Involvement of E. coli 6S RNA in Oxidative Stress Response.

6S RNA, a small non-coding RNA present in almost all bacteria, inhibits transcription via direct binding to RNA polymerase holoenzymes. The mechanism of 6S RNA action was investigated to a large extent in E. coli, however, lack of 6S RNA (ΔssrS) was demonstrated to be unfavorable but not essential for cell survival under various growth conditions. In the present study, we revealed, for the first time, a lethal phenotype of the ΔssrS strain in the presence of high concentrations of H2O2. This phenotype was rescued by complementation of the ssrS gene on a plasmid. We performed comparative qRT-PCR analyses on an enlarged set of mRNAs of genes associated with the oxidative stress response, allowing us to identify four genes known to be involved in this pathway (soxS, ahpC, sodA and tpx) that had decreased mRNA levels in the ΔssrS strain. Finally, we performed comparative proteomic analyses of the wild-type and ΔssrS strains, confirming that ΔssrS bacteria have reduced levels of the proteins AhpC and Tpx involved in H2O2 reduction. Our findings substantiate the crucial role of the riboregulator 6S RNA for bacterial coping with extreme stresses.

The Potential of Telomeric G-quadruplexes Containing Modified Oligoguanosine Overhangs in Activation of Bacterial Phagocytosis and Leukotriene Synthesis in Human Neutrophils.

Human neutrophils are the first line of defense against bacterial and viral infections. They eliminate pathogens through phagocytosis, which activate the 5-lipoxygenase (5-LOX) pathway resulting in synthesis of leukotrienes. Using HPLC analysis, flow cytometry, and other biochemical methods, we studied the effect of synthetic oligodeoxyribonucleotides (ODNs) able to fold into G-quadruplex structures on the main functions of neutrophils. Designed ODNs contained four human telomere TTAGGG repeats (G4) including those with phosphorothioate oligoguanosines attached to the end(s) of G-quadruplex core. Just modified analogues of G4 was shown to more actively than parent ODN penetrate into cells, improve phagocytosis of Salmonella typhimurium bacteria, affect 5-LOX activation, the cytosol calcium ion level, and the oxidative status of neutrophils. As evident from CD and UV spectroscopy data, the presence of oligoguanosines flanking G4 sequence leads to dramatic changes in G-quadruplex topology. While G4 folds into a single antiparallel structure, two main folded forms have been identified in solutions of modified ODNs: antiparallel and dominant, more stable parallel. Thus, both the secondary structure of ODNs and their ability to penetrate into the cytoplasm of cells are important for the activation of neutrophil cellular effects. Our results offer new clues for understanding the role of G-quadruplex ligands in regulation of integral cellular processes and for creating the antimicrobial agents of a new generation.