| Summary: | Thesis (M.Sc.)--Memorial University of Newfoundland, 1994. Medicine Bibliography: leaves [102]-118. Aeromonas hydrophila secretes many proteins into the extracellular milieu. Strain C5.84 is a Tn5-751 insertion mutant of this bacterium which is unable to secrete extracellular proteins, instead accumulating them in the periplasm (Jiang and Howard, 1991). A 3.5 kb Bgl II fragment which complements the mutation in C5.84 was isolated from the chromosome of the wild type strain and sequenced. Computer analysis of this fragment revealed an operon-like structure with two complete genes, exeA and exeB, a promoter 5' to the exeA gene and a 13 base pair 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 further established the requirement for both gene products for extracellular secretion. In addition, immunoblots with an anti-ExeB serum demonstrated that the Tn5-751 insertion in exeA had a polar effect on the expression of exeB. -- In vivo and in vitro expression studies confirmed that there is a promoter 5' to exeA and that exeA and exeB form an operon. 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 hydropathy analysis of these proteins and preliminary localization studies suggested that both ExeA and ExeB may be anchored to the inner membrane. -- These results demonstrate the requirement 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.
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