Construction Of Mesorhizobium Huakuii CtrA Mutant And Preliminary Screening For Its Host Interacting Protein

The ctrA gene exits in alpha-proteobacteria widespreadly. It’s a regulatory factor for the cell-cycle progression and the generation of asymmetry, and acts as a response regulator (RR). The growth of bacterioid involves rhizobium’s cell division and differentiation. It is considered that ctrA may takes part in rhizobium bacterioid differentiation or symbiotic nitrogen fixation, or even proberboly being interactive with possible unknown protein of the host plant, but the relevant studies and literatures are quite few.In this work, firstly, we tried to construct Mesorhizobium huakuii ctr A-inactivated mutant by constructing homologues replacement plasmid pJQ200SK-ctr A, but unfortunately, we did not obtain the expected mutant. Considering that the ctrA gene might be indispensible for the living of rhizobia7653R, the mutant would not be screened out directly, so we changed the approach to reconstruct the ctrA low-level-expression plasmid pHN-ctr A and another double-cross replacement plasmid, these two constructs have laid a foundmentary basis for the further screening of the target mutant.Meanwhile, we reconstructed a constitutive-expression plasmid pBBR-ctrA and introduced some extra copies of the ctrA into M. huakuii7653R. It was found that the growth rate remained the same, but the symbiotic phenotypes showed less root nodule formation and lower nitrogen fixation enzymatic activity. Real time fluorescent quantitation PCR showed that the ctrA gene was constitutively expressed in the free-living cells and symbiotic bacteroids, and the expression levels displayed no significant difference for the bacteroids in the different development staged root nodules, but with a bit increase in the ctrA expression level with some extra copies introduced by pBBR-ctrA plasmid.In addition, we used ctrA as a bait to screen potential target proteins in the Astragalus sinicus AD-cDNA library. Four positive candidate clones were obtained; one of them encoded a JUN kinase activation domain binding protein, which was important in the singal transduction pathway. Finally, basing on the conservative CtrA Box sequences, we did a search of the potential genes of M. huakuii7653R whose transcription would be regulated by the CtrA. As a result, a total of64genes were found in the7653R genome. A new phenomenon was observed that CtrA could regulate a few (novel) genes involved in the symbiotic root nodule formation and nitrogen fixation, such as opa22, minCD, ftsZ and etc.In summary, the results generated from this work have laid a solid foundation, and provides some new information to the future investigation on the exact function and mechanism of ctrA in the regulation of bacterioid differation and nitrogen fixation.