β-barrel membrane proteins play important structural and functional roles in Gram negative bacteria and in mitochondria and chloroplasts of eukaryotes. A conserved machine is responsible for the folding and insertion of β-barrel membrane proteins but its mechanism remains largely unknown. In E. coli, a five protein β-barrel assembly machine (Bam) assembles β-barrel proteins into the outer membrane (OM). Among all β-barrel membrane proteins in E. coli , the β-barrel component of the OM LPS translocon is one of only two essential β-barrels, the other being the central component of the Bam machinery itself. The OM LPS translocon, which consists of OM β-barrel protein LptD (lipopolysaccharide transport) and OM lipoprotein LptE, is responsible for the final export of LPS molecules into the outer leaflet of the OM, resulting in an asymmetric bilayer that blocks the entry of toxic molecules such as antibiotics. This thesis describes the characterization of the biogenesis pathway of the OM LPS translocon and its folding and insertion into the OM by the Bam machinery. An in vivo S35-Methionine pulse-labeling assay was developed to identify intermediates along the biogenesis of the OM LPS translocon. Seven intermediates were identified along the pathway. We show that proper assembly of the OM LPS translocon involves an oxidative disulfide bond rearrangement from a nonfunctional intermediate containing non-native disulfides. We also found that the rate determining step of OM LPS translocon biogenesis is β-barrels folding process by the Bam machinery. Using in vivo chemical crosslinking, we accumulated and trapped a mutant form of LptD on BamA, the central component of the Bam machinery. We extended the S35-Methionine pulse-labeling method to allow chemical crosslinking of substrates on the Bam complex and trapped LptD while it was being folded on the Bam machine. We demonstrated that the interaction between LptD and BamA is independent of LptE, while that between LptD and BamD, the other essential component of the Bam complex beside BamA, is LptE dependent. Based on these findings, we proposed a model of Bam-assisted folding of the OM LPS translocon in which LptE templates the folding of LptD. / Chemistry and Chemical Biology