Effects Of Metformin On The Chemotaxis And Motility Of Escherichia Coli And Its Mechanism Exploration

In the natural circumstances,microorganisms could change their behavior in response to the change of the external environment,which is a significant condition for their survival.However,the process of microbial behavior change is completed by the coordination of chemotactic signal transduction network and motor structure in vivo,which is very complex.Escherichia coli is used as the research object,and comprehensively use multifarious biophysical methods such as optical microscopic imaging,genetic modification techniques and biological statistical models to study the process of behavior change of E.coli at the molecular and cellular levels,including chemotaxis and motility.Studying the process of behavioral change in E.coli is an important scientific issue for basic research,and it is also conducive to developing application scenarios.The chemotactic motility of bacteria may affect human health and lead to some diseases.On the one hand,studying the above behavior changes of bacteria will enrich the theoretical knowledge of microbiology.On the other hand,it will provide new research ideas on the therapy of bacterial diseases.The study of the dynamic structure of bacteria will facilitate the design and application of molecular machines in the future.Metformin is a biguanide family molecule which considered to be the most prescribed drug therapy for the treatment of type 2 diabetes and other metabolic syndrome for the reason that it is relative safe,low cost,and has beneficial effects on cardiovascular mortality and blood glucose.Metformin has the potential benefits in reducing the risk of cancer in mammalians and promoting lifespan in nematode Caenorhabditis elegans by altering gut microbial metabolism.So we wonder if metformin would affect some of the behavioral characteristics of E.coli,including chemotaxis and motility.Based on the above considerations,we used bead assay and microfluidic technology to explore the effects of metformin at concentrations ranging from 0 m M to 20 m M on the chemotaxis and motor behavior of E.coli.The research results mainly consist of several points:(1)We investigated the growth of E.coli in medium containing different concentrations of metformin.In the first step,we constructed the E.coli strains required by the relevant experiments through genetic modification technology.Strains that could express plasmid p KAF131 were successively prepared: JY26,JY27,HCB429 and HCB1.In the second step,to monitor the change of optical density(OD)value of E.coli by adding various concentrations of metformin in TB medium,it was found that metformin could inhibit the growth of E.coli,and the inhibition was dose-dependent.(2)We investigated the chemotactic changes of E.coli in the presence of metformin at different concentrations.In the first step,we built a microfluidic device.In the second step,strains that could express plasmid p KAF131 were successively prepared: JY26,JY27,HCB429 and HCB269.We cut the long flagella of the bacteria to make them shorter,and then attached latex beads with a radius of 0.5 μm to the short flagella of E.coli.The motility of the beads were monitored by switching the direction of the triangle valve to switch different solutions.We found that metformin was sensed by Tsr chemoreceptor as a repellent to E.coli and that E.coli mutants without chemoreceptors and Che Y protein could not sense metformin,and we also found that metformin prolonged the recovery time of E.coli chemotactic response to attractant.(3)We investigated the changes of motility of E.coli in the presence of metformin at different concentrations.In the first step,we designed and built the experimental equipment of bright field and dark field.In the second step,strains that could express plasmid p KAF131 were successively prepared: JY26 and JY27.Strain JY26 carry plasmids p FD313 and p JY7,strain JY26 carry plasmids p FAF131 and p Trc99 a PR,strain HCB1 carry plasmid p JY7 and strain HCB1 were constructed.Finally,the rotation of the latex beads attached to the E.coli flagellar motor was measured by an inverted light microscope,and the angular rotation of the cell head and flagella of E.coli was odserved by a high throughput darkfield flicker microscopy.We found that metformin inhibited the motility of E.coli and facilitated the accumulation of E.coli near the glass surface.We further demonstrated that the motility inhibition is due to reduced torque of the flagellar motor.