Isolation and characterization of a second exe operon required for extracellular protein secretion in Aeromonas...

Strain C5.84 is a Tn5-751 insertion mutant of Aeromonas hydrophila which is unable to secrete extracellular proteins, instead accumulating them in the periplasm (B. Jiang and S.P. Howard, J. Bacteriol. 173:1241-1249, 1991). A 3.5-kb BglII fragment which complements this mutation was isolated from the chromosome of the parent strain. Analysis of this fragment revealed an operon-like structure with two complete genes, exeA and exeB, a functional promoter 5' to the exeA gene, and a 13-bp inverted repeat immediately 3' to the exeB gene. Although the transposon had inserted in exeA, provision of a wild-type copy of this gene alone in trans did not restore competence for export to C5.84. Complementation required the presence of both exeA and exeB, and marker exchange mutagenesis confirmed the requirement for both gene products for secretion. In vitro expression as well as analysis of the deduced amino acid sequence of ExeA indicated that it is a hydrophilic 60-kDa protein with a consensus ATP binding site. ExeB is a 25-kDa basic protein which shares limited homology with PulB, a protein of unknown function associated with the maltose regulon of Klebsiella oxytoca, and OutB, a protein which has been shown to be required for efficient secretion in Erwinia chrysanthemi. The hydrophilic character of these proteins and preliminary localization studies suggested that they are anchored to the inner membrane. These results demonstrate the involvement of a second operon encoding a putative ATP-binding protein in the secretion of extracellular proteins from gram-negative bacteria and further suggest that the cytoplasmic compartment may play a greater role in protein translocation across the outer membrane from the periplasm than previously thought.