RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coli
ABSTRACT Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-sile...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-12-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.02662-22 |
| _version_ | 1828262946257502208 |
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| author | Christine M. Hustmyer Michael B. Wolfe Rodney A. Welch Robert Landick |
| author_facet | Christine M. Hustmyer Michael B. Wolfe Rodney A. Welch Robert Landick |
| author_sort | Christine M. Hustmyer |
| collection | DOAJ |
| description | ABSTRACT Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters, but how H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA strand sequence called operon polarity suppressor (ops). RfaH–ops interactions establish long-lived RfaH–EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify eight RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination, whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS–binding features, explaining minor-paralog targeting. IMPORTANCE Bacterial pathogens adapt to hosts and host defenses by reprogramming gene expression, including by H-NS counter-silencing. Counter-silencing turns on transcription initiation when regulators bind to promoters and rearrange repressive H-NS nucleoprotein filaments that ordinarily block transcription. The specialized NusG paralog RfaH also reprograms virulence genes but regulates transcription elongation. To understand how elongation regulators might affect genes silenced by H-NS, we mapped H-NS, StpA (an H-NS paralog), RfaH, σ70, and RNA polymerase (RNAP) locations on DNA in the uropathogenic E. coli strain CFT073. Although H-NS–StpA filaments bind only 18% of the CFT073 genome, all loci at which RfaH binds RNAP are also bound by H-NS–StpA and are silenced when RfaH is absent. Thus, RfaH represents a distinct class of counter-silencer that acts on elongating RNAP to enable transcription through repressive nucleoprotein filaments. Our findings define a new mechanism of elongation counter-silencing and explain how RfaH functions as a virulence regulator. |
| first_indexed | 2024-04-13T04:03:37Z |
| format | Article |
| id | doaj.art-b1f0eae1456c4204903bd54899b2728e |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-13T04:03:37Z |
| publishDate | 2022-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-b1f0eae1456c4204903bd54899b2728e2022-12-22T03:03:23ZengAmerican Society for MicrobiologymBio2150-75112022-12-0113610.1128/mbio.02662-22RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coliChristine M. Hustmyer0Michael B. Wolfe1Rodney A. Welch2Robert Landick3Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, USADepartment of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, USADepartment of Medical Microbiology and Immunology, University of Wisconsin–Madison, Madison, Wisconsin, USADepartment of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, USAABSTRACT Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters, but how H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA strand sequence called operon polarity suppressor (ops). RfaH–ops interactions establish long-lived RfaH–EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify eight RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination, whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS–binding features, explaining minor-paralog targeting. IMPORTANCE Bacterial pathogens adapt to hosts and host defenses by reprogramming gene expression, including by H-NS counter-silencing. Counter-silencing turns on transcription initiation when regulators bind to promoters and rearrange repressive H-NS nucleoprotein filaments that ordinarily block transcription. The specialized NusG paralog RfaH also reprograms virulence genes but regulates transcription elongation. To understand how elongation regulators might affect genes silenced by H-NS, we mapped H-NS, StpA (an H-NS paralog), RfaH, σ70, and RNA polymerase (RNAP) locations on DNA in the uropathogenic E. coli strain CFT073. Although H-NS–StpA filaments bind only 18% of the CFT073 genome, all loci at which RfaH binds RNAP are also bound by H-NS–StpA and are silenced when RfaH is absent. Thus, RfaH represents a distinct class of counter-silencer that acts on elongating RNAP to enable transcription through repressive nucleoprotein filaments. Our findings define a new mechanism of elongation counter-silencing and explain how RfaH functions as a virulence regulator.https://journals.asm.org/doi/10.1128/mbio.02662-22bacterial chromatinChIP-seqcounter-silencinggene silencingH-NSRNAP |
| spellingShingle | Christine M. Hustmyer Michael B. Wolfe Rodney A. Welch Robert Landick RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coli mBio bacterial chromatin ChIP-seq counter-silencing gene silencing H-NS RNAP |
| title | RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coli |
| title_full | RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coli |
| title_fullStr | RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coli |
| title_full_unstemmed | RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coli |
| title_short | RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coli |
| title_sort | rfah counter silences inhibition of transcript elongation by h ns stpa nucleoprotein filaments in pathogenic escherichia coli |
| topic | bacterial chromatin ChIP-seq counter-silencing gene silencing H-NS RNAP |
| url | https://journals.asm.org/doi/10.1128/mbio.02662-22 |
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