Abstract
Hfq is a bacterial master regulator which promotes the pairing of nucleic acids. Due to the high molecular weight of the complexes formed between nucleic acids and the amyloid form of the protein, it is difficult to analyze solely by a gel shift assay the complexes formed, as they all migrate at the same position in the gel. In addition, precise kinetics measurements are not possible using a gel shift assay. Here, we used a synchrotron-based biophysical approach, synchrotron radiation circular dichroism (SRCD), to probe the interaction of the Escherichia coli Hfq C-terminal amyloid region with nucleic acids involved in the control of ColE1-like plasmid replication. We observed that this C-terminal region of Hfq has an unexpected and significant effect on the annealing of nucleic acids involved in this process and, more importantly, on their alignment. Functional consequences of this newly discovered property of the Hfq amyloid region are discussed in terms of the biological significance of Hfq in the ColE1-type plasmid replication process and antibiotic resistance.
Highlights
IntroductionAntibiotic resistance of bacteria has become one of the major medical problems in recent years [1]
Recent investigations demonstrated that perturbations with ColE1-like plasmid functions in E. coli hfq mutants resulted in significantly altered levels of bacterial resistance to different antibiotics that were not related to effects of Hfq on transcripts derived from antibiotic-resistance genes [18]. These results indicated that the effects of Hfq on ColE1-like plasmid biology might be important for medically-significant features of bacteria, like antibiotic resistance
Hfq and its C-terminal region (CTR) can interact with and stabilize G-quadruplex structures [29,30], influence the mechanical properties of the DNA [31], modulate genomic instability [29,30,32] and drive bacterial heterochromatin formation [28,33]. Because of such diverse activities of the CTR, and the previously reported functions of Hfq in the control of ColE1-like plasmid DNA replication, the aim of this work was to investigate precisely, using synchrotron radiation circular dichroism (SRCD), the interactions between RNA and DNA molecules involved in the regulation of replication
Summary
Antibiotic resistance of bacteria has become one of the major medical problems in recent years [1]. The appearance of many bacterial strains resistant to one or many antibiotics makes this crucial therapy used to combat various infectious diseases less and less effective, resulting in alarming predictions on the possible lack of cures in such cases in the near future [2]. The spreading of antibiotic resistance is caused mainly by the overuse of these therapeutics and the selection of resistant strains. Antibiotic-resistant bacteria occur naturally in the environment, predominantly because they bear genes coding for proteins able to inactivate antimicrobial compounds, to prevent the penetration of antibiotics to cells or to remove them from cells, or because of mutations that change the antibiotic targets [3]
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