PubMed · 12555080
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Ditlev E Brodersen, V Ramakrishnan. 2003. Shape can be seductive.. https://doi.org/10.1038/nsb0203-78
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Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogeneic 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 their expression 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 that is enriched in the locus of enterocyte effacement (LEE⁺) E. coli strains and present in up to nine copies in EHEC and Enteropathogenic Escherichia coli (EPEC) genomes. HnrS base-pairs with the hha ribosome-binding site to inhibit translation, thereby modulating Hha-H-NS repression of virulence loci including the LEE type III secretion system. Loss of HnrS alters motility, T3SS expression, and a subset of Hha-regulated genes. These findings reveal an RNA-based counter-silencing strategy encoded by prophage to relieve xenogenic silencing.
The mechanisms by which cells respond to growth inhibitory redox-cycling agents is only partially understood. In Escherichia coli K12, the IscR regulon, which includes the ISC and SUF Fe-S cluster biogenesis machineries, is differentially expressed in response to these agents. Here, we report how one redox-cycling agent, phenazine methosulfate (PMS), regulates IscR activity via its [2Fe-2S] cluster cofactor. A direct role for IscR in mediating the response to PMS was inferred from the PMS-dependent weakening of [2Fe-2S]-IscR binding to an isc operon type 1 DNA site in vitro. This decrease in DNA binding was attributed to the accompanying oxidation of its [2Fe-2S]1+ cluster. Exposure of anaerobic cultures to PMS leads to increased isc expression, as expected from IscR cluster oxidation and impaired binding to type 1 sites in the isc promoter. However, this same anaerobic PMS treatment did not change expression of type 2 site promoters, such as suf, which require IscR that lacks an Fe-S cluster (apo-IscR) for effective transcriptional regulation. In contrast, PMS exposure under aerobic conditions significantly increased both isc and suf expression, indicating the formation of both [2Fe-2S]2+-IscR and apo-IscR. This effect was partially attributed to superoxide generation by PMS under aerobic conditions, as evidenced by a superoxide dismutase-deficient mutant showing a modest impact on isc and suf expression. Together, these findings provide new insights into redox-cycling dependent regulation of IscR activity and highlight the distinct activities of apo-IscR, [2Fe-2S]2+-IscR and [2Fe-2S]1+-IscR in controlling the IscR regulon.
Purine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2'-deoxypurine nucleosides to the free base and ribose (or 2'-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4'- and 5'-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency.