Antibacterial Mechanism Of 3-carene Against Pseudomonas Fluorescens

3-Carene is the main component of plant essential oils such as pepper oil,which has antibacterial effect and broad prospects in the field of food antisepsis.The antimicrobial mechanism of 3-carene on Pseudomonas fluorescens was studied,mainly from the antibacterial activity,morphological structure,breathing and energy metabolism combined with metabolomics,transcriptomics and related enzyme activities,to revealed the inhibition mechanism and offered a theoretical knowledge when evolving 3-carene as a food preservative.Results showed as follows:(1)The minimum inhibitory concentration(MIC)and growth curve revealed the inhibitory effect of 3-carene.(2)Electron microscope observation and leakage of cell contents were also investigated to reveal the effects on the cell structure and permeability.The results revealed that 3-carene destroyed the morphology and structure of P.fluorescens,the increase of membrane permeability,which causeed leakage of intracellular electrolytes and macromolecular substances such as proteins.(3)The interaction between 3-carene and P.fluorescens DNA was studied by fluorescence spectroscopy.The results indicated that 3-carene may bind to bacterial DNA and effect the conformation and structure of genomic DNA.(4)By measuring the membrane potential(MP),metabolic activity and the enzyme activity in metabolic pathways such as citrate cycle(TCA cycle),we found that the treatment of 3-carene caused the MP and the intracellular protein content of P.fluorescens continued to decrease,and led to the metabolic activity and key enzyme in the TCA cycle decreased significantly.The disorder of ATP content and Na~+K~+ATPase indicated that3-carene caused metabolic disorder and inhibited energy synthesis.(5)Metabolomics was used to identify and screen differential metabolites and metabolic pathways,and explored the metabolic mechanism of 3-carene against P.fluorescens.The results indicated that treated with 3-carene for 12 h led to significant changes in 42 metabolites,among which 14 and 28 metabolites were upregulated and downregulated,respectively.Moreover,3-carene treatment resulted in disturbances in metabolic processes,including amino acid metabolism,pantothenate and coenzyme A(Co A)biosynthesis and the tricarboxylic acid(TCA)cycle.(6)A genome-wide transcriptional analysis of of P.fluorescens that responded to3-carene was performed using RNA-seq technology.Results revealed that,the effect of3-carene on P.fluorescens resulted in 785 differentially expressed genes(DEGs),among which 307 were up-regulated and 478 were down-regulated.The presence of significant DEGs enrichment in several pathways:oxidative phosphorylation,TCA cycle and ATP-binding cassette(ABC)transporters.(7)Combined analysis of metabolome and transcriptome revealed that oxidative phosphorylation,ABC transporters and some amino acid metabolisms were the main metabolic pathway changed with the treatment of 3-carene.In summary,3-carene had antibacterial effect that can change and destroye the normal morphology of P.fluorescens and caused intracellular material leakage,triggered cell membrane de-mutation,caused abnormal cell metabolic activity;3-carene can also bind to bacterial DNA,causing DNA conformation and structure changes;3-carene inhibited respiratory and energy metabolism of P.fluorescens and the respiratory chain;3-carene can exert its bacteriostatic effect by causing gene differential expression and metabolic dysfunction in bacterial cells.