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Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in

B A Braaten1, L B Blyn, B S Skinner

  • 1Department of Pathology, University of Utah Medical Center, Salt Lake City 84132.

Journal of Bacteriology
|March 1, 1991
PubMed

Insights

A novel methylation-blocking factor (mbf) controls pyelonephritis-associated pilus (pap) operon transcription in Escherichia coli. This mbf locus is essential for pap pilin gene expression and pilus formation, regulating phase variation.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Escherichia coli utilizes phase variation to regulate pyelonephritis-associated pilus (pap) operon transcription, alternating between active (phase-on) and inactive (phase-off) states.
  • This regulation involves differential methylation of GATC sites within the pap operon's regulatory region, specifically GATC1028 and GATC1130.

Purpose of the Study:

  • To identify the genetic factors responsible for regulating pap operon methylation states and controlling phase variation.
  • To elucidate the mechanism by which methylation influences pap pilin gene transcription and pilus expression.

Main Methods:

  • Transposon mutagenesis using mTn10 to identify mutations affecting pap operon regulation.
  • Construction of papBAp-lac operon fusions to quantify pap pilin gene transcription via beta-galactosidase activity.
  • Northern blotting and primer extension analyses to confirm transcriptional changes.
  • Isolation and complementation analysis of the identified locus using cosmid cloning.

Main Results:

  • A new chromosomal locus, mbf (methylation-blocking factor), was identified as essential for protecting both GATC1028 and GATC1130 sites from methylation.
  • Mutations in mbf led to a significant decrease (approximately 30-fold) in pap pilin gene transcription and Pap pilus expression, similar to the phase-off state.
  • Complementation analysis confirmed that all seven isolated mbf mutants contained insertions within the same gene or operon.

Conclusions:

  • The mbf locus is a critical regulator of pap operon transcription and is necessary for maintaining the phase-on state.
  • The findings support a model where pap phase variation is controlled by alternating DNA methylation states, mediated by the mbf locus.
  • mbf plays a crucial role in pilus expression, impacting bacterial virulence mechanisms.

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