05. Epigenetic regulation of bacterial gene expression (including virulence factors)

1. DNA Methylation regulates Initiation of Replication

Initiation of Chromosome replication requires protein DnaA to bind to oriC

DnaA binds when multiple GATC sites at oriC are fully methylated After replication GATC is hemi-methylated

SeqA protein binds hemi-methylated GATC

SeqA binding inhibits methylation – prevents new replication cycle beginning

Only when SeqA dissociates (after a some time) can Dam complete methylation

This allows DnaA to bind and initiate a new replication cycle.

Methylation controls the binding of DnaA to OriC, which controls the initiation of new DNA replication

2. DNA Methylation directs DNA Mismatch Repair

Mistake during replication, Mismatched bases – which one is the mutant?

Most chromosomal GATC sites in E. coli are fully methylated.

But immediately after DNA replication the two new dsDNA strands are each hemi-methylated (see figure above)

If the mismatch repair system (MutSLH) detects a mutation then the new DNA (non-methylated strand) is removed and re-synthetised.

Methylation distinguishes OLD DNA (correct) from NEW DNA (mutant).

< 5 min after replication Dam methylates the non-methylated GATC site, regenerating a fully methylated GATC site.

3. Role of DNA Methylation in Genome Defence

R-M (Restriction-Modification) systems are an important bacterial defence against foreign DNA (e.g. phage).

Bacteria modify specific sequences on their own DNA (recognise self) and also produce a restriction enzyme that cuts unmodified sequences (foreign DNA)

4. Role of DNA Methylation in Gene Regulation

Methylation influences protein binding to DNA (e.g., RNA polymerase or other transcriptional regulators) thus regulating gene expression

Dam (deoxy adenine methylase) methylates GATC → GA*TC

GATC may be present within the binding site for a regulatory protein Regulatory regions often have multiple GATC sites

  1. Regulator Protein binding can prevent methylation

  2. Methylation can prevent regulator protein binding

  3. New DNA (directly after replication) is initially hemi-methylated

  4. Competition for GATC between Dam and potential regulator protein

    • If Dam wins the GATC will be methylated

    • If the regulator protein wins the GATC remains hemi-methylated

    The remainder of this lecture contains different examples of gene expression regulated by methylation state (epigenetic regulation)

Principle of Epigenetic control of gene expression → Different phenotypes can result: Example: gene expressed or not expressed

Epigenetic Regulation of Gene Expression - Overview

Two protein-binding sites, each containing a GATC (at or near promoter region).

Two different binding patterns are possible (State A, State B), each prevents methylation of a specific GATC site.

If non-methylation increases the affinity of the protein for its binding site, this creates a positive feedback loop that stabilizes the DNA methylation pattern and makes it heritable.

The system can be reset when hemi methylated DNA substrates are formed upon DNA replication.

Switching occurs if the protein moves from one cognate site to the other (it may have greater affinity for one site over the other).

Ancillary factors can skew switching, or it may be stochastic, or in response to environmental signals.

Methylation influences protein binding → Protein binding influences promoter activity

Epigenetic Regulation of Virulence Gene Expression

Example: pap fimbrial operon in E. coli

P yelonephritis-associated pili

Pap17 pilus phase variation of uropathogenic E. coli visualized with anti-Pap17 antibodies labelled with 10-nm colloidal gold particles.

The bacterium at the left is in the ON-phase for Pap-17 expression. The two bacteria at the right are in the OFF phase.

  • No change in DNA nucleotide sequence

  • Changes in methylation of DNA sequences

    • in the regulatory regions of the pap operon

Epigenic regulation of the pap fimbrial operon in E. coli

Pap fimbrial adhesin is a virulence factor that binds to the host mucosa.

Expression is controlled by phase variation (ON / OFF)

The OFF→ON switching frequency is 5.5 × 10−4 per cell and generation (LB)

The ON→OFF switching frequency is 2.3 × 10−2 per cell and generation (LB)

Switching frequencies are skewed by environmental inputs via Crp and H-NS

Determined by the formation of alternative nucleoprotein complexes that either activate ('on') or suppress ('off') transcription of the pap operon.

Nucleoprotein complexes protect GATC sites from Dam methylation.

Complex formation is inhibited by methylation of the GATC sites. Subject to both positive and negative feedback control.

Epigenic regulation of the pap fimbrial operon in E. coli

Top. Methylation patterns associated with the OFF and ON states. Nonmethylation of GATCproximal in the OFF state, and of GATCdistal in the ON state is a consequence of Lrp binding. Bottom. Feedback loops propagate the OFF and ON states of the pap operon. 1. Binding of Lrp to GATCproximal reduces Lrp affinity for GATCdistal propagating the OFF state. 2. Synthesis of PapB boosts PapI synthesis, and propagates the ON state