Phenotypic Plasticity Study Of Staphylococcus Aureus By Dynamic Genomewide Association Analysis

Phenotypic plasticity is the capacity of a single genotype in respond to environmental changes to produce multiple phenotypes,which is ubiquitous in nature.The phenotypic plasticity of plants and animals have been well studied,but less research regarding microbial phenotypic plasticity.Phenotypic plasticity plays an important role in the adaptive evolution of microbiology.Under antibiotic pressure,plasticity genes are likely to affect the resistance evolution of strains.In this study,in order to investigate the phenotypic plasticity of S.aureus under vancomycin,99 strains of S.aureus with different minimum inhibitory concentrations of vancomycin were cultured at vancomycin concentrations of 0 μg/m L,2 μg/m L and 4 μg/m L,respectively.The growth data of all strains within 48 h were collected,and the growth curves and maximum growth rates were plotted.With the increase of vancomycin concentration,the growth patterns of 99 strains was significantly different.The growth of only a few strains was unaffected,and there was no significant difference in their maximum growth rates,showing better phenotypic plasticity.In 4 μg/m L vancomycin-treated group,the maximum growth rate of several strains was significantly reduced,indicating poor phenotypic plasticity.In order to analyze key genes affecting phenotypic plasticity of S.aureus,the whole genome of all strains was sequenced in this study.To locate significant single-nucleotide polymorphisms(SNPs)closely related to phenotypic plasticity,the molecular markers of all strains were correlated with growth data by static genome-wide association analysis(GWAS)based on case-control study and dynamic GWAS based on growth parameters.The significant SNPs obtained from GWAS analysis were genetically annotated.The case-control study was used to analyze the growth of vancomycin at a concentration of 4 μg/m L,and npt A,lip2,ula A,emp,yid J and other genes related to its growth were mapped.Under dynamic GWAS analysis based on growth parameters,some genes that may affect different growth stages of bacteria were located,such as rlu A,fem AB,gln Q,etc.Among them,lip2,ssp A,pro S,and ebh genes are significantly related to the phenotypic plasticity of S.aureus in different analyses,and it is speculated that these genes play an important role in the regulation of phenotypic plasticity of S.aureus.In this study,static GWAS and dynamic GWAS were used to locate significant genes related to phenotypic plasticity of S.aureus from different angles,which laid a theoretical foundation for the study of the regulatory mechanism of phenotypic plasticity in S.aureus.Compared with traditional genome editing technology,CRISPR/Cas9 system can improve the integration efficiency of the recombinant plasmid and the host genome,thereby efficiently editing the target genes of S.aureus.In our study,CRISPR/Cas9 system was used to carry out site-directed mutagenesis of the gra S gene,and a mutant strain S8’-gra S(I59L)was obtained.The establishment of functional verification platform for phenotypic plasticity genes of S.aureus provides a practical basis for further research.