Single Cell Tracking Strategy And The Research Of Bacteria Evolution

Biometric parameters are often indicated with specific population averages for sev-eral years.With these traditional techniques,such as optical density,average intensity of fluorescent proteins,Western blot,and Northern blot,we have gained an in-depth understanding of the process of transcription,translation,and the growth of organisms.However,the traditional technology based on the population mean will "clear" key ki-netic processes and subtle gaps in growth.In the last two decades,the combination of time-lapse microscopy,quantitative image analysis,and fluorescent protein reporter genes has enabled us to directly obeserve multiple bacterial components over time in a single bacterium.Combined with theoretical modeling,these techniques now provide powerful insights into the behavior of genetic circuits in different microbial systems.This paper focus on the research of Pseudomonas aeruginosa,and proposes a novel bac-terial tracking algorithm with the topological tracking and chain segmentation,which eventually results in a highly accurate physical image of the binary tree of bacteria.Using this algorithm,we have done a lot of inductive and summary work on the sur-face twitching motility of P.aeruginosa.At the same time,this algorithm can help us to further explore the distribution of single bacterial phenotypes and the challenges in bacterial evolution.We have done some explorative experiments on the network of horizontal gene transfer and some works of single cell analysis.Details are as follows:Dual colors fluorescent timer enables us to detect persisters:We present a method capable of detecting single slow-growing and growth-arrested cells in a bacterial culture composed of physiologically and phenotypically different cells.Unlike the use of transcriptional reporters to gauge the metabolic activities in cells,here,we fuse two different fluorescent proteins with distinctive maturation rates to construct a timer to directly detennine the growth rate of single Pseudomonas aerug-inosa cells.We demonstrate that the dual-color fluorescent timer can indicate the slow-growing and growth-arrested cells from bacterial cultures in the presence of various environmental stresses,including nutrient starvation or antibiotic treatments,which greatly expand the methods for detecting and isolating persister cells.Conditional privatization of a public siderophore in P.aeruginosa:Understanding the mechanisms that promote cooperative behaviors of bacteria in their hosts is of great significance to clinical therapies.Environmental stress is gener-ally believed to increase competition and reduce cooperation in bacteria.Here,we show that bacterial cooperation can in fact be maintained because of environmental stress.We show that Pseudomonas aeruginosa regulates the secretion of iron-scavenging siderophores in the presence of different environmental stresses,reserving this public good for pri-vate use in protection against reactive oxygen species when under stress.We term this strategy 'conditional privatization'.Using a combination of experimental evolution and theoretical modeling,we demonstrate that in the presence of environmental stress the conditional privatization strategy is resistant to invasion by non-producing cheaters.These findings show how the regulation of public goods secretion under stress affects the evolutionary stability of cooperation in a pathogenic population,which may assist in the rational development of novel therapies.