Three dimensional structure of bacterial pili

Crystallographic and associated biochemical and structural studies are in progress on the fiber-forming pilin proteins of the gonococcal pilus. Preparative scale purification procedures have been developed for the gonococcal pilin protein, which appear generally applicable to bacterial pilins. For three gonococcal pilin protein strains, we have obtained both reassembled pilus fibers and three-dimensional crystals. One needle-shaped crystal form of gonococcal C30 pilin diffracts beyond 3 Å resolution using synchrotron x-ray radiation. A diffraction data set to 3.5 Å resolution has been collected on these needle-shaped crystals (lattice spacings a=125.4(3) b=120.4(3), c=26.61(4) Å) in which the packing arrangement of the pilin subunits appears to resemble that seen in the pilus fibers using electron microscopy. X-ray diffraction data confirm our proposed model for the overall polypeptide fold of a pilin subunit, which is an antiparallel 4- helix bundle similar to tobacco mosaic virus coat protein and myohemerythrin.     

X-ray diffraction studies of bacterial pili

A sample of bacterial pili was prepared from Escherichia coli. An X-ray diffraction pattern was obtained from an oriented wet gel specimen in 0.01 m-phosphate buffer (pH 7.0) packed in a capillary tube. Sixteen independent spots were observed with the spacing of the outermost being 4.2 Å. Analysis of the diffraction pattern shows that the arrangement of subunits in pili rods is strictly simple-helical with 3.14₅ units being present in one turn of the helix and the axial rise per unit being 8.09_o Å.     

Analysis of factors determining sex differences in stress reactions of white rats

Female rats demonstrate more considerable increasing of corticosterone synthesis and secretion in comparison with male ones under the conditions of emotional and emotion-pain stress. These differences are not disappeared after castration. The sexual differences in stress reactions of infants are accompanied by lower sensitivity of their adaptation system in relation to stressors. The adult neonatal androgenized females show the same reactivity as normal females under the condition of emotion-pain stress. It is concluded that the sexual differences in stress reactions are genetically determined.     

Unveiling molecular interactions that stabilize bacterial adhesion pili

Adhesion pili assembled by the chaperone-usher pathway are superelastic helical filaments on the surface of bacteria, optimized for attachment to target cells. Here, we investigate the biophysical function and structural interactions that stabilize P pili from uropathogenic bacteria. Using optical tweezers, we measure P pilus subunit-subunit interaction dynamics and show that pilus compliance is contour-length dependent. Atomic details of subunit-subunit interactions of pili under tension are shown using steered molecular dynamics (sMD) simulations. sMD results also indicate that the N-terminal “staple” region of P pili, which provides interactions with pilins that are four and five subunits away, significantly stabilizes the helical filament structure. These data are consistent with previous structural data, and suggest that more layer-to-layer interactions could compensate for the lack of a staple in type 1 pili. This study informs our understanding of essential structural and dynamic features of adhesion pili, supporting the hypothesis that the function of pili is critically dependent on their structure and biophysical properties.     

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.     

Effect of chlorpromazine on conjugal plasmid transfer and sex pili

The major tranquillizer chlorpromazine (Cpz) inhibited the conjugal transfer of R and F'lac plasmids. The frequency of transfer of R-144 and R-100 plasmids was reduced with 2-3 log by Cpz at a concentration of 50-100 microgram/ml, while the frequency of RM-98 plasmid did not change under the same conditions. Cpz at 100 microgram/ml was an effective inhibitor of the transfer of F'lac plasmid. By means of electron microscopy and plaque assay, 100 microgram/ml Cpz was shown to reduce the adsorption rate of male specific ribonucleic acid phages MS-2 to the sides of F-pili. Common pili and flagellae seemed to be intact, but sex pili probably retracted in the presence of Cpz.     

Ribosomes from a relC mutant strain of Escherichia coli show altered activity with bacterial release factor-1

Ribosomes from a relC mutant of Escherichia coli, JF505, are altered in the large subunit protein L11. This protein has abnormal mobility on gel electrophoresis. The ribosomes have a lowered specific activity for release factor-1 which is intermediate between that found for ribosomes containing normal L11 and that for L11 lacking ribosomes. JF505 ribosomes are as sensitive to inactivation of in vitro termination by thiostrepton as normal ribosomes when the antibiotic is added in dimethylsulphoxide but less sensitive when it is added in ethanol.     

Twin ushers guide pili across the bacterial outer membrane

The chaperone/usher pathway is responsible for the assembly of adhesive pili on the surface of gram-negative pathogenic bacteria. In this issue, Remaut et al. (2008) present the crystal structure of the PapC usher translocation domain and images of the FimD usher bound to a pilus translocation intermediate. These new structures provide the first detailed view of a translocase in action.     

Pleiotropic effects of de-repressed bacterial sex factors on colicinogeny and cell wall structure

Summary Colonies of Escherichia coli or Salmonella typhimurium that form colicin I often produce larger inhibition zones when the sex factor of their plasmid is de-repressed. In liquid culture, virtually all colicin I is cell-bound; colicin titres with de-repressed factors are only slightly greater than with the wild type; no more covalently-closed plasmid DNA is present; and no more plasmid-determined enzyme is formed. The large zones are therefore unlikely to reflect an increase in the number of plasmid genomes per cell. De-repressed factors make the cells susceptible to lytic agents, indicating a change in the cell wall, which may result in greater release of cell-bound colicin and so increase the size of inhibition zones. Salmonella typhimurium LT2 carries a plasmid of unknown function.     

The role of bacterial pili in protein and DNA translocation.

Gram-negative bacteria have surface appendages that assemble via different secretion machineries. Recently, new experimental approaches have contributed to a better understanding of the molecular mechanisms of flagellar and pilus assembly, and protein secretion. These findings can be applied to plant pathogenic bacteria, which probably transfer effector proteins directly into their eukaryotic host cells. Here, it is suggested that assembly of Hrp pili occurs in the periplasm and that unfolded effector proteins attach to pilins within the pili, thus effecting protein translocation. A two-domain structure for the HrpA pilin from Pseudomonas syringae is also predicted.     

Structure and assembly of Gram-positive bacterial pili: unique covalent polymers

Bacterial pili are long, multi-subunit protein assemblies that extend from bacterial surfaces, mediating adhesion and colonisation. The recently characterised pili expressed by Gram-positive pathogens represent a novel variation; completely covalent polymers in which sortase-mediated isopeptide bonds link successive pilin subunits. Recent structural studies of the component pilins have revealed a common pattern of tandem immunoglobulin (Ig)-like domains, joined end-on-end. This long thin assembly is further stabilised by autocatalytically generated isopeptide bond crosslinks within the domains, joining Lys and Asn(or Asp) side chains. Specialised subunits at the tip and the base complete the assembly, with the tip pilins presenting novel adhesive structures.