I footprint analyses. (A and B) The DNase I footprint of the top strand of gutABD regulatory region DNA with CAP (A) or GutR (B) is shown. Results obtained with nonmethylated gutABD regulatory region DNA (A, lanes 1 to 7; B, lanes 1 and 2) and methylated gutABD regulatory region DNA (A, lanes 8 to 14; B, lanes 3 and 4) are shown. In panel A, the relative levels of CAP added are shown, with 1 being equal to 280 nM. In panel B, a level of GutR sufficient for binding saturation was used. In panel A, lanes 2 and 9, the asterisk indicates that the reactions took place in the presence of 840 nM CAP but in the absence of cAMP. (C) Protection by GutR and CAP of the top DNA strand. Open circles below a base designate DNase I-hypersensitive sites, and thin lines designate protection from DNase I digestion. The asterisk indicates the base (G−46) that was sensitive in methylated DNA but not in nonmethylated DNA. The GATC-44.5 sequence is boxed, and the predicted CAP binding site is underlined with a thick line (30). The numbering of the gut DNA is that of Yamada and Saier (32).

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