Preliminary Study On The Induced Mechanism And Function Of Outer Membrane Vesicles Of Novosphingobium Pentaromativorans US6-1 Under BaP Stress

Outer membrane vesicles(OMVs)are extracellular spherical vesicle structures which are ubiquitous in Gram-negative bacteria,and participate in physiological processes such as secretion,nutrient acquisition,oxidative stress,defense and resistance.Novosphingobium pentaromativorans US6-1 is one of the degradation model bacteria that can efficiently degrade high-weight polycyclic aromatic hydrocarbons(PAHs).And US6-1 products many OMVs under the stress of benzo[a]pyrene(BaP).In order to explore the production mechanism and function of the OMVs in US6-1 under BaP stress,the US6-1 under BaP stress was taken as object in this research.To demonstrate the mechanism and function of OMVs,the US6-1 OMVs and cells under different conditions were analyzed by transmission electron microscopy,qualitative protein mass spectrometry,fluorescence quantitative PCR,cell reactive oxygen species level determination and transcriptome analysis.The main research results are as follows:1.Compared with the simple outer membrane vesicles produced under 2216E culture conditions,N.pentaromativorans US6-1 produced more OMVs with a multilayered membrane under BaP stress and the OMVs have a diameter of 200-700 nm.The results of transmission electron microscopy showed that the multilayer membrane structure in the OMVs also existed in the cytoplasm and the density of the OMVs was similar to the cytoplasm.Compared with the 2216E culture conditions,the US6-1 cell wall showed a wavy curl under BaP stress.These results suggest that the OMVs produced by US6-1 under BaP stress may originate from the cytoplasm rather than the periplasmic space;2.Compared with Pyruvate treatment,in the results of transcriptome,the transcription level of US6-1 Tol-Pal system genes significantly decreased under BaP stress,and the phospholipid metabolism gene glpA which was related to cell membrane synthesis was up-regulated.In the US6-1 under BaP stress,the transcription level of RegA/RegB system about oxidative phosphorylation decreased,the overall transcription level of complex Ⅰ,Ⅱ,Ⅲ,Ⅳ,and complex Ⅴ decreased,and the transcription level of key enzymes for Poly-3-β-hydroxybutyrate synthesis decreased.The transcription level of some tbdt genes increased,and the transcription level of the tbdt gene NSU_pLA1112 was extremely high under BaP stress.These results indicate that US6-1 cell wall stability decreases,energy metabolism is inhibited,and the TBDT transport system is active under BaP stres;3.Compared with the 2216E culture conditions,the level of reactive oxygen species and the number of OMVs produced by US6-1 under BaP stress increased significantly.The OMV protein profile results showed that the OMVs produced by US6-1 under BaP stress had a higher proportion of cytoplasmic protein than that in 2216E culture.Under BaP stress,more types of OMVs protein produced by US6-1 include glutathione peroxidase,glutathione S transferase,thioredoxin,and thioredoxin reduction enzymes,catalase and other proteins related to reactive oxygen stress.There was low content of TolA protein,an important protein involved in stabilizing the cell wall,in the OMVs produced by US6-1 under BaP stress.These results indicate that the OMVs originate from the cytoplasm,which contain DNA repair enzymes and a large number of oxidative stress-related enzymes,which may be involved in the production of OMVs and the detoxification process of PAHs;4.Compared with 2216E culture conditions,there were more related proteins about TBDT transport system,Tat protein transport system,Sec protein transport system and RND efflux system in the OMVs produced by US6-1 under BaP stress.In the OMVs,there were enzymes in the naphthalene degradation pathway and BaP ring-opening dioxygenase.These results indicate that OMVs may be involved in the transport,detoxification and degradation of PAHs.The above experimental results indicate that reactive oxygen species induce the production of OMVs.In the process of US6-1 degradation and detoxification of BaP,oxy-BaP might damage DNA and generated large amounts of reactive oxygen species.To maintain homeostasis,US6-1 reduced cell wall stability and secreted specific cytoplasmic components by OMVs.The OMVs produced by US6-1 under BaP stress might participate in the degradation and detoxification of PAHs.