Membrane Proteomics Of Moderately Halophilic Bacterium Bacillus. Sp I121 Under Salt Stress

In order to investigate changes of plasma membrane proteome under salt stress and the mechanisms of membrane proteins responding to salt stress,we choose moderately halophilic bacterium Bacillus sp.â… 121.as research material.The main results are as follows:1.The study of physiology of moderately halophilic Bacillus sp.I121We examined the characteristics of Bacillus.sp I121 responding to salt stress.Our results indicate that Bacillus.sp I121 could grow in the range of 0.2M to 3.6M NaCl concentration and the optimal NaCl concentration is 0.9M.The growth rate Would be greatly inhibited when the NaCl concentration is 3.6M.We detect the compatible solutes accumulated by Bacillus sp.I121 that were grown under different salinity.Our results show that Bacillus.sp I121 do not accumulate trehalose and glycin betaine.When cells grow in low NaCl concentration,proline would be accumulated to cope with the salt stress and when the NaCl concentration is high,ectoine would be accumulated.We also examined carbon sources of Bacillus sp.I121.Our results indicate that Bacillus sp.1121 could use maltose and sucrose as fast carbon sources and could also use glutamate quickly.However,Bacillus sp.I121 do not use glucose,fructose effectively.We examined the effects of exogenous compatible solutes on the growth rate of Bacillus sp.I121.Our results show that glutamate could highly improve the growth rate of Bacillus.sp 1121.Proline and ectoine have no effects on the growth of Bacillus sp.I121. These results indicate that accumulation of proline and ectoine in Bacillus sp.I121 Under salt stress was mainly dependent on de novo synthesis and there might be a glutamate uptake sys(?)em in Bacillus sp.I1212.The differential membrane proteomics of moderately halophilic Bacillus sp.I121 We study the membrane proteome of Bacillus sp.I121 using 5%-20%gradient SDS-PAGE and the results indicate that eight protein bands show great distinctions under different salinity.The MS results indicate that most of the identified proteins are trans-membrane proteins which testify the effectiveness of SDS-PAGE on the exploration of membrane proteins.However,because SDS-PAGE is a low resolution method and the bands usually contain several proteins,SDS-PAGE could not be used to study change in proteome.We explore the membrane protein complexes and subunits of these protein complexes by the method of BN/SDS-PAGE.The results of BN-PAGE show that expressions of membrane protein complexes were affected by salt stress and at least 10 distinct bands were resolved.These bands cover the range of 20KDa to 1000KDa.The BN/SDS-PAGE results show that nine proteins spots were greatly affected by salinity.We analyzed these protein spots by MS and eight spots were successfully identified.Up-regulated proteins under salt stress include:ABC transporter permease,glycerol-3-phosphate permease,pyrimidine nucleotide transporter,formate dehydrogenase,and down-regulated proteins include: succinate dehydrogenase iron-sulfur subunit,F₁F₀-ATP synthase a andβsubunits,and a hypothetical protein OB1959。The bioinformatics combining with physiology results indicate that ABC transporter permease is responsible for maltose transport.High salt concentration up regulated the expression of ABC transporter permease and glycerol-3-phosphate permease and activated sugar metabolism through the uptake of maltose and glycerol-3-phosphate.Down-regulated of succinate dehydrogenase and ATP synthase indicate that TCA cycle was suppressed,thus,proline and ectoine could be synthesized through suppressed TCA cycle and the process is highly related to nucleotide metabolism.