Organic Solvent Tolerance Mechanisms Of Pseudomonas Putida Idaho

In non-aqueous phase biotransformation and environmental microbiology technology related to organic solvents,organic solvents are harmful to microorganisms,damaging membrane,destroying cellular structure and functional integrity,inhibiting cell growth and even leading to cell death.However,organic solvent tolerant bacteria can adapt to organic solvents with different mechanisms, such as degradation of toxic compounds,solvent active pump systems,rapid repairation of membrane,reduction of membrane permeability,changes of lipid acids, reduction of celluar hydrophobic surface,formation of membrane vesicles,and so on. So organic solvent tolerant bacteria can avoid the damage of organic solvents and have important application foreground.Pseudomonas putida Idaho is an oganic solvent tolerant bacteria and has special solvent tolerant characteristics different from other bacteria.It has genes which can degrade many aromatic compounds and can be induced by organic solvents.But it has no degrading plasmids and the degrading genes are in its chromosome.It has no solvent active pump systems,but can be tolerant to many organic solvents of high concertrations,which can be above 50%(v/v).It can also grow in p-xylene,toluene or other alkylbenzene as sole carbon source and energy source.P.putida Idaho has important application value in biocatalysis and bioremediation fields.Researches on the organic solvent tolerant mechanism of P.putida Idaho can bring the knowledge of the bacteria and provide new methods of genetical modification of microorganisms.It is a great complement to the organic solvent tolerant mechanisms of microorganisms and has great significance of basic theories. In the aspect of application,it is in favor of screening important emzymes for industry and is the important basis for whole cell application in biocatalysis and bioremediation.Based on the literatures and previous work of our laboratory,this paper studied the organic solvent tolerant mechanism of P.putida Idaho,using biological methods such as real time fluorescent quantitative PCR and two-dimensional gel electrophoresis.1.Researches on glycerol phosphate acyltransferase which catalyses the first step of phospholipid synthesis pathway,to understand the relationship between organic solvent tolerant characteristics and membrane synthesis.PlsB are used to form the primery initial membrane phospholipids using complete lipid acid chains and plays an important role in membrane biosynthesis.The gene plsB of Escherichia.coli Top 10 and P.putida Idaho was amplified and expressed in E. coli.Organic solvent tolerant characteristics of the recombinants were also tested.The results showed that the transformants containing external plsB has no enhanced ability of organic solvent tolerance,which implied that PlsB can not increase the cell ability of organic solvent tolerance.2.The changes of several key emzymes of phospholipid synthesis in P.putida Idaho under the pressure of organic solvents.Quantitative analysis of these emzymes were studied on transcriptional level using real time fluorescent quantitative PCR and one-step RT-qPCR.The results showed that genes of these emzymes were not changed on transcriptional level under different concertrations of organic solvents,which further certified that the rate of membrane synthesis was not enhanced under the pressure of organic solvents and not determined the ability of organic solvent tolerance.3.The changes of protein expression of P.putida Idaho under the pressure of organic solvents was examed by two-dimensional electrophoresis(2DE). Sample preparation of P.putida Idaho was optimized and established finally. Repeatability of sample preparation and two-dimensional electrophoresis was also achieved.This sample preparation method was also applicative to other bacteria of Pseudomonas putida.Software analysis of 2DE gel map revealed that protein expression of P.putida Idaho was different with and without organic solvents.4.Relevant data of the differentially expressed proteins were obtained through analysis of 2DE gel map with PDQuest.These proteins were identified by MOLDI-TOF mass spectrometry and classified according to their functions. The relevant information of these proteins was also abtained.Analysis and explanation of the changes of the proteins under organic solvents lead to further understanding of organic solvent tolerant characteristics of P.putida Idaho.