RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS-StpA Nucleoprotein Filaments in Pathogenic...

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Pathogenic *Escherichia coli* utilize virulence genes for host adaptation, with expression often silenced by the transcription silencer H-NS and its paralogs.
• H-NS forms nucleoprotein filaments that inhibit transcription initiation and elongation, posing a challenge for understanding virulence gene regulation.
• Activators can displace H-NS from promoters to counter-silence initiation, but mechanisms for overcoming H-NS-mediated elongation inhibition remain unclear.

Purpose of the Study:

• To investigate how the elongation regulator RfaH overcomes H-NS-mediated silencing of virulence operons in uropathogenic *E. coli* (UPEC).
• To map the genome-wide distribution of key regulatory proteins and RNA polymerase in UPEC to understand their interplay.
• To define the role of RfaH as an elongation counter-silencer and its interaction with H-NS and its paralogs.

Main Methods:

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was employed to map the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ⁷⁰ in UPEC strain CFT073.
• Genetic deletions of RfaH, H-NS, and StpA were created to assess their impact on transcription and gene expression.
• Analysis of RfaH-regulated operons and their interaction with H-NS binding features was performed.

Main Results:

• Eight RfaH-activated operons were identified, all of which were bound by H-NS and StpA, with four representing new additions to the RfaH regulon.
• Deletion of RfaH led to premature transcription termination, while deletion of H-NS and StpA allowed elongation independently of RfaH.
• RfaH acts as an elongation counter-silencer of H-NS, with StpA's DNA bridging activity contributing to H-NS-mediated elongation inhibition.

Conclusions:

• RfaH represents a distinct class of counter-silencer that specifically targets elongating RNA polymerase to enable transcription through repressive nucleoprotein filaments.
• The findings define a novel mechanism of elongation counter-silencing, explaining how RfaH functions as a critical virulence regulator in UPEC.
• Minor H-NS paralogs like Hfp contribute to residual RfaH effects, particularly in operons with strong H-NS binding characteristics.