| Acetic acid is a metabolite well-known for its cellular toxicity at concentrations as low as0.5%(v/v).Acetobacter pasteurianus has been used for industrial vinegar production because of its high resistance to acetic acid. In the present work, fed-batch fermentation was carried out to acclimate Acetobacter pasteurianus in high concentration of acetic acid. We investigated the effects of high concentration of acetic acid on the morphology and microstructural changes relevant to the cell wall and membrane,and the potential mechanism of acetic acid resistance on the cellular level.We established a proper system of two-dimensional gel electrophoresis to distinguish the total protein expression profiles of Acetobacter pasteurianus under high acetic acid resistance,in order to reveal the potential molecular mechanisms of the resistance to acetic acid. The implementation of this work lay the foundation for improveing vinegar production in vinegar industry,reducing the cost of consumption, providing a scientific basis of the resistance of Acetobacter pasteurianus to acetic acid.The influence of Acetobacter pasteurianus under different concentrations of acetic acid were investigated.The effects of different concentrations of acetic acid on the microstructural change of cell wall and membrane,and Na+/K+-ATPase activity were examined.The results showed that higher the concentration of acetic acid,the higher Na+/K+-ATPase activity.The significant effects on cell morphology were photographed under high concentrations of acetic acid pressure.This study indicated that high concentrations of acetic acid can cause the microstructural variation,and cell wall and membrane to adapt to the high acidity.We established and optimized a two-dimensional gel electrophoresis (2-DE) protocol to assay the total protein of Acetobacter pasteurianus. The total proteins extracted from Acetobacter pasteurianus were performed on2-DE, dyed and scanned to gain gel image. The gel images were analysised by PD-Quest sofrware.The2-DE protocol were optimized by comparative tests on some important factors.This work provided a technical basis for the further study on differential proteomics of Acetobacter pasteurianus.By combination of2-DE and mass spectrometry techniques,we identified26typical proteins in Acetobacter pasteurianus under different concentration of acetic acid.After analysis of peptide fingerprinting obtained by MALDI-TOF-MS,proteins spots related to cell wall and membrane linked to the respiratory chain were identified to be alcohol dehydrogenase,PQQ-dependent alcohol dehydrogenase, transketolase,aldo-keto reductase,fructose-bisphosphate aldolase, peroxiredoxin,heat shock protein,outer membrane protein,and transcriptional regulator.Of which,alcohol dehydrogenase class is mainly used to convert ethanol into acetic acid,PQQ can promote ADH transport in transmembrane.Aldo-keto reductase catalyzes reactions reduction of aldehydes and ketones during acetic acid bacteria fermentation.Fructose bisphosphate aldolase exists in the biosynthesis pathway such as gluconeogenesis pathway and pentose phosphate cycle for the organism in anabolism providing energy and ATP.Peroxiredoxin is an antioxidant protein,can protect cells from attacking by hydrogen peroxide,alkyl peroxide and other reactive oxygen. Heat shock proteins are stress proteins,and their function is to prevent protein from denaturation.Outer membrane protein is an important component of the outer membrane of gram-negative bacterial,playing an important role in the cell membrane formation and maintenance of bacterial morphology,metabolism and other functions.Transcriptional regulator is involved in regulating the metabolism of bacteria,cell division,quorum sensing,resistance to harsh environments,such as the purpose of virulence genes.Therefore,it was confirmed that high concentration of acetic acid can affect cell membrane structure and morphology, especially the expression changes of cell wall and membrane-related and oxidoredutase activity proteins. |
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