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J. Bacteriol. doi:10.1128/JB.01103-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Kinetic analysis of the assembly of the outer membrane protein LamB in Escherichia coli mutants each lacking a secretion or targeting factor in a different cellular compartment

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

	Abstract

Outer membrane -barrel proteins in Gram-negative bacteria such as Escherichia coli must be translocated from their site of synthesis in the cytoplasm to the periplasm and finally delivered to the outer membrane. At least a dozen proteins located in the cytoplasm, the periplasm, and both the inner and outer membranes are required to catalyze this complex assembly process. At normal growth temperature and conditions the transport and assembly processes are so fast that assembly intermediates cannot be detected. Using cells grown at low temperature to slow the assembly process, and pulse-chase analysis with immuno-detection methods, we followed newly synthesized LamB molecules during their transit through the cell envelope. The quality and reproducibility of the data allowed us to calculate rate constants for three different sub-assembly reactions. This kinetic analysis reveals that secB and secD mutants exhibit nearly identical defects in precursor translocation from the cytoplasm. However, subsequent sub-assembly reaction rates provide no clear evidence for an additional role for SecD in LamB assembly. Moreover, we find that surA mutants are qualitatively indistinguishable from yfgL mutants, suggesting that both of these gene products share a common function in the assembly process, most likely the delivery of LamB to the YaeT assembly complex in the outer membrane.

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