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The bacterial pathogen Staphylococcus aureus forms multicellular communities known as biofilms in which cells are held together by an extracellular matrix principally composed of repurposed cytoplasmic proteins and extracellular DNA. These biofilms assemble during infections or under laboratory conditions by growth on medium containing glucose, but the intracellular signal for biofilm formation and its downstream targets were unknown. Here, we present evidence that biofilm formation is triggered by a drop in the levels of the second messenger cyclic-di-AMP. Previous work identified genes needed for the release of extracellular DNA, including genes for the cyclic-di-AMP phosphodiesterase GdpP, the transcriptional regulator XdrA, and the purine salvage enzyme Apt. Using a cyclic-di-AMP riboswitch biosensor and mass spectrometry, we show that the second messenger drops in abundance during biofilm formation in a glucose-dependent manner. Mutation of these three genes elevates cyclic-di-AMP and prevents biofilm formation in a murine catheter model. Supporting the generality of this mechanism, we found that gdpP was required for biofilm formation by diverse strains of S. aureus. We additionally show that the downstream consequence of the drop in cyclic-di-AMP is inhibition of the "accessory gene regulator" operon agr, which is known to suppress biofilm formation through phosphorylation of the transcriptional regulator AgrA by the histidine kinase AgrC. Consistent with this, an agr mutation bypasses the block in biofilm formation and eDNA release caused by a gdpP mutation. Finally, we report the unexpected observation that GdpP inhibits phosphotransfer from AgrC to AgrA, revealing a direct connection between the phosphodiesterase and agr. [ABSTRACT FROM AUTHOR]

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a [beta]-barrel structure.sup.1,2. [beta]-Barrels are sheets of [beta]-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane.sup.3-5. One subunit of the machines is itself a [beta]-barrel protein that has a central role in folding other [beta]-barrels. In Gram-negative bacteria, the [beta]-barrel assembly machine (BAM) consists of the [beta]-barrel protein BamA, and four lipoproteins.sup.5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP).sup.9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid [beta]-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies.sup.10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete. Cryo-electron microscopy structures of a folding intermediate on the BAM complex of Escherichia coli reveal how interactions between the BamA catalyst and substrate permit stable association during folding, followed by rapid turnover., Author(s): David Tomasek [sup.1] [sup.2] , Shaun Rawson [sup.3] , James Lee [sup.1] [sup.2] [sup.5] , Joseph S. Wzorek [sup.2] [sup.6] , Stephen C. Harrison [sup.3] [sup.4] , Zongli Li [...]

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide.sup.1. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface.sup.1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown.sup.2. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient. Crystal structures of a five-protein complex comprising the inner-membrane components of the bacterial lipopolysaccharide transporter provide insight into the mechanism of extraction of lipopolysaccharide from the inner membrane and its transport to the outer membrane., Author(s): Tristan W. Owens [sup.1] , Rebecca J. Taylor [sup.1] , Karanbir S. Pahil [sup.1] , Blake R. Bertani [sup.2] , Natividad Ruiz [sup.2] , Andrew C. Kruse [sup.3] , [...]

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time‐resolved Förster resonance energy transfer (TR‐FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non‐polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors. [ABSTRACT FROM AUTHOR]