Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.

Highlights d Phospholipid transport of Vibrio cholerae is silenced upon host entry d Reduced transporter activity triggers OMV release and increases colonization fitness d Increased vesiculation accelerates modulation of cell surface composition

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Outer membrane vesicle (OMV) release by Gramnegative bacteria has been observed and studied for decades. First considered as a by-product of cell lysis, it soon became evident that OMVs are actively secreted from the outer membrane (OM) of Gram-negative bacteria. Accordingly, these small particles (~ 10-300 nm in diameter) consist mainly of OM components like phospholipids (PLs), OM proteins, and lipopolysaccharides or lipooligosaccharides. However, OMVs may also comprise periplasmic, inner membrane, or cytoplasmic components. Since the shedding of substantial amounts of OM material represents a significant energy cost to the bacterial cell, OMV production must have some vital biological functions for Gram-negative bacteria. Indeed, intense research on that topic revealed that OMVs play important roles in bacterial physiology and pathogenesis, ranging from secretion and delivery of biomolecules (for example, toxins, DNA, or quorum sensing molecules) over stress response and biofilm formation to immunomodulation and adherence to host cells. Only recently researchers have begun to elucidate the mechanistic aspects of OMV formation, but a general mechanism for the biogenesis of these vesicles is still lacking. Here we review the findings and implications of our recent study published in Nature Communications (Roier S, et al. (2016) Nat. Commun. 7:10515), where we propose a novel and highly conserved bacterial OMV biogenesis mechanism based on PL accumulation in the outer leaflet of the OM. This mechanism might not only have important pathophysiological roles in vivo, but also represents the first general mechanism of OMV formation applicable to all Gram-negative bacteria.Current OMV biogenesis models are based on either loss or relocation of covalent linkages between the OM and the underlying peptidoglycan layer, require an accumulation of peptidoglycan fragments or misfolded proteins in the periplasmic space, or need the enrichment of speciesspecific membrane curvature-inducing molecules. However, these models are limited by the fact that they either require genetic manipulations, the presence of stress, or are only applicable to a single bacterial species hitherto. In order to discover a general and conserved mechanism of OMV formation within Gram-negative bacteria, we screened transposon mutants of Haemophilus influenzae, an opportunistic pathogen of the human respiratory tract with a relatively small genome size of 1.83 Mb, for altered OMV production. Amongst others, we identified gene disruptions in yrbB and yrbE that seem to increase vesiculation. These two genes are part of a gene cluster comprising three additional genes (yrbC, yrbD, and yrbF). A previous study by Malinverni and Silhavy suggested that in Escherichia coli, homologs of these five gene products, together with the VacJ protein, are important for maintaining the lipid asymmetry in the Gram-negative OM. This highly conserved ABC (ATPbinding cassette) transport system is thought to prevent PL accumulation in the outer leaflet of the OM by retrogra...