Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer.
Aeromonas hydrophila secretes protein toxins via the type II pathway, involving the products of at least two operons, exeAB (gspAB) and exeC-N (gspC-N). In the studies reported here, aerolysin secretion was restored to C5.84, an exeA::Tn5-751 mutant, by overexpression of exeD alone in trans. Expression studies indicated that these results did not reflect a role of ExeAB in the regulation of the exeC-N operon. Instead, immunoblot analysis showed that ExeD did not multimerize in C5.84, and fractionation of the membranes showed that the monomeric ExeD remained in the inner membrane. Expression of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion. Pulse-chase analysis also suggested that the induction of ExeAB allowed multimerization of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized even in the absence of ExeAB and, although most remained in the inner membrane, an amount similar to that in wild-type outer membranes fractionated with the outer membrane of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.
Ast
Vivian M
VM
Department of Biology, University of Regina, Saskatchewan, Canada.
Schoenhofen
Ian C
IC
Langen
Geoffrey R
GR
Stratilo
Chad W
CW
Chamberlain
M Dean
MD
Howard
S Peter
SP
D016980:Aeromonas hydrophila / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D017931:DNA Primers; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009876:Operon; D010957:Plasmids
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002352:Carrier Proteins; D017931:DNA Primers; C090402:ExeA protein, Aeromonas hydrophila; C090403:ExeB protein, Aeromonas hydrophila; C094204:ExeD protein, Aeromonas; D008565:Membrane Proteins; D026901:Membrane Transport Proteins