Dam- and OxyR-dependent phase variation of agn43

Essential elements and evidence for a new role of DNA methylation

Anu Wallecha, Vincent Munster, Jason Correnti, Teresa Chan, Marjan Van der Woude

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Phase variation of the outer membrane protein Ag43 in E. coli requires deoxyadenosine methylase (Dam) and OxyR. Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene, agn43, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding. Here we report further characterization of agn43 transcription and its regulation. Transcription was initiated from a σ7O-dependent promoter at the G residue of the upstream GATC sequence. Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription. Analyses of transcription in vivo of agn'-lacZ with mutated Dam target sequences support this conclusion. Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating agn43 transcription. In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR. Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the agn43 regulatory region from -16 to +42, which includes the three GATC sequences. Deletion analyses of the regulatory region showed that a 101-nucleotide region of the agn43 regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation.

Original languageEnglish (US)
Pages (from-to)3338-3347
Number of pages10
JournalJournal of Bacteriology
Volume184
Issue number12
DOIs
StatePublished - 2002

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DNA Methylation
Nucleic Acid Regulatory Sequences
Methylation
Deoxyribonuclease I
DNA-Directed DNA Polymerase
DNA-Directed RNA Polymerases
Digestion
Membrane Proteins
Nucleotides
2'-deoxyadenosine
Escherichia coli
Genes
In Vitro Techniques

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Dam- and OxyR-dependent phase variation of agn43 : Essential elements and evidence for a new role of DNA methylation. / Wallecha, Anu; Munster, Vincent; Correnti, Jason; Chan, Teresa; Van der Woude, Marjan.

In: Journal of Bacteriology, Vol. 184, No. 12, 2002, p. 3338-3347.

Research output: Contribution to journalArticle

Wallecha, Anu ; Munster, Vincent ; Correnti, Jason ; Chan, Teresa ; Van der Woude, Marjan. / Dam- and OxyR-dependent phase variation of agn43 : Essential elements and evidence for a new role of DNA methylation. In: Journal of Bacteriology. 2002 ; Vol. 184, No. 12. pp. 3338-3347.
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