A Study On Two-component Signal Systems Of Kocuria Rhizophila DC2201

The two-component system(TCS)is an universal mechanism for bacterial sensing and responsing environments.It consists of histidine kinase(HK)and response regulator(RR).They enable the body to respond appropriately to changes in environmental conditions.Kocuria rhizophila is a commonly used quality control strain for antibiotic activity testing,but current studies on its TCS are still rare,and it had only been reported in DC2201 strain.In this paper,we used the bioinformatics and modern microbial molecular biology methods and techniques to study the TCS of K.rhizophila.The processes and results are as follows:1.The numbers,structures and functions of TCSs of 8 strains of K.rhizophila with known sequences were analyzed by various bioinformatics methods.The results showed that there were 13 TCSs in K.rhizophila,and HKs and RRs were modular proteins involved in external pressure response,cell wall synthesis,enzyme activity,material metabolism and transportation.2.Eukaryotic expression vectors pGBT9-HK8700 and pGAD10-RR8701 were constructed by yeast two-hybrid system using HK8700 and RR8701 as bait proteins and prey proteins respectively.The vectors were transformed into yeast AH109 by PEG/LiAc method.The interaction between HK8700 and RR8701 was confirmed by screening various amino acid deletion mediums.3.Prokaryotic expression vectors pET-29a(+)-HK8700* and pPGH-RR8701 were constructed by specific amplification of hk8700* and rr8701 by PCR.A large number of soluble proteins HK8700* and RR8701 were obtained by IPTG induction and affinity chromatography column elution.The purified protein HK8700* was first autophosphorylated and then phosphorylated with RR8701.The phosphorylated protein on acrylamide was stained with methyl green,which confirmed that HK8700-RR8701 was a pair of TCS.4.The response regulatory protein gene rr8701 in the TCS HK8700-RR8701 was used as the target gene,and the temperature-sensitive plasmid pKC1139 was used as the vector to amplify the upstream and downstream fragments of rr8701 and the chloramphenicol gene cat on the plasmid pIJ790.The knockout box UCD was obtained by fusion PCR,and the knockout vector pKC1139-UCD of rr8701 gene was constructed.The above results will provide a basis for the construction of its TCS regulatory network.The constructed knockout vector pKC1139-UCD laid a foundation for exploring the function of HK8700-RR8701,establishing the genetic transformation system of K.rhizophila,and promoting its industrial application.