Molecular Cloning And Characterization Of MtrAB In Arthrobacter Sp. A3

The MtrA-MtrB two-component signal transduction system was first found in human pathogens Mycobacterium tuberculosis,and then found in many other bacteria and most of them are actinobacteria.Previous study have found that these signal transduction system,highly conserved in sequence and genomic organization, affects the stress protection,cell wall stability and proliferation of most bacteria. Arthrobacter sp.A3,high-G+C Gram-positive psychrotrophic bacterium,was isolated from the Number 1 Glacier permafrost.In this experiment we researched the MtrA-MtrB two-component signal transduction system of this bacterium,including cloning and sequenced analysising of mtrAB,expressing the fusion protein,and preparing the polyclonal antibodies.All of these contribute to the follow-up work. The results are as follows:(1) The mtrA,mtrB of Arthrobacter sp.A3 were obtained through TAIL-PCR (Thermal Asymmetric Interlaced),and highly conservation in sequences and genomic organization were found.Analysis of bioinformatics was done,including the prediction of gene ORF(Open Reading Frame) and protein secondary structure. These results suggest that this two-component signal transduction system is essential for the bacteria survival in harsh conditions.(2) Constructing expression plasmid pET-28-MtrA and pGEX-MtrB_(865-971). Recombinant protein MtrA-HiS₆ and gst-MtrB_(865-1971) were overexpressed in E.coli express system and purified by His and GST affinity column chromatography.(3) Polyclonal antibodies against His₆-MtrA and gst-MtrB_(865-1971) were obtained from rabbit and the specificity of the antibodies was checked by ELISA(Enzyme Linked Immunosorbent Assay) and Western blot analysis.The results allow the conclusion that the polyclonal antibodies are specific for the respective antigen and the antibody titer was more than 1:100 000. (4) Western blot analysis of total protein extracted from this bacterium at different temperatures was immunodecorate using the polyclonal antibodies produced in this work.The results show that the level of the intercellular MtrA and MtrB did not change significantly,but changed largely when H₂O₂ added in culture medium. In conclusion,we constructed the prokaryotic expression vectors of mtrA and mtrB from Arthrobacter sp.A3,expressed and purified His₆-MtrA and gst-MtrB_(865-1971) in vitro,and prepared the polyclonal antibodies.The concentration of MtrA and MtrB did not change significantly during the different temperature conditions but changed largely when H_O2 added,in the culture medium.Our results laid the foundation for the mechanism research of MtrA-MtrB two-component signal transduction system from Arthrobacter sp.A3.