Molecular and biochemical characterization of ExeAB, two components involved in the type II secretion pathway of Aeromonas hydrophila

The objective of this study was to investigate the role of ExeA in the Aeromonas hydrophila type II secretion pathway. Pulse-chase analysis indicated that ExeA was stabilized by coexpression with ExeB. From in vivo cross-linking analyses, both proteins were found to participate in a complex of approximately 85 kDa, each requiring the presence of the other protein. Radiolabeling followed by cross-linking, immunoprecipitation and then reduction of the cross-links confirmed the presence of the two proteins in the same complex. The complex could also be extracted intact from cell membranes with non-ionic detergents. In addition, gel filtration analysis performed with octyl-glucoside soluble membrane extracts suggested the existence of a native heterododecameric ExeAB complex.; Examination of a collection of ExeA substitution derivatives, altered in consensus ATP binding/hydrolysis motifs located near its N-terminus, suggested that ATP binding by ExeA is required for ExeAB complex formation, while hydrolysis is required for its function in secretion. To directly examine ExeA function, the N-terminal cytoplasmic domain of ExeA with the addition of a C-terminal hexahistidine tag (cytExeA) was overproduced in Escherichia coli and purified by metal chelate affinity chromatography followed by anion-exchange chromatography. Purified preparations of cytExeA were found to exhibit ATPase activity dependent on the presence of a divalent cation. Enzymatic characterization of cytExeA revealed an optimum reaction temperature of approximately 37–40°C and a pH of 7–8. As well, Mg2+ was the preferred cation; cytExeA exhibited an apparent K_m for Mg-ATP of 1.2 mM and a V_max of 1.1 nmoles min−1 mg−1 of protein. cytExeA was also able to utilize CTP, GTP and UTP as substrates. As well, inhibitors of F-type ATPases were found to significantly reduce cytExeA ATPase activity. Finally, ATPase and GTPase activities were approximately 6-fold higher for cytExeA containing the amino acid substitution K56R.; Lastly, the multimerization of the putative secretion port ExeD was found to be dependent on both exeA and exeB expression, which further correlated with the formation of functional ExeAB complex and secretion. These findings support a model whereby ExeAB delivers ATP hydrolysis energy to the type II secreton facilitating the correct localization and multimerization of ExeD.