| Rhodococcus sp.R04 can effectively degrade a variety of aromatic compounds,especially biphenyl(BP)and polychlorinated biphenyl(PCB),which are potentially harmful to the organism.It is considered to be a promising method for the degradation of aromatic compounds,which is less destructive to the environment and is economically effective and will not cause additional pollution.At present,the research of Rhodococcus is mainly focused on the way to metabolize aromatic compounds and enzymes in metabolic pathway,but few people have studied the influence of these aromatic compounds on Rhodococcus.Our team has studied the degradation of BP/PCB by R.R04 for many years.Previous studies have found that the division and morphology of R.R04 cells have changed greatly during the degradation of BP by R.R04 cells.However,in the process of BP degradation,which metabolites have affected the division and morphology of the R.R04 cells,it has not been reported.In this paper we used BP and its metabolite DHBP and HOPDA as carbon sources respectively for the culture of wild-type R.R04 cells,corresponding metabolic enzyme defective R.R04 cells and E.coli BL21 cells,then observed cell morphology and division by fluorescence microscopy.The results showed that when DHBP was used to culture the wild-type and corresponding defective R.R04 cells,the cell boundaries were blurred,can not seen the clear and complete septum and only fluorescent spots in cytoplasm were observed.when DHBP cultured E.coli BL21 cells,although the cell membrane was clearly stained and a clear and intact septum was seen,the cells becames significantly smaller.It showed that DHBP was a main substance that affected the morphology and division of R.R04 cells.In addition,in order to further understand the effects of BP and its metabolites on cell morphology,cytoskeleton protein MreB was selected for this study.Prokaryotic cytoskeletal protein MreB is similar to eukaryoticactin.In the process of prokaryotic cell growth and division,MreB protein forms helix structure on the inner side of cell membrane,which can guide the synthesis of new peptidoglycan,so that cells can maintain specific morphology.We used the prepared MreB antibody to detect the expression level of MreB protein in R.R04 cells at different growth periods under different culture conditions by western-blot.The results showed that either glucose or BP was used as the carbon source for culture,from the exponential phase to the conversion phase to the stable phase,the expression level of MreB protein in R.R04 cells was continuously down-regulated.In addition,the expression level of MreB protein was decreased in the three growth stages detected under the BP culture conditions compared to glucose culture conditions.Meanwhile,it was observed that from the exponential phase to the stable phase,cell morphology changed from rod to spherical,and BP inhibited the division of R.R04 cells under fluorescence microscope,indicating that the MreB protein is related to the maintenance of cell morphology and cell division.Finally,we constructed recombinant vector pGEX-6P-1-mreB and transformed E.coli BL21 cells.Observe the effect of MreB overexpression on the morphology of E.coli cells by fluorescence microscopy.As a result,the obvious morphological changes of E.coli cells were not observed.In addition,the recombinant vector pET-m3c-mreB-gfp was constructed and transformed into E.coli BL21 cells.The location of MreB-GFP fusion protein in E.coli was observed by fluorescence microscopy.The results showed that the MreB-GFP fluorescence was localized in the cytoplasm,or the fluorescence gathered in the cell division site and the two poles.This may be due to the formation of inclusion bodies. |
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