The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport.

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin.

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

Unlabelled: The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface.

Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane.

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts.

Lipopolysaccharide transport to the cell surface: periplasmic transport and assembly into the outer membrane.

Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM.

Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis.

The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available.

D-amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis.

The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall.

Forming cross-linked peptidoglycan from synthetic gram-negative Lipid II.

The bacterial cell wall precursor, Lipid II, has a highly conserved structure among different organisms except for differences in the amino acid sequence of the peptide side chain. Here, we report an efficient and flexible synthesis of the canonical Lipid II precursor required for the assembly of Gram-negative peptidoglycan (PG). We use a rapid LC/MS assay to analyze PG glycosyltransfer (PGT) and transpeptidase (TP) activities of Escherichia coli penicillin binding proteins PBP1A and PBP1B and show that the native m-DAP residue in the peptide side chain of Lipid II is required in order for TP-catalyzed peptide cross-linking to occur in vitro.

Dimethyl sulfoxide and ethanol elicit increased amyloid biogenesis and amyloid-integrated biofilm formation in Escherichia coli.

Escherichia coli directs the assembly of functional amyloid fibers termed "curli" that mediate adhesion and biofilm formation. We discovered that E. coli exhibits a tunable and selective increase in curli protein expression and fiber assembly in response to moderate concentrations of dimethyl sulfoxide (DMSO) and ethanol.