Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A

摘要

Background: Type four secretion systems (TFSS) are bacterial macromolecular transport systems responsible fortransfer of various substrates such as proteins, DNA or protein-DNA complexes. TFSSs encode two or three ATPasesgenerating energy for the secretion process. These enzymes exhibit highest sequence conservation among typefour secretion components.Results: Here, we report the biochemical characterization of three ATPases namely TraE, TraJ and TraK (VirB4,VirB11 and VirD4 homologs of the Agrobacterium tumefaciens transfer system, respectively) from the transfer systemof Aeromonas veronii plasmid pAC3249A. ATPases were expressed as His-tag fusion proteins in E. coli and purifiedby affinity chromatography. ATP binding and ATP hydrolysis experiments were performed with the purifiedATPases. TraE and TraK showed strong binding to TNP-ATP and TNP-CTP (fluorescent analogs of ATP and CTPrespectively) whereas TraJ showed weak binding. The optimum temperature range for the three ATPases wasbetween 42°C and 50°C. Highest ATP hydrolysis activity for all the ATPases was observed in the presence of Mg~(2+)and Mn~(2+). However, TraJ and TraK also showed activity in the presence of Co~(2+). TraJ exhibited the highest specificactivity of all the three ATPases with vmax 118 ± 5.68 nmol/min/mg protein and KM 0.58 ± 0.10 mM.Conclusions: This is the first biochemical characterization of conjugative transport ATPases encoded by aconjugative plasmid from Aeromonas. Our study demonstrated that the three ATPases of a newly reported TFSS ofA. veronii plasmid pAc3249A are functional in both ATP hydrolysis and ATP binding.