The Effect Of Mechanosensitive Channels On Acoustic Stress Response In Escherichia Coli

Sound waves are widely present in nature and interact with all kinds of biological as an important environmental factor.At present,the research on acoustic biological effect is in-depth.However,the research on how to transform the biological signal from the physical stimulus to the intracellular biological signal,and then induce the intracellular cascade effect is relatively scarce.In this paper the single cell prokaryotes of E.coli was treated with calcium channel inhibitor,mechanosensitive channel?Msc?inactivation and transcriptome analysis to study the effect of calcium ions in the acoustic stress,analyze the function of Msc in the mechanical signal transduction,and the effects of acoustic exposure on energy metabolism and intracellular cell metabolic network structure.This study made a great contribution to understand the adjustment and adaptation mechanism of the organism which including the microorganism.It plays a guiding role in the development and utilization of sound biological effect.The main results of this study are as follows:1.The effects of sound exposure on the growth of population were experiment at8000 Hz and 80 Db by using E.coli K12,E.coli Jw3252??MscL?and E.coli Jw2891??MscS?as materials.The results showed that,in stationary phase the sound exposured group's bacterial density of the three strains was higher than the control group,and the biomass of K12 was increased by 19.64±0.18%,Jw3252 was increased by 8.47±0.06%and Jw2891 increased by 8.35±0.14%.We speculated that mechanosensitive channels may play an important role in E.coli sound stress,and the deletion of the mechanosensitive channel of large conductance?MscL?and the mechanosensitive channel of small conductance?MscS?can weaken the sound biological effects.These two kinds of mechanosensitive channel may have synergistic effect in acoustic stress.2.A two-channel deletion mutant strain was constructed by using the E.coli knockout kit.The calcium channel inhibitor LaCl3 was added to the medium of K12.Both the two-channel deletion mutant strain and K12 with calcium channel inhibitor LaCl3 were treated with the same sound exposure.The results showed that bacterial concentration in sound treatment group was lower than the control group in these two experiments.The characteristics of secondary logarithmic phase in two-channel deletion mutant E.coli were disappeared and the decline rate of two-channel deletion mutant was about 5.00±0.03%,while the decline rate of treatment group with La Cl3 was16.50±0.13%.The results confirmed that MscL has synergistic reaction with MscS in the process of acoustic transmission,and their deletion may cause the decline in ability of resistance or adaptability to environmental changes.The growth of bacteria was inhibited when the calcium channel was inhibited.3.Fura2-AM fluorescence detection method was used to detect the intracellular Ca²⁺ concentration of wild-type and mutant E.coli.The results showed that intracellular Ca²⁺concentration in the sound exposure group was higher than that in the control group after 12 hours both in wild-type and mutant E.coli,and the content of Ca²⁺ in wild-type E.coli was significantly higher than that in the mutant stain.The decreases tend of intracellular Ca²⁺concentration in the sound exposure group decreased slower than control group after 36 hours.Thus we thought that Msc may be the initial receptor response to sound stress which located on the bacterial membrane.At the same time,Msc mediated the bacterial transmembrane behavior of the calcium signal and induces the bacterial sound stress response.4.Based on GO enrichment and KEGG analysis to study the impact of sound on the metabolic network structure and energy metabolism partten of E.coli K12.The results of gene expression showed that the effect of sound exposure was significant in early phase and stationary phase.At 6 hours,12 hours,24 hours and 36 hours,there were 110,43,108,87 up-regulated genes and 179,37,93,106 down-regulated genes in the treatment group,respectively.The further analysis revealed that the bacteria chemotaxis pathway was significantly increased at the beginning of the acoustic exposure,and it raised the trend of bacterial movement to the favorable environment.When the growth entered the mid-log phase,the acoustic exposure significantly increased the level of glycerol phospholipid metabolism,the rapid division and proliferation of the cells to provide sufficient material and energy protection.When the growth entered into middle exponential phase,it meant that the acoustic exposure significantly increased the glycerol phospholipid metabolism level,and it provided the sufficient material and energy protection for the rapid division and metabolism of the bacteria under sound exposure.When the sound exposure lasted into the late exponential phase,in treatment group the secretion of the extracellular production was lower and the transcriptional translation and nucleic acid synthesis ability were higher than the control group which is prepare for the sustained sound stress response.In the stationary phase,in order to improve the stress resistance and achieve the continuousgrowth of bacteria in treatment group,the ability of treatment group to use complex compounds for energy was stronger than control.