Establishment And Applications Of A New Method For Prophage Induction Based On Microcalorimetry

Lysogenic bacteria exist widely in nature and play an important role in different ecosystems.Once lysogenic bacteriophages are activated,they affect biogeochemical cycles by releasing nutrients and bacteriophages that will lysis bacteria.As a result,they can alter the composition and structure of microbial communities.Prophages can be activated by a variety of chemicals.Therefore,it is very important to evaluate the prophage inducing activities of chemicals,especially those that may be released into the environment.Traditional detection methods all have certain limitations,so it is urgent to establish a rapid,simple,accurate and efficient method to detect the prophage inducing activities of chemicals.In addition,the nano materials because of their excellent optical and electrical properties are widely used in many fields.Numerous studies have focused on the adverse effect on ecosystem health after the release of nanomaterials into the soil and water.Many studies have revealed the negative effect of nanomaterials on bacterial growth and biodegradation process,and toxicity targets have defined.However,the potential impact of nanomaterials on ecosystems by activating prophage has been largely ignored.In this thesis,based on microcalorimetry,we have established a rapid and accurate method for the detection of prophage inducing activities of chemicals.Then,the sensitivity of the method was improved by changing the detection strain from single lysogenic bacteria to mixed bacteria.Moreover,the prophage inducing activities of triazine Ag(I)complexes,two different surface ligands CdTe quantum dots and three different sizes of GOs with illumination were measured,and the inducing mechanism was studied by a series of cell biological methods.These results not only provide a method for rapid and accurate detection of prophage inducing activities of chemicals,but also provide a theoretical basis for the application and biosafety evaluation of nanomaterials.This thesis consists of six chapters.Chapter 1:This chapter introduced a more comprehensive overview of lysogenic,the application of calorimetry in the life process and the biosafety of nanomaterials.Chapter 2:We developed a fast and reliable method for detecting the prophage inducing properties of chemicals.The proposed method is based on the difference of metabolic heat between E.coli(?+)and E.coli(?-)under the same chemicals.Compared with anaerobic calorimetry,anaerobic calorimetry is more suitable for the determination of prophage inducing activities of different chemicals.The reliability of this method was confirmed by detecting the prophage inducing activities of a series of chemicals.The main advantages of this method are i)real-time monitoring of the activation and phage production process,ii)a fast analysis time of only a few hours,iii)no need for sample preparation,iv)the suitability to automation and parallel measurements,and v)the use of already available multi-channel instruments.Chapter 3:The sensitivity of the detection method was improved by changing the detection strain from single lysogenic bacteria to mixed bacteria.The detection sensitivity improved was demonstrated by detecting a series of inductive chemicals.The calorimetry method of solid medium with oxygen and liquid medium without oxygen,both can be used to detect prophage inducing activities of chemicals.By comparing the different calorimetry method,we found that the liquid without oxygen calorimetry test has higher detection sensitivity.Chapter 4:The prophage inducing activities of triazine Ag(I)complexes was detected.Moreover the activity of SOD and the amount of MDA in bacterial cells was detected.It was confirmed that the main reason for the prophage inducing activities was the increased oxidation level in bacteria.Chapter 5:Water soluble CdTe QDs with an average size of?2 nm coated with mercaptopropionic acid(MPA)and glutathione(GSH)were prepared and characterized.Prophage inducing activities of the CdTe QDs were investigated by growth curve analysis,plaque-forming unit measurements and calorimetric methods.Our data indicated that the QDs can break the lysogenic cycle into lytic development in response to DNA damage.The oxidative stress induced by the CdTe QDs acted as the major reason for the oxidative damage to the bacterial DNA,while the contribution of Cd²⁺release could also not be neglected.The prophage induction activity of the two CdTe QDs could be greatly inhibited with the addition of ROS scavenger.Alleviating performance for metal ion chelator were relative weaker.The prophage induction activity of MPA-CdTe QDs was significant stronger than that of GSH-CdTe QDs,due to the significant higher oxidative stress induced by MPA-CdTe QDs.Chapter 6:We investigated the size-dependency of GOs on the activation of silent viruses inside of bacteria.We found that photoexcited smaller GOs could induce lysogenic transition,while the large one could hardly activate prophage.The difference in prophage inducing activities of the GOs was attributed to their different interaction modes with bacterial membrane.The smaller GOs could penetrate into the membrane and raise the cellular oxidative stress by photo-generated ROS.The larger GO mainly contact with the membrane by basal planes,which could not increase the cellular ROS level.