Jeffrey Meisner

The linear homopolymer poly-beta-1,6-N-acetyl-D-glucosamine (beta-1,6-GlcNAc; PGA) serves as an adhesin for the maintenance of biofilm structural stability in diverse eubacteria. Its function in Escherichia coli K-12 requires the gene products of the pgaABCD operon, all of which are necessary for biofilm formation. PgaC is an apparent glycosyltransferase that is required for PGA synthesis. Using a monoclonal antibody directed against E. coli PGA, we now demonstrate that PgaD is also needed for PGA formation. The deletion of genes for the predicted outer membrane proteins PgaA and PgaB did not prevent PGA synthesis but did block its export, as shown by the results of immunoelectron microscopy (IEM) and antibody adsorption assays. IEM also revealed a conditional localization of PGA at the cell poles, the initial attachment site for biofilm formation. PgaA contains a predicted beta-barrel porin and a superhelical domain containing tetratricopeptide repeats, which may mediate protein-protein interactions, implying that it forms the outer membrane secretin for PGA. PgaB contains predicted carbohydrate binding and polysaccharide N-deacetylase domains. The overexpression of pgaB increased the primary amine content (glucosamine) of PGA. Site-directed mutations targeting the N-deacetylase catalytic activity of PgaB blocked PGA export and biofilm formation, implying that N-deacetylation promotes PGA export through the PgaA porin. The results of previous studies indicated that N-deacetylation of beta-1,6-GlcNAc in Staphylococcus epidermidis by the PgaB homolog, IcaB, anchors it to the cell surface. The deletion of icaB resulted in release of beta-1,6-GlcNAc into the growth medium. Thus, covalent modification of beta-1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative and gram-positive species, dictated by cell envelope differences.

We demonstrate a new method of achieving rectification in single molecule devices using the high-bias properties of gold-carbon bonds. Our design for molecular rectifiers uses a symmetric, conjugated molecular backbone with a single methylsulfide group linking one end to a gold electrode and a covalent gold-carbon bond at the other end. The gold-carbon bond results in a hybrid gold-molecule "gateway" state pinned close to the Fermi level of one electrode. Through nonequilibrium transport calculations, we show that the energy of this state shifts drastically with applied bias, resulting in rectification at surprisingly low voltages. We use this concept to design and synthesize a family of diodes and demonstrate through single-molecule current-voltage measurements that the rectification ratio can be predictably and efficiently tuned. This result constitutes the first experimental demonstration of a rationally tunable system of single-molecule rectifiers. More generally, the results demonstrate that the high-bias properties of "gateway" states can be used to provide additional functionality to molecular electronic systems.

Electronic factors in molecules such as quantum interference and cross-conjugation can lead to dramatic modulation and suppression of conductance in single-molecule junctions. Probing such effects at the single-molecule level requires simultaneous measurements of independent junction properties, as conductance alone cannot provide conclusive evidence of junction formation for molecules with low conductivity. Here, we compare the mechanics of the conducting para-terminated 4,4'-di(methylthio)stilbene and moderately conducting 1,2-bis(4-(methylthio)phenyl)ethane to that of insulating meta-terminated 3,3'-di(methylthio)stilbene single-molecule junctions. We simultaneously measure force and conductance across single-molecule junctions and use force signatures to obtain independent evidence of junction formation and rupture in the meta-linked cross-conjugated molecule even when no clear low-bias conductance is measured. By separately quantifying conductance and mechanics, we identify the formation of atypical 3,3'-di(methylthio)stilbene molecular junctions that are mechanically stable but electronically decoupled. While theoretical studies have envisaged many plausible systems where quantum interference might be observed, our experiments provide the first direct quantitative study of the interplay between contact mechanics and the distinctively quantum mechanical nature of electronic transport in single-molecule junctions.

A series of tripodal receptors preorganize electron-deficient aromatic rings to bind halides in organic solvents using weak sigma anion-to-arene interactions or C-H...X- hydrogen bonds. 1H NMR spectroscopy proves to be a powerful technique for quantifying binding in solution and determining the interaction motifs, even in cases of weak binding.

The peptidoglycan (PG) sacculus, a meshwork of polysaccharide strands cross-linked by short peptides, protects bacterial cells against osmotic lysis. To enlarge this covalently closed macromolecule, PG hydrolases must break peptide cross-links in the meshwork to allow insertion of new glycan strands between the existing ones. In the rod-shaped bacterium Bacillus subtilis, cell wall elongation requires two redundant endopeptidases, CwlO and LytE. However, it is not known how these potentially autolytic enzymes are regulated to prevent lethal breaches in the cell wall. Here, we show that the ATP-binding cassette transporter-like FtsEX complex is required for CwlO activity. In Escherichia coli, FtsEX is thought to harness ATP hydrolysis to activate unrelated PG hydrolases during cell division. Consistent with this regulatory scheme, B. subtilis FtsE mutants that are unable to bind or hydrolyse ATP cannot activate CwlO. Finally, we show that in cells depleted of both CwlO and LytE, the PG synthetic machinery continues moving circumferentially until cell lysis, suggesting that cross-link cleavage is not required for glycan strand polymerization. Overall, our data support a model in which the FtsEX complex is a remarkably flexible regulatory module capable of controlling a diverse set of PG hydrolases during growth and division in different organisms.