Phosphorylation Of Brucella Two-Component Regulatory System In Vitro

Two-component systems (TCS) are typically composed of a membrane-located sensor with histidine kinase activity and a cytoplasmic transcriptional regulator, response regulator. TCSs are widely used signal transduction devices which respond to changing growth conditions in bacteria. Generally, stimuli detected by these systems are transformed into a cellular signal by autophosphorylation of the sensor proteins at a conserved histidine residue. The phosphorylated histidine of these sensor proteins then transform it into an aspartic acid residue in the so-called 'receiver domain' of the response regulator. Phosphorylation of the regulatory proteins have functions of transcription factor which regulate expression of some genes to adapt the surroundings. During pathogenic bacteria infectious cycle, TCSs are frequently used to efficiently adapt to different niches inside and outside of their host organisms, therefore TCSs can be considered as an essential prerequisite for their pathogenicity.Although a little progress has been made in studying the contribution of TCS to brucella virulence, in the elucidation of basic principles of the signal transduction process itself is insufficiently recognized. In the study, Five histidine kinases and eight response regulators of Brucella melitensis were selected from NCBI, expressed and purified and then study their autophosphorylation in vitro. The five histidine kinases were BMEI0374, BvrS, TceS, TcfS and CenK. the forward primer used for PCR amplification of a histidine kinase were designed to purify the cytoplasmic, soluble kinase domain of a histidine kinase. Clone PCR amplicons into the pET-28a vector and transform them into E. coli BL21 cells for IPTG induced protein expression. The expressed product were identified by SDS-PAGE and captured and purified using a His-tagged Ni affinity column. BMEI0374, BvrS, TceS and TcfS were purified for autophosphorylation, but only bvrS could be tested for autophosphorylation. These results provided a good groundwork for further research on the phospho transfer profiling of Brucella melitensis two-component signal transduction systems and screening novel histidine inhibitor.Full-length response regulators can be cloned, expressed, and purified as done for the histidine kinases. Except for RR4 and RR10,16 other proteins were successfully expressed and purified in Escherichia coli. After phosphorylation with acetylphosphate, we found that eleven (RR1, RR2, RR3, RR5, RR6, RR7, RR8, RR11, RR13, RR14, RR15 and RR16) of sixteen exhibited an enhanced resistance towards proteolysis, while the other four did not. The results suggested that RR1, RR2, RR3, RR5, RR6, RR7, RR8, RR11, RR13, RR14, RR15 and RR16 preincubated with acetylphosphate induced a conformational change, but RR9, RR12, RR17 and RR18 did not. The generation of phosphorylated response regulators with acetylphosphate will facilitate further selection of the genes which regulated by the response regulators in the absence of auxiliary kinases in vitro.