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 temperatures and conditions the transport and assembly processes are so fast that assembly intermediates cannot be detected. Using cells grown at a low temperature to slow the assembly process and pulse-chase analysis with immunodetection 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 subassembly reactions. This kinetic analysis revealed that secB and secD mutants exhibit nearly identical defects in precursor translocation from the cytoplasm. However, subsequent subassembly reaction rates provided no clear evidence for an additional role for SecD in LamB assembly. Moreover, we found that surA mutants are qualitatively indistinguishable from yfgL mutants, suggesting that the products of both of these genes share a common function in the assembly process, most likely the delivery of LamB to the YaeT assembly complex in the outer membrane.The cell envelope of gram-negative bacteria, such as Escherichia coli, is composed of an outer membrane, an inner (cytoplasmic) membrane, and an aqueous space termed the periplasm that lies between them. The outer membrane bilayer is asymmetric; the outer leaflet contains lipopolysaccharides (LPS), and the inner leaflet contains phospholipids. Generally speaking, two types of proteins are found in the outer membrane. Lipoproteins have an amino-terminal lipid modification that anchors the proteins in the inner leaflet of the outer membrane. Integral outer membrane proteins (OMPs) adopt a three-dimensional -barrel conformation as a result of the packing of amphipathic antiparallel -strands (16,17,23). We want to understand outer membrane biogenesis, a process that occurs outside the cytoplasm in an environment that lacks obvious energy sources, such as ATP. In particular, we want to understand how OMPs are targeted to and assembled in the outer membrane.OMPs are synthesized initially as precursors (preproteins) with an amino-terminal extension known as the signal sequence (Fig. 1A). During synthesis, OMP polypeptides emerging from the ribosome are bound by the cytoplasmic chaperone SecB. SecB functions to maintain preproteins in an unfolded, translocation-competent state. In addition, SecB functions together with the signal sequence to direct the preprotein to the Sec translocation machinery in the inner membrane (18). The Sec machinery includes the membrane-associated ATPase, SecA (27), the channel-forming proteins SecYEG (26), and the associated proteins SecDF and YajC (5, 7). Although the exact role of the SecDF-YajC complex is not clea...