Physiological Mechanism Of Response To Acid Stress By XRE Family Transcription Regulator XrpA In Serratia Marcescens

As an important industrial microorganism,Serratia marcescens,the accumulation of by-products in the industrial production process will lead to the formation of acid stress environment in the fermentation system,which will not only inhibit the growth of the bacteria but also reduce the production of the products.This dissertation chose S.marcescens JNB5-1obtained from the previous screening of our laboratory was taken as model system to analyze the physiological mechanism of S.marcescens in response to acid stress.Therefore,the regulation mechanism of transcriptional regulatory factor XrpA in response to acid stress was studied in detail with the method of molecular bioinformatics and biology.Biodiversity analysis of XrpA showed that transcription regulator XrpA and Xrp A-like proteins were widely found in a variety of microorganisms,among which more than 90%were distributed in the Enterobacteriaceae.The main results were described as follows.1.The method of Tn5G transposon insertion mutation was used to construct the mutant library of S.marcescens,and an acid-sensitive mutant strain SX1-25 was screened from its mutant library.To analyze the genetic basis of mutant strain SX1-25 decreased acid tolerance,this study conducted reverse PCR to identify the Tn5G insertion site of the mutant strain SX1-25.The obtained sequences were compared with the NCBI database to determine the insertion site of Tn5G transposon.It was found that the insertion site of Tn5G transposon was the gene BVG90?23400,which encodes the transcription regulator with unknown function.In this study,the transcription regulatory factor was named Xrp A.2.The knockout strain JNB5-1?XrpA and the revertant strain JNB5-1?XrpA/pSX1314were constructed.At pH 4.0,the cell biomass and cell viability of the mutated strain JNB5-1?XrpA were 93.6%and 60.9%lower than that of the wild strain JNB5-1,respectively,while the cell biomass and cell viability of the revertant strain JNB5-1?Xrp A/pSX1314 did not significantly change compared with the wild strain JNB5-1,indicating that the transcription regulator XrpA was necessary for the resistance of S.marcescens to acid stress.3.By transcriptome data analysis,compared with the wild strain WT,the expression of genes related to acid stress in mutant strain JNB5-1?XrpA were significantly different at pH4.0,including significant differences in the transcription levels of many genes involved in lipid synthesis and metabolism of cell membranes,and the synthesis of fatty acids might be inhibited,and the ratio of saturated fatty acids to unsaturated fatty acids in cell membrane decreased.The expression of acid shock protein decreased significantly.Transcriptome data were verified by RT-qPCR and found to have good accuracy.The change of the transcriptional level of genes related to lipid synthesis and metabolism of cell membranes affect the membrane performance.At pH 4.0,compared with the WT strain,the cell membrane integrity and proton pump H~+-ATPase activity of mutant JNB5-1?XrpA decreased by 78.8%and 25.7%respectively,and the cell membrane permeability increased by 56.3%.However,compared with the WT strain,the cell membrane integrity,permeability and proton pump H~+-ATPase activity of revertant strain was no significant difference.