Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer

Molecular Microbiology - Tập 44 Số 1 - Trang 217-231 - 2002
Vivian M. Ast1, Ian C. Schoenhofen1, Geoffrey R. Langen1, Chad W. Stratilo1, M. Dean Chamberlain1, S. Peter Howard1
1Department of Biology; University of Regina; Saskatchewan; Canada

Tóm tắt

Summary 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 sug‐gested 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.

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Tài liệu tham khảo

Ausubel F.M., 1990, Current Protocols in Molecular Biology

10.1046/j.1365-2958.1998.00677.x

10.1093/emboj/17.20.6061

Bleves S., 1999, Structure‐function analysis of XcpP, a component involved in general secretory pathway‐dependent protein secretion in Pseudomonas aeruginosa, J Bacteriol, 181, 4012, 10.1128/JB.181.13.4012-4019.1999

10.1046/j.1365-2958.1999.01546.x

10.1006/jmbi.1999.3340

10.1016/0378-1119(84)90136-7

10.1074/jbc.271.5.2703

10.1099/00221287-146-3-639

10.1111/j.1365-2958.1992.tb01775.x

10.1046/j.1365-2958.1997.3531727.x

10.1006/jmbi.1996.0866

10.1128/jb.171.7.3673-3679.1989

10.1046/j.1365-2958.1999.01636.x

10.1128/jb.178.12.3544-3549.1996

10.1093/emboj/19.24.6697

10.1002/j.1460-2075.1996.tb00434.x

10.1046/j.1365-2958.1996.01539.x

10.1038/35020000

10.1111/j.1365-2958.1993.tb01949.x

Holtke H., 1992, Sensitive chemiluminescent detection of digoxigenin‐labelled nucleic acids: a fast and simple protocol and its applications, Biotechnology, 12, 104

10.1128/jb.175.20.6695-6703.1993

10.1046/j.1365-2958.1996.d01-1713.x

Ishidate K., 1986, Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope, J Biol Chem, 261, 428, 10.1016/S0021-9258(17)42490-2

10.1128/jb.176.22.6819-6826.1994

10.1128/jb.173.3.1241-1249.1991

10.1111/j.1365-2958.1992.tb00856.x

Katayama C., 1990, Single‐stranded DNA sequencing: a simplified protocol for single stranded DNA rescue, Strategies, 4, 56

10.1002/j.1460-2075.1993.tb05969.x

10.1046/j.1365-2958.1997.6141981.x

10.1128/JB.182.10.2761-2770.2000

10.1111/j.1462-5822.2007.00901.x

10.1074/jbc.272.17.11109

10.1111/j.1365-2958.1996.tb02499.x

10.1126/science.278.5343.1635

10.1126/science.284.5419.1516

10.1073/pnas.161170398

10.1111/j.1365-2958.1993.tb01744.x

10.1128/jb.170.6.2575-2583.1988

10.1016/0378-1119(91)90007-X

10.1073/pnas.96.14.8173

10.1093/emboj/19.10.2229

10.1016/S0021-9258(19)45127-2

10.1038/367292a0

10.1046/j.1365-2958.1996.431958.x

10.1038/35054089

10.1046/j.1365-2958.1997.3451726.x

Possot O.M., 1999, Membrane association and multimerization of secreton component PulC, J Bacteriol, 181, 4004, 10.1128/JB.181.13.4004-4011.1999

10.1046/j.1365-2958.2001.02403.x

10.1007/BF02818976

10.1093/embo-reports/kve042

10.1006/jmbi.1998.1791

10.1126/science.1411544

Sambrook J., 1989, Molecular Cloning: a Laboratory Manual

10.1046/j.1365-2958.2001.02403.x

10.1002/j.1460-2075.1995.tb07155.x

10.1073/pnas.74.12.5463

10.1046/j.1365-2958.1997.t01-1-00190.x

10.1093/emboj/19.10.2221

10.1046/j.1365-2958.1998.01011.x

10.1093/emboj/16.11.3007

10.1099/00221287-144-11-3219

Simon R., 1983, A broad host range mobilisation system for in vivo genetic engineering: transposon mutagenesis in gram‐negative bacteria, Biotechnology, 1, 784, 10.1038/nbt1183-784

10.1021/bi00453a018

10.1126/science.2814486

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Molecular Microbiology

Tập 44 Số 1

217-231

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