Heterologous expression of Microlunatus phosphovorus regulatory protein Mlp21700 and its binding to promoters of Poly-P metabolic genes in vitro

Abstract:

[Background] Microlunatus phosphovorus is one of the important phosphorus accumulating organisms. It can accumulate polyphosphate (Poly-P) aerobically and hydrolyze Poly-P anaerobically, a process fine-tuned by specific regulatory genes. [Objective] To investigate Poly-P metabolic genes bound by Mlp21700, electrophoretic mobility shift assay (EMSA) was performed. [Methods] The coding sequence of Mlp21700 was amplified and cloned into pET28a to generate a recombinant plasmid pET28a-21700 that was then transformed into Transetta(DE3) to express the recombinant protein Mlp21700. Mlp21700 was purified under non-denaturing conditions. Promoter sequences of Poly-P metabolic genes were amplified by PCR, labeled with biotin, and used as probes in EMSA assays to investigate binding of Mlp21700 to the above probes. [Results] pET28a-21700 was confirmed by DNA sequencing and enzyme digestion. SDS-PAGE analysis indicated that Mlp21700 was highly expressed in soluble form. The purity of Mlp21700 surpassed 90% and the concentration reached 0.64 mg/mL. Our EMSA assays showed that Mlp21700 bound to the promoters of Mlp26610 (ppgk) and Mlp44770 (ppx). [Conclusion] Mlp21700 may directly regulate Mlp26610 and Mlp44770, and thus regulate Poly-P metabolism.