Growth Stress And Division Inhibition Of Biphenyl Metabolism On Microorganisms

Rhodococcus sp.R04 is a Gram-positive bacterium that can effectively degrade biphenyl/polychlorinated biphenyl.The research team has done a lot of research on its function of degrading biphenyl/polychlorinated biphenyl.In the study,it was found that biphenyl also reacted with R04 cells during the metabolism of biphenyl,which caused the cell division to be blocked,unable to form a normal dividing septum,and a series of metabolites were produced during metabolism,such as 2,3-Dihydroxybiphenyl(DHBP),2-hydroxy-6-keto-6-phenyl-2,4-hexadienoic acid(HOPDA),etc.It is unclear which metabolite or biphenyl itself has a strong inhibitory effect on the division of Rhodococcus sp.R04 cells.At the same time,FtsZ plays an important role in cells as an important division protein in the process of cell division,whether the abnormal division of Rhodococcus sp.R04 cells is directly related to FtsZ protein under biphenyl stress is a key problem to be solved in this study.In this study,the FtsZ protein of Rhodococcus sp.R04 was successfully heterologously expressed in Escherichia coli BL21,and the expression level was high and it was soluble protein.The purification of FtsZ protein was obtained by affinity chromatography on cobalt column or nickel column and preparing polyclonal antibodies.The successful preparation of polyclonal antibody provides a powerful material for further study on the localization and expression of FtsZ protein in Rhodococcus sp.R04.The expression of FtsZ protein in different phases of Rhodococcus sp.R04 cells in biphenyl culture can be detected by western blot.Immunofluorescence technique can also be used to observe the localization of FtsZ protein in logarithmic phase of cells cultured with biphenyl and its metabolism,which provides a theoretical basis for further study of the interaction between FtsZ protein and Rhodococcus sp.R04 cell division and septum formation.In order to further study the expression and localization of Rhodococcus sp R04 FtsZ protein in E.coli and its effect on the morphology of E.coli,we constructed the fusion expression plasmid p ET-m3c-fts Z-rfp and induced E.coli containing the fusion expression plasmid by IPTG,it was found that with the increase of IPTG concentration,E.coli filamentous was obvious and cell division was blocked.It was found that FtsZ was located in Z ring during cell division by FtsZ protein was fluoresced,which indicated that FtsZ protein participated in the formation of septum ring and affected cell morphology and division as a cell division related protein.Secondly,we cultured E.coli containing fusion expression plasmids with biphenyl and its metabolites and it was found that under DHBP culture conditions,E.coli cells shrank significantly,growth arrested,and FtsZ protein was almost not expressed.Therefore,we speculated that biphenyl metabolite 2,3-dihydroxybenzene(DHBP)had a strong inhibitory effect on E.coli division and FtsZ protein expression.In order to study the inhibitory effect of biphenyl itself or its metabolites on cell division,we cultured metabolic enzyme-deficient Rhodococcus sp.R04 and several model strains with biphenyl respectively.Compared with the precursor-biphenyl and its metabolite HOPDA,DHBP showed strong inhibition and morphological changes in all subjects,whether G+ or G-bacteria,or Rhodotorula cell division.DHBP can make the incomplete septum of metabolic enzyme deficient Rhodococcus sp.R04 when it divides,and swelling on one side or the end of the division will affect its normal division;DHBP causes E.coli BL21 to grow slowly,the cell volume shrinks,the cell surface recesses,and the cytoplasmic content loses;DHBP inhibits the division of Bacillus subtilis WB600,and the cell volume shrinks obviously when biphenyl exists.The septum of Bacillus subtilis was abnormal,and the length of the bacteria was about 2~3 times longer than that of the control.The division of Staphylococcus aureus and Rhodotorula treated with DHBP was affected.The number of Staphylococcus aureus cells with septum was significantly reduced,and the percentage of Rhodotorula cells germinated and reproduced decreased from 64.2% to 19.3%.