Sy, B. M. | Search bioRxiv

bioRxiv2025

Phage-encoded sRNA counteracts xenogenic silencing in pathogenic E. coli

Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogenic silencing by nucleoid-associated proteins such as H-NS and Hha. In Enterohaemorrhagic Escherichia coli (EHEC), prophages make up a large fraction of the accessory genome and encode many virulence factors, yet to be expressed they must overcome this silencing. We identify a prophage-encoded small RNA (sRNA), HnrS, that functions as an anti-silencing factor by targeting the H-NS paralogue Hha. HnrS is a short (66-nt) sRNA present in multiple copies (up to nine) in EHEC and Enteropathogenic Escherichia coli (EPEC) genomes and is enriched in E. coli strains that carry the locus of enterocyte effacement (LEE). We show that HnrS directly base-pairs with the ribosome-binding site of the hha mRNA, repressing its translation and thereby reducing Hha-enhanced H-NS silencing. This counter-silencing de-represses the LEE type III secretion system and concomitantly represses motility. Transcriptomic profiling further revealed that HnrS indirectly activates genes involved in nitrate/nitrite respiration and nitric oxide resistance, metabolic pathways that contribute to survival in the inflamed gastrointestinal tract. Deletion of hnrS reduced expression of nitrate reductase genes and impaired actin pedestal formation on host epithelial cells. Our results indicate that prophage-encoded, multicopy hnrS provides a counter-silencing mechanism that reduces Hha-H-NS repression at specific virulence loci. This likely enables expression of horizontally acquired genes without broadly disrupting the core H-NS regulon. HnrS illustrates how mobile genetic elements deploy sRNAs to counteract xenogenic silencing and promote virulence gene expression, enhancing colonisation of the host. Importance statementHorizontally acquired genes are often silenced to protect bacterial genomes, but this defence also limits the expression of new traits. We identify a prophage-encoded small RNA, HnrS, that counteracts this restriction by repressing the xenogenic silencer Hha, lifting Hha-H-NS-mediated repression of virulence and metabolic genes. HnrS activates the locus of enterocyte effacement, nitrate/nitrite respiration, and nitric oxide resistance--pathways that help E. coli survive and colonise the inflamed gut. Our findings reveal an RNA-based counter-silencing mechanism encoded by mobile genetic elements, showing how phages can reprogram bacterial regulatory networks to promote adaptation and pathogenicity.

microbiology↗

bioRxiv2021

Enterohaemorrhagic E. coli utilizes an AND-OR logic gate to regulate expression of an outer membrane haem receptor

To sense the transition from environment to host, bacteria use a range of environmental cues to control expression of virulence genes. Iron is tightly sequestered in host tissues and in the human pathogen enterohaemorrhagic E. coli (EHEC) iron-limitation induces transcription of the outermembrane haem transporter encoded by chuAS. ChuA expression is post-transcriptionally activated at 37{degrees}C by a FourU RNA thermometer ensuring that the haem receptor is only expressed under low iron, high temperature conditions that indicate the host. Here we demonstrate that expression of chuA is also independently regulated by the cAMP-responsive sRNA CyaR and transcriptional terminator Rho. These results indicate that chuAS expression is regulated at the transcription initiation, transcript elongation, and translational level. The natural dependence of these processes creates a hierarchy of regulatory AND and OR logic gates that integrate information about the local environment. We show that the logic of the chuA regulatory circuit is activated under conditions that satisfy low iron AND (low glucose OR high temperature). We speculate that additional sensing of a gluconeogenic environment allows further precision in determining when EHEC is at the gastrointestinal epithelium of the host. With previous studies, it appears that the chuAS transcript is controlled by eight regulatory inputs that control expression through six different transcriptional and post-transcriptional mechanisms. The results highlight the ability of regulatory sRNAs to integrate multiple environmental signals into a conditional hierarchy of signal input.

microbiology↗