Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A.
BACKGROUND
Type four secretion systems (TFSS) are bacterial macromolecular transport systems responsible for transfer of various substrates such as proteins, DNA or protein-DNA complexes. TFSSs encode two or three ATPases generating energy for the secretion process. These enzymes exhibit highest sequence conservation among type four 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 system of Aeromonas veronii plasmid pAC3249A. ATPases were expressed as His-tag fusion proteins in E. coli and purified by affinity chromatography. ATP binding and ATP hydrolysis experiments were performed with the purified ATPases. TraE and TraK showed strong binding to TNP-ATP and TNP-CTP (fluorescent analogs of ATP and CTP respectively) whereas TraJ showed weak binding. The optimum temperature range for the three ATPases was between 42 degrees C and 50 degrees C. Highest ATP hydrolysis activity for all the ATPases was observed in the presence of Mg2+ and Mn2+. However, TraJ and TraK also showed activity in the presence of Co2+. TraJ exhibited the highest specific activity 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 a conjugative plasmid from Aeromonas. Our study demonstrated that the three ATPases of a newly reported TFSS of A. veronii plasmid pAc3249A are functional in both ATP hydrolysis and ATP binding.
Molecular Biology Unit, National Centre for Cell Science, Pune 411007, India. yogesh@nccs.res.in.
Ashraf Y
AY
Rangrez
Mohammad Y
MY
Abajy
Walter
W
Keller
Yogesh
Y
Shouche
Elisabeth
E
Grohmann
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000333:Aeromonas / Q000201:enzymology*; D003035:Cobalt / Q000737:chemistry; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D055571:Secretory Pathway; D013696:Temperature
D011994:Recombinant Proteins; D003035:Cobalt; D008345:Manganese; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium