The cell wall envelope of Gram-positive bacteria can be thought of as a surface organelle for the assembly of macromolecular structures that enable the unique lifestyle of each microorganism. Sortases - enzymes that cleave the sorting signals of secreted proteins to form isopeptide (amide) bonds between the secreted proteins and peptidoglycan or polypeptides - function as the principal architects of the bacterial surface. Acting alone or with other sortase enzymes, sortase construction leads to the anchoring of surface proteins at specific sites in the envelope or to the assembly of pili, which are fibrous structures formed from many protein subunits. The catalysis of intermolecular isopeptide bonds between pilin subunits is intertwined with the assembly of intramolecular isopeptide bonds within pilin subunits. Together, these isopeptide bonds endow these sortase products with adhesive properties and resistance to host proteases.

/pdf/architects-at-the-bacterial-surface-sortases-and-the-1llv073jm6.pdf

Architects at the bacterial surface — sortases and the assembly of pili with isopeptide bonds

The immunoglobulin (Ig) protein domain is widespread in nature having a well-recognized role in proteins of the immune system. In this review, we describe the proteins containing Ig-like domains in Escherichia coli and enterobacteria, reporting their structural and functional properties, protein folding, and diverse biological roles. In addition, we cover the expression of heterologous Ig domains in E. coli owing to its biotechnological application for expression and selection of antibody fragments and full-length IgG molecules. Ig-like domains in E. coli and enterobacteria are frequently found in cell surface proteins and fimbrial organelles playing important functions during host cell adhesion and invasion of pathogenic strains, being structural components of pilus and nonpilus fimbrial systems and members of the intimin/invasin family of outer membrane (OM) adhesins. Ig-like domains are also found in periplasmic chaperones and OM usher proteins assembling fimbriae, in oxidoreductases and hydrolytic enzymes, ATP-binding cassette transporters, sugar-binding and metal-resistance proteins. The folding of most E. coli Ig-like domains is assisted by periplasmic chaperones, peptidyl–prolyl cis/trans isomerases and disulfide bond catalysts that also participate in the folding of antibodies expressed in this bacterium. The technologies for expression and selection of recombinant antibodies in E. coli are described along with their biotechnological potential.

http://digital.csic.es/bitstream/10261/75605/1/FEMSREV%20final.pdf

Immunoglobulin domains in Escherichia coli and other enterobacteria: from pathogenesis to applications in antibody technologies.

Biogenesis and adhesion of type 1 and P pili.

Pilus biogenesis at the outer membrane of Gram-negative bacterial pathogens.

Gram-negative bacteria assemble a variety of surface structures, including the hair-like organelles known as pili or fimbriae. Pili typically function in adhesion and mediate interactions with various surfaces, with other bacteria, and with other types of cells such as host cells. The chaperone/usher (CU) pathway assembles a widespread class of adhesive and virulence-associated pili. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and integral outer membrane protein termed the usher, which forms a multifunctional assembly and secretion platform. This review addresses the molecular and biochemical aspects of the CU pathway in detail, focusing on the type 1 and P pili expressed by uropathogenic Escherichia coli as model systems. We provide an overview of representative CU pili expressed by E. coli and Salmonella, and conclude with a discussion of potential approaches to develop antivirulence therapeutics that interfere with pilus assembly or function.

/pdf/pili-assembled-by-the-chaperone-usher-pathway-in-escherichia-22y5odmjmc.pdf

Pili Assembled by the Chaperone/Usher Pathway in Escherichia coli and Salmonella.

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

/pdf/biomolecular-solid-state-nmr-spectroscopy-at-1200-mhz-the-3zf8u5lnk1.pdf

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution

The outer membrane usher guarantees the formation of functional pili by selectively catalyzing donor-strand exchange between subunits that are adjacent in the mature pilus.

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins and viral capsids and others. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

/pdf/biomolecular-solid-state-nmr-spectroscopy-at-highest-field-19vm0fvez1.pdf

Rudolf Glockshuber

Papers

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution

The outer membrane usher guarantees the formation of functional pili by selectively catalyzing donor-strand exchange between subunits that are adjacent in the mature pilus.

Biomolecular solid-state NMR spectroscopy at highest field: the gain in resolution at 1200 MHz