Study Of The Growth Characteristics Under Low Temperature And Expression Of Cold Shock Protein Of Acinetobacter Johnsonii Strain DBP-3

The denitrifying bacterium Acinetobacter johnsonii strain DBP-3 which was capable of removing pollutants of phosphate, nitrate, and ammoniacal salt is psychrotolerant, and it have important implications for eutrophic water treatment of cold region. Currently, most researches about Acinetobacter johnsonii are focusing on its biodegraded characteristic, but the cold shock response mechanism was unclear. Cold shock protein(Csp), whose functioning is the cold adaptation mechanism of bacterium, can help cells to adapt to the low-temperature environment in different degrees.In this article, for better understanding the mechanisms of cold adaption for the psychrotolerant denitrifying bacterium Acinetobacter johnsonii DBP-3, the growth characteristics under different temperatures were measured by nephelometry, and an Arrhenius plot of the growth of strain DBP-3 was established for setting the cold shock temperature purpose. The csp genes were searched via NCBI and Universal Protein database, and identified by PCR and sequencing. To preliminarily find the key Csps in cold shock response, the expression characteristics at transcriptional level of csps under cold shock conditions were investigated using q RT-PCR. Furthermore, SDS-PAGE and mass spectrometry were performed to detect the Csps induced under cold shock conditions at translational level. To further verify the expression and effect of the key Csps at low temperatures, preparation of polyclonal antibody, Western Blot and overproducing of Csps in E. coli were performed. Additionally, the complete deduced protein sequences of the Csp3 and Csp4, together with the Csps of the strains possessing similar function with strain DBP-3 and representative bacterium, were used for the multiple sequence alignment and phylogenetic tree construction.The results showed that the optimal and the minimal temperature for the growth of A. johnsonii were 25 °C and below 5 °C, respectively. A temperature of 7.5 °C in the sub-range from 5 °C to 10 °C, which has a significant impact on the growth of strain DBP-3, was chosen for the cold shock experiments. In genome of Acinetobacter johnsonii, seven csps(temporarily named csp1 to csp7) were found and identified, and the m RNA level of csp3 and csp4 were significantly enhanced in the cold shock treatment group, which had an almost 400 fold change(P < 0.001). Furthermore, SDS-PAGE and Western Blot results showed that Csp3 and Csp4 proteins overproduced under cold shock treatment. In addition, phylogenetic analysis showed that the similarity percent between Csp3 and Csp4 was 76.85 %, and Csp3 and Csp4 belonged to Csp E family. Expression vectors of Csp3 and Csp4 were constructed successfully, the results showed that Csp4 can make E. coli grow under 7.5 °C.All the results above shows that Csp4 may play an important role in low temperature resistance of Acinetobacter johnsonii DBP-3, which are significant because they will serve as a theoretical underpinning for the studies on application of Acinetobacter johnsonii at low temperature and cold resistance mechanism of bacterium.