Proteomics Research Of Mechanism Of Alkane Transport And Uptake By Pseudomonas Aeruginosa SJTD-1

SJTD-1 is a new strain of Pseudomonas aeruginosa which were isolated from oil contaminated soil.Our previous studies showed that SJTD-1 can metabolize n-alkanes?C12^C30?in inorganic salt environment with alkanes as the sole carbon source.Thus SJTD-1 has very broad application prospects in treating oil pollution.The efficiency of the utilization of alkanes by microorganism is not only dependent on the metabolic rate of alkane,but also closely related to the transport and absorption efficiency of alkanes.Studies of the mechanism on transport and absorption of alkane is helpful to improve the efficiency of the utilization of alkanes,which is great significant both in the basic mechanism and environmental management.Although we have a certain understanding of proteins related with metabolic pathways of the degradation of alkanes by P.aeruginosa,yet the mechanism on uptake,transport and absorption of alkane is still unkown.It is urgent to conduct research on absorption and transport mechanism of alkane by Pseudomonas.Therefore,we study the phosphorylation of P.aeruginosa SJTD-1 and the expression difference of the stains cultivated in two carbon source?C16 and C18?to find some target proteins to explain the mechanism in uptake,transport and absorption of alkanes.The phosphorylation of P.aeruginosa SJTD-1 was to be studied at first.The proteins of P.aeruginosa SJTD-1 was prepared and digested to produce peptides,then TiO₂ was used to enrich phosphopeptides from peptides.Nano liquid chromatography-mass spectrometry were used to analysis and identify the phosphorylation sites.13 phosphopeptides,belonging to 12phosphoproteins were characterized.This is the first time we use nano liquid chromatography-mass spectrometry to study the phosphorylation of P.aeruginosa SJTD-1.In our study,it is worth noting that among those phosphoproteins we identified,7 phosphoproteins were first found in Pseudomonas.Analysis of phosphoproteins,we found that most of them were related to DNA replication,material transport and energy metabolism.Our experimental results play the foundation for the study of DNA replication,meterial and energy metabolism and cell membrane transport of Pseudomonas.And it has guiding significance for the further study on the mechanism of uptake and transport of alkanes by P.aeruginosa SJTD-1.Although phosphorylation involved in many biological processes of P.aeruginosa SJTD-1,such as DNA replication,meterial and energy metabolism by activating or inhibiting of protein in the cell,the phosphorylation of P.aeruginosa SJTD-1 was not enough to explain the mechanism on uptake,transport and absorption of alkane by P.aeruginosa SJTD-1.iTRAQ and label free comparative proteomics analysis was used to identify the proteins of alkane uptake in response to n-octadecane?C18?comparing with n-hexadecane?C16?in P.aeruginosa SJTD-1.A total of1258 and 1239 proteins were identified by label free and iTRAQ-based quantitative methodologies,respectively.By application of 1.5?iTRAQ?or2-fold?label free?for upregulated and 0.65?iTRAQ?or 0.5-fold?label free?for downregulated cutoff values,114 and 99 proteins were found to be differentially expressed comparing SJTD-1 cultivated on C18 with C16respectively.GO clustering and KEGG Pathway were used to analysis those differentially expressed proteins.The main biological process was involved in metabolism and cell process for the differentially expressed proteins.KEGG pathway analysis showed that the mainly important metabolic pathway were bacterial chemotaxis and two-component system besides the synthesis and metabolism of amino acid.The number of common differentially expressed proteins of both methods was 7 proteins.Notably,the common differential expressed proteins by two relative quantitative technologies identified was less,the main function and metabolic pathway analysis of differential proteins involved was highly consistent with each other.“Two-component system”are widespread in bacteria and involved many biological process.These results indicated that these proteins may be associated with the uptake and absorption of alkanes by SJTD-1.Additionally,quantitative real-time PCR?RT-qPCR?results confirmed C18-induced change in transcription levels of FleQ,FliC,NirS,FadL,FadD,PfpI,MucD,PA1228 and PA1767 proteins.Although the fold change of differential expressed proteins in transcription level were different with iTRAQ or Label free,the trend is similar.Our research indicated that these differentially expressed proteins play an important role in chemotaxis and swimming of bacteria,uptake and transport of alkanes by SJTD-1,providing a reliable basis for the further study on uptake and transport of alkane by P.aeruginosa SJTD-1.