Study On The Polymerization Of Rhodococcus Sp.R04 FtsZ Under Biphenyl Stress

As an important division protein,FtsZ participates in the formation of the diaphragm and regulates cell division.The study found that the series of metabolites produced during the metabolism of biphenyl（BP）by Rhodococcus sp.R04,2,3-dihydroxybiphenyl（DHBP）,2-hydroxy-keto--6phenyl-2,4-hexadienoic acid（HOPDA）inhibited the division of Rhodococcus sp.R04 cells to varying degrees,causing the cells to fail to form a normal dividing septum.Is the influence of biphenyl and its metabolites on the division of Rhodococcus R04 cells directly related to the FtsZ protein?What kind of relationship does it have?This is the key problem to be solved in this research.In this study,through affinity chromatography on a cobalt chloride column,a high purity R04 FtsZ protein was successfully obtained.The malachite green phosphorus method was used to detect the GTP enzyme activity,and different concentrations of biphenyl,DHBP,HOPDA and FtsZ were determined.The results show that biphenyl and its metabolites have almost no effect on enzyme activity.We used light scattering method to determine the polymerization of FtsZ protein,and determined the effect of different concentrations of biphenyl and its metabolites on protein polymerization.The polymerization of FtsZ protein was observed under transmission electron microscope.Studies have shown that biphenyl and its metabolites mainly affect the polymerization of FtsZ protein and substrate.As the concentration of metabolites increases,the number of polymers formed decreases and the depolymerization time shortened,at the same concentration,DHBP and HOPDA have a greater impact on polymerization,and biphenyl has a small impact on polymerization.Based on the previous research results of the research group,we speculate that the effect of DHBP on FtsZ protein polymerization is the damage of cell division membrane one of the factors.In order to study the real-time dynamic changes of the FtsZ protein in the body during the metabolism of biphenyl by Rhodococcus sp.R04,we labelled the Rhodococcus sp.R04 FtsZ protein,and used homologous recombination to insert the red fluorescent protein rfp gene after the fts Z gene sequence.The physiological characteristics of the recombinant strains under different culture conditions（LB medium,basic medium with glucose as the sole carbon source,and basic medium with biphenyl as the sole carbon source）were analyzed.The results of the study showed that the strain after inserting red fluorescence The original growth characteristics are hardly changed,and the growth status under different culture conditions is very small from the wild-type strain.This provides a powerful material for us to study the dynamic changes of intracellular FtsZ protein under the action of biphenyl and its metabolites.In order to explore whether the effects of biphenyl,DHBP,and HOPDA on FtsZ protein in vivo and in vitro are universal,we extracted Escherichia coli（E.coli）and Bacillus subtilis（B.subtilis）and Staphylococcus aureus（S.aureus）genomes,and their fts Z genes were PCR amplified to construct recombinant plasmids p ET21a-fts Z_E.coli,p ET21a-fts Z_B.subtilis,p ET21a-fts Z_S.aureus,The soluble expression of FtsZ protein induced by IPTG after transforming into Escherichia coli,and the protein is purified by cobalt chloride affinity chromatography column to obtain the active protein with higher purity.Studies have shown that biphenyl and its metabolites affect the polymerization of FtsZ protein,which in turn affects the formation of cell compartments.The obtained RFP-labeled Rhodococcus strains,Escherichia coli FtsZ,Bacillus subtilis FtsZ,and Staphylococcus aureus FtsZ active proteins have laid the foundation for the next step of studying the influence of biphenyl and its metabolites on FtsZ.