| Most bacteria in nature do not live in the form of single cells,but in the form of multi-cell aggregation in biofilms wrapped by multiple substrates,resisting adverse factors such as temperature,osmotic pressure,pH,ultraviolet rays,metal ions,etc.The biofilm is a complex composed of extracellular polymers(Extracellular Polymeric Substances,EPS)surrounded by micro colonies.EPS account for 90% of the biofilm biomass and is an important physical barrier to protect cells in the biofilm.It consists of encapsulating bacterial cells in dense EPS matrix components and attaching them firmly to biological and non-biological surfaces.Therefore,biofilm is an important part of the survival of bacteria in the natural environment,and is a widely existing self-protective survival strategy.The formation of bacterial biofilms is a dynamic development process,which includes a series of development stages including initial surface attachment,micro colonial formation,maturation,diffusion,and propagation,and finally forms a complex,organized multicellular system.The development of the biofilm is regulated by the quorum sensing system and cyclic di-GMP.c-di-GMP is the second messenger molecule ubiquitous in bacteria and plays an important role in the adaptation of bacteria to the natural environment.c-di-GMP is synthesized by two-molecule GTP catalyzed by a class of diguanylate cyclase(DGC)containing GG(D/E)EF domain.The synthesized c-di-GMP is hydrolyzed to5'-phosphoguanylyl-(3'-5')-guanosine(pGpG)by phosphodiesterase(PDE)containing EAL or HD-GYP domain,and then further decomposed into guanosine monophosphate(GMP).In bacterial cells,the increase in c-di-GMP content canpromote the formation of biofilm and reduce the movement level of cell movement;the decrease of c-di-GMP content will inhibit the formation of biofilm and improve the level of cell movement.Therefore,c-di-GMP plays a role in regulating bacteria from free state to biofilm state to a large extent.Rhodococcus sp.NJ-530 is an actinomycete isolated from the Antarctic sea ice floes.It is a representative bacterium for studying the adaptation mechanism of marine bacteria to extreme environments.Although the study of biofilm forming bacteria is very extensive,the investigation of actinomycetes is still very limited.After whole genome sequence of Rhodococcus sp.NJ-530,it was found that the bacterium contains many genes that catalyze the synthesis and degradation of c-di-GMP.It is speculated that Rhodococcus sp.NJ-530 adapts to the extreme Antarctic environment is related to its special life activity--biofilm formation.In this paper,Rhodococcus sp.NJ-530 from Antarctic sea ice was isolated as the target strain,and the following research contents were mainly carried out:(1)finding and comparing the diguanylate cyclase gene dgc in Rhodococcus sp.NJ-530 whole genome,synthesizing the gene by PCR technology,and conducting bioinformatics analysis.The total length of the gene was 948 bp,encoding 315 amino acids,the predicted molecular weight of the protein was 34.6 KDa,the isoelectric point was 5.58,and the GenBank registration number was MN419917.(2)using real-time qRT-PCR and micro plate detection technology,the change trend of the dgc gene transcription level and biofilm formation amount of in Rhodococcus sp.NJ-530 cultured under temperature and salinity stress were investigated.qRT-PCR results showed that the dgc gene was expressed at low temperature(-5?,0 ? and5?),low salinity(16 ‰)and normal salinity(32 ‰)conditions.With the increase in temperature and salinity,the expression of the dgc gene was gradually suppressed.At the same time,the micro plate detection experiment showed that the dgc gene positively regulates the content of c-di-GMP and the amount of biofilm formation.When Rhodococcus sp.NJ-530 was cultured at the highest temperature(25?),the amount of biofilm formation was the least.As the temperature gradually decreases,the cumulative amount of biofilm continued to increase.Similarly,under low salinity(16 ‰)and normal salinity(32 ‰),the biofilm formation was more.As salinity increases,the amount of biofilm formation was obviously inhibited.(3)The dgc gene synthesized in vitro was linked to the expression vector and transferred to E.coli engineering bacterium BL21(DE3)for prokaryotic expression andpurification,and the DGC protein was obtained.The results of Western Blot analysis showed that the target protein was present in the supernatant of cell fragmentation and the molecular weight was the same as predicted.(4)Activity in vivo was studied for the E.coli engineering bacterium BL21(DE3),explore the biofilm formation of recombinant protein in E.coli was adjusting action.In vivo activity experiment showed that engineered bacteria containing DGC recombinant protein produced more biofilm than wild-type E.coli,proving that DGC recombinant protein catalyzed the synthesis of more c-di-GMP in E.coli and the biofilm contented was also to rise accordingly.In summary,the research contents and results in this paper were shown,the formation of biofilm played an important role in protecting Rhodococcus sp.NJ-530 from adapting to the extreme environment of Antarctica,and helped to understand the adaptation mechanism of marine bacteria such as actinomycetes in the extreme environment of Antarctica.At the same time,through to the dgc gene bioinformatics analysis and dynamic monitoring of the biofilm formation,to master the dynamic development of bacterial biofilm process,to effectively remove the spoilage effect of low-temperature spoilage bacteria on food and the infection and invasion effect of pathogenic bacteria on medical devices and human body provided an important train of thought. |
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