外文翻译--The Research on Proteomic Changes in Two Bioflocculant Producing Bacteria When Grown in Minimal Media versus Bioflocculant Media

The Research on Proteomic Changes in Two Bioflocculant Producing Bacteria When Grown in Minimal Media versus Bioflocculant Media Bing Yu1,2,Fang Ma1,Peishi Qi1,Ang Li1,Lili Wang1 1.State Laboratory of Urban Water Resources and Environment,School of Municipal and Environmental Engineering,Harbin Institute of Technology,Harbin,China 2.Key Laboratory of Molecular Biology,College of Life Science,Heilongjiang University,Harbin,China Email:, AbstractIn order to reveal the molecular mechanisms and the metabolic pathway of producing bioflocculant,the changes in protein complexes of F2 grown in minimal media versus bioflocculant media and those of F6 grown in minimal media versus bioflocculant media were respectively researched by SDS-PAGE.SDS-PAGE results indicated that the relative band volumes of three bands were obviously decreased in F2,the relative band volume of one band was obviously increased in F2 as well as the relative band volumes of two bands were obviously decreased in F6,the relative band volume of one band was obviously increased in F6.The conclusion was that seven protein complexes,which are differentially expressed and important to induce and produce the bioflocculant,were obtained and need further indentify the protein components in them.Keywords-Bioflocculant producing bacteria;the relative band volume;protein complexes;SDS-PAGE.I.INTRODUCTION The bioflocculant is a high polymer metabolic product of microorganism which can make the suspended particles,thalline cells and colloid particles aggregate and precipitate 1.The bioflocculant is produced through microbial fermentation and is widely used in the field of water supply and waste water disposal because of its many advantages such as high efficiency,nontoxicity,non-secondly pollution,easy biodegradability and healthy security.The compound bioflocculant(CBF)2 is a novel bioflocculant produced by cellulose degrading bacteria and bioflocculant producing bacteria,using straw-cellulose as initial carbon source and energy source through two-step fermentation.The flocculation efficiency of CBF is generally above 90%.F2 and F6 are two bioflocculant producing bacteria,which are fermented to produce CBF.Biochemical properties and 16S rDNA of F2 and F6 were analyzed.The results indicated that F2 was a strain of Rhizobium radiobacter and F6 was a strain of Bacillus sphaeicus 3.Results of flocculating activity distribution tests indicated that the effective components of CBF existed in fermentation solution and were extracellular product.Moreover,through methods of coomassie blue reaction,ultraviolet spectroscope and infrared spectrum,the effective components of CBF were determined as polysaccharide and the output of polysaccharide in the crude CBF was 2.14g/L 4.Analysis of changes in bacterial proteomes has been an area of intense research in the last several years.Changes in bacterial growth media or conditions can have significant effects on protein expression5.When F2 and F6 were respectively grown in minimal media,the output of the bioflocculant was nearly zero,when F2 and F6 were respectively grown in bioflocculant media,the output of the bioflocculant was increased greatly.F2 and F6 may express differential proteins in bioflocculant media which are important to induce and produce the bioflocculant.In order to reveal the metabolic pathways of producing bioflocculant,the differentially expressed proteins in two bioflocculant producing bacteria when grown in minimal media versus bioflocculant media should be analyzed.This work compared the changes in protein expression of F2 grown in minimal media versus bioflocculant media in SDS-PAGE gel and F6 was researched by the same method,which can play an important role to understand and regulate the metabolic pathway of producing bioflocculant and improve the yield of the CBF.II.MATERIALS AND METHODS A.Materials 1)Bacterial strains Bioflocculant producing bacteria F2 and F6(from the Heilongjiang Environment Biotechnology Key Laboratory).2)Main equipments Electrophoresis Chamber(Beijing Six One DYCZ-24DN);Electrophoresis Apparatus(Bio-Rad PAC1000);3)Main Media The compositions of two media used in this study were shown as Table I.The minimal media was autoclaved at 121C for 20 min.The bioflocculant media was autoclaved at 112C for 30 min.978-1-4244-4713-8/10/$25.00 2010 IEEETABLE I.MEDIA COMPOSITIONS Name Compositions Concentration(g/L)pH The minimal media Peptone Beef extract NaCl 10 3 5 7.0 The bioflocculant media Glucose K2HPO4 KH2PO4 Urea MgSO47H2O NaCl Yeast extract 10 5 2 0.5 0.2 0.1 0.5 7.2 B.Methods F2 was cultivated both on minimal media and on bioflocculant media for overnight at 30C,140rpm;F6 was cultivated both on minimal media and on bioflocculant media for overnight at 30C,140rpm.F2 and F6 cells were respectively harvested from 10mL of minimal media and from 10mL of bioflocculant media by centrifugation(12000 r/min,3min at 4C).The pellets of F2 and F6 were respectively washed twice with 20 mmol/L Tris-HCl buffer(pH 8.0).Then the washed pellets were resuspended in 3 mL of the same buffer and lysed by freezing and thawing followed by sonication(225W,at 4C for 30 min,Ultrasonic processor CPX 750,USA).After centrifugation(12000 r/min,10 min at 4C),the supernatants were used as crude extracts.The cytosol protein concentrations of the crude extracts were determined using the Bradford method 6 and the cytosol protein concentrations of F2 in minimal media and in bioflocculant media were adjusted to the same value.The cytosol protein concentrations of F6 in minimal media and in bioflocculant media were also adjusted to the same value.The acrylamide concentrations for the concentrating and separating gels were 5%and 15%,respectively.Four kinds of 7.5 g cytosol proteins from F2 and F6 grown in minimal media and bioflocculant media were run on two SDS-PAGE gels.The gels were stained with Coomassie Brilliant Blue R-250.The SDS-PAGE gels were scanned by Image Scanner(HP Labscan).III.RESULTS A.SDS-PAGE of cytosol proteins from F2 As Figure 1 was shown that the relative band volumes of band I,II,III were obviously decreased,the relative band volume of band IV was obviously increased and the relative band volumes of the other bands were similar when cytosol proteins from F2 in minimal media were compared to those in bioflocculant media.Each band represents one protein complex of cytosol proteins from F2.B.SDS-PAGE of cytosol proteins from F6 As Figure 2 was shown that the relative band volumes of band I,II were obviously decreased,the relative band volume of band III was obviously increased and the relative band volumes of the other bands were similar when cytosol proteins from F6 in minimal media were compared to those in bioflocculant media.Each band represents one protein complex of cytosol proteins from F6.Lane 1,2:SDS-PAGE of cytosol proteins from F2 grown in minimal media;Lane 3,4:SDS-PAGE of cytosol proteins from F2 grown in bioflocculant media;Lane M:protein molecular weight marker(Broad)Figure 1.The images of SDS-PAGE from F2 Lane 1、2:SDS-PAGE of cytosol proteins from F6 grown in minimal media;Lane 3、4:SDS-PAGE of cytosol proteins from F6 grown in bioflocculant media;Lane M:protein molecular weight marker(Broad)Figure 2.The images of SDS-PAGE from F6 200KDa 97.2KDa 66.4KDa 44.3KDa 29.0KDa 20.1KDa 14.3KDa 6.5KDa M 1 2 3 4 Complex I Complex II Complex III Complex IV 200KDa 97.2KDa 66.4KDa 44.3KDa 29.0KDa 20.1KDa 14.3KDa 6.5KDa M 1 2 3 4 Complex I Complex III Complex II IV.DISSCUSTIONS A.Semi-quantitative estimation of the relative band volumes in SDS-PAGE With the development of the techniques for investigation of protein function,people have realized that protein complexes play a critical role in biological processes 7.Through SDS-PAGE,the changes of the relative band volumes of bands in F2 and F6 in two media were semi-quantitatively compared and seven protein complexes were obtained.These protein complexes will be excised,digested and analyzed with MS for the identification of protein components for the future.Many proteins will be ascertained in these complexes,which will give us insights into the mechanism of producing bioflocculant.B.Identification of protein components from the complexes by shotgun proteomic experiments The shotgun expression proteomics will be used to determine the protein components from the protein complexes.By determining which proteins are differentially expressed in bioflocculant media(which proteins are up-regulated or down-regulated),critical information on how F2 and F6 can produce the bioflocculant may be provided and proved further.V.CONCLUSIONS F2 and F6,two bioflocculant producing bacteria,are fermented to produce CBF which has wide and efficient use in the field of water supply and waste water disposal.According to the greatly increased output of the bioflocculant in two bioflocculant producing bacteria when grown in minimal media versus bioflocculant media,proteomic changes of F2 and F6 grown in two media were researched.Proteome of F2 and F6 grown in minimal media versus bioflocculant media were run in two SDS-PAGE gels.The SDS-PAGE results indicated that the relative band volumes of three bands were obviously decreased in F2,the relative band volume of one band was obviously increased in F2,the relative band volumes of two bands were obviously decreased in F6 and the relative band volume of one band was obviously increased in F6.Each band represents one protein complex.It is probably these differentially expressed protein complexes in gels that induce and produce the bioflocculant.Finally,seven protein complexes were obtained to identify the protein components in the future.The results in the paper can build a solid foundation to research the molecular mechanisms and the metabolic pathway of producing bioflocculant.In the future,the metabolic pathway of the CBF can be regulated and the yield of the CBF can be increased further.ACKNOWLEDGMENT We gratefully acknowledge the National High Technology Research and Development Program of China(863 Program)(Granted No.SQ2009AA06XK1482412)and The Science and Technology Development Program of Heilongjiang Province Department of Education(Granted No.11541284)for their financial supports.REFERENCES 1 Fang Ma,Yu jie Feng,Nan qi Ren,“Environment biotechnology,”Chemistry Industry Press,pp.150-162,Beijing,China,2003.2 Fang Ma,Jun liang Liu,Shu geng Li,“The development of the compound bioflocculant,”Chinese Water and Wastewater,Vol.19,No.4,pp.1-4,2003.3 Yan bin Zhu,Min Feng,Ji xian Yang,Fang Ma,Bo Wu,Shu geng Li,“Screening of complex bioflocculant producing bacterium and their flocculating mechanism,”Journal Of Harbin Institute Of Technology,Vol.36,No.6,pp.759-762,2004.4 Qin Wang,“The flocculation mechanism and production technology of the compound bioflocculant,”Ph.D dissertation of Harbin Institute of Technology,pp.19-26,2005.5 John D.Lippolis,Darrell O.Bayles,Timothy A.Reinhardt,“Proteomic changes in Escherichia coli when grown in fresh milk versus laboratory media,”Journal of Proteome Research,Vol.8,No.1,pp.149-158,2009.6 BradfordMM,“A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding,”Analytical Biochemistry,Vol.72,pp.54-248,1976.7 Bo Meng,Zhong Qian,Fan Wei,“Proteomic analysis on the temperature-dependent complexes in Thermoanaerobacter tengcongensis,”Proteomics,Vol.9,pp.31893200,2009.