Gram-negative bacteria represent a major public health concern due to their high resistance to antibiotics resulting in millions of human deaths world-wide each year. Their multilayered envelope contains an outer membrane (OM) that forms an effective permeability barrier shielding against noxious molecules, including several antibiotics. Being exposed to the cell surface, the OM represents a promising target for the development of new antimicrobials that can act from the exterior of the cell. The design of new antimicrobial stategies urges a better understanding of the molecular pathways of OM biogenesis. Integral OM proteins are crucial for envelope homeostasis. The beta-barrel assembly machinery (BAM) plays an essential role in OM protein assembly. The activity of BAM is regulated in space and time ensuring the constant supply of protein components to active sites of OM biogenesis. Many questions remain unresolved concerning the protein folding reaction mediated by BAM and the regulation of its activity throughout the OM. Motivated by the need to better understand the biogenesis of the bacterial OM, we have discovered that in the enterobacterium Escherichia coli, a member of gamma-proteobacteria, the lipoprotein DolP associates with the BAM complex and plays a critical role in OM homeostasis and integrity. DolP is widely conserved in gamma-, beta- and some alpha-proteobacteria contributing to the virulence of several pathogens, as well as to their ability to survive in the presence of some antibiotics. Our preliminary data reveal that DolP directly interacts with BamA, the catalytic subunit of BAM, promoting BamA folding and function. Inactivation of DolP phenocopies BamA depletion and makes cells sensitive to antibiotics that are normally excluded by Gram-negative bacteria. DolP localizes at active sites of OM biogenesis, ideally positioned to support BAM activity. The molecular mechanisms by which DolP contributes to OM assembly by the BAM complex and ensures OM integrity remain to be established. Our project uses an interdisciplinary approach to determine how DolP interacts with the BAM complex, influences its organization with partner complexes and regulatory factors, and supports its OM protein assembly activity. By employing a multiscale experimental strategy, we are investigating the molecular processes mediated by DolP i) at the cell envelope-wide scale, ii) in a chemically defined in vitro system, and iii) at the structural level. We will conduct these studies in the enterobacterial model organism E. coli and test our results in other pathogens of the gamma- and beta-proteobacterial classes. Our results will be important for the research of new antibacterial compounds that can interfere with OM integrity in Gram-negative bacterial pathogens.