High Salt Laboratory Evolutionary Study Of Synechocystis PCC 6803

Experimental evolution is the study of evolutionary processes involving experimental populations in response to conditions imposed by the experimenters.It's an effective method to study how the organisms adapt to the ever changing environments.Cyanobacteria can live in most eco-environments of the earth.It was proposed that cyanobacteria can adapt to the environment with the strategy of “bet hedging”(random mutation),indicating that the different cells/sub-populations under the same experimental stress may use different strategies to survive and adapt.At present,most of the studies in experimental evolution are about Escherichia coli.So far,there's no study on experimental evolution of cyanobacteria under long term high salinity stress(salt stress).As a model blue-green microalga for gene engineering,the genome of unicellular model cyanobacterium Synechocystis sp.strain PCC 6803(hereafter referred to Synechocystis 6803)is very plastic.Synechocystis sp.PCC 6803 was selected as the primer material to study adaptation strategies and evolution directions of cyanobacteria under salt stress.This study started from a monoclonal strain thus all cells have the same genome from the begining.After cultivation,the monoclonal cells were inoculated in BG11 medium(3% NaCl).There are 10 parallel experimental strains in salt containing media and 2 strains are always inoculated in normal BG11 medium as control.The experiment had continued for 435 days,as 43 generations.The influences on cell morphology,physiology and metabolome of Synechocystis6803 strains were investigated.Also,the tolerance mechanisms and possible evolutionary directions of Synechocystis 6803 under salt stress had been studied.The results are listed as following:(1)Cell morphology.(1)The cells of the 3rd,12 th and 43 rd generations did not show damage under salt stress.Their cell diameters increased comparing to those of the controls,and the ratios of the dividing cells of acclimatized strain cells increased.The diameters of the 3rd generation acclimatized strain cells were were significantly bigger than wild type.The cells of the 12 thand 43rd acclimatized strains were smaller than the 3rd generation.The results indicated that the volume of cells for Synechocystis 6803 increased and the cell division was inhibited under salt stress.Furthermore,the diameters of control strains were in normal Gaussian distribution.There was only one major peak in the distribution graph of control cell diameters,while several peaks for the 3rd,12 th and 43 rd generation acclimatized strains.There were probably several subpopulations in each acclimatized strain.(2)Ten acclimatized strains of the 12 th generation and 2 control strains were inoculated in BG11 medium(3% NaCl).The 10 acclimatized strains grew better than the two controls.For instance,the diameters of cells from the two controls increased significantly.In contrast,there was no significant change in the diameters of cells from the 10 acclimatized strains,since no obvious difference was observed in mean diameters among cells from acclimatized strains.(3)Ten acclimatized strains of the 28 th generation were inoculated in normal BG11 medium for 7 days,which made the initial cultivation conditions of the 10 acclimatized strains more similar with the two control strains.Then the 10 acclimatized strains and the 2control strains were inoculated in BG11 medium supplemented with 3% NaCl.The acclimatized strains grew better than the 2 controls.The diameters of the 2 controls increased significantly.There was no visible change in the diameters of the 10 acclimatized strains cells.However,interestingly there were obvious differences in mean diameters between 10 acclimatized strains cells.(2)Growth rate.(1)The growth rates of the 28 th acclimatized strains were bigger than that of the 2controls under salt stress.What's more,there were obvious differences observed between 10 acclimatized strains.(2)After high salt cultivation for 3 days,the growth rates of the 33 rd acclimatized strains were bigger than that of the 2 controls under salt stress.(3)Photosynthetic efficiency.The photosynthetic efficiency(Fv/Fm)of the 28 th acclimatized strains were higher than that of the control.There were visible differences in photosynthetic efficiency(Fv/Fm)among10 acclimatized strains.The trend of Y(II)for 12 strains was the same as Fv/Fm.We speculated that 10 acclimatized strains of Synechocystis 6803 can adapt to high salt stress,and the ability of adaptation may be heritable.There may be some differential adaptation among different parallel acclimatized strains.(4)Metabolome.Ten acclimatized strains of the 33 rd generation were inoculated in BG11 medium with3% NaCl,and the control strains were cultivated in both normal BG11 and BG11 with 3%NaCl.According to the results,there were 59 metabolites detected in the cyanobacteria metabolome.In the graphs of heatmap and PCA,10 acclimatized strains were sub-divided into 6 or more groups.The significant metabolites,glyceryl-glycoside,glycerol and hexadecanoic acid,may be the key metabolites for Synechocystis 6803 to adapt to high salt stress.The synthesis of compatible solutes such as glyceryl-glycoside and the change of composition for fatty acids such as hexadecanoic acid may be important mechanisms for Synechocystis 6803 to adapt to high salt stress.In conclusion,by means of analyzing the influences of salt stress to the cell morphology,physiology and metabolome for Synechocystis 6803,we proposed its tolerance mechanisms and potential differential evolutionary directions under salt stress.It will be helpful for intensive study about experimental evolution of Synechocystis 6803 under salt stress,and understanding better for the extensive adaptation ability of cyanobacteria to survive in the ever-changing environments.