Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 mu M acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473-497) medium with 13.3 mu M benzylaminopurine, 2.6 mu M indole-3-acetic acid, 1 g l(-1) activated charcoal, 1.5 mg l(-1) phosphinothricin, and 300 mg l(-1) cefotaxime. Phosphinothricin at 1.5 mg l(-1) was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 mu M 6-benzylaminopurine and 1.5 mg l(-1) phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l(-1) phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta

Structure of the Full-Length Major Pilin from Streptococcus pneumoniae: Implications for Isopeptide Bond Formation in Gram-Positive Bacterial Pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili

Structural Differences between the Streptococcus agalactiae Housekeeping and Pilus-Specific Sortases: SrtA and SrtC1

The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a ‘lid’ in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the ‘lid’ mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis

Numerical computation of turbulent incompressible flow around aerofoils

The present study aims at calculation of 2D turbulent13; flow around aerofoil and its wake by solving the non-13; -linear coupled system of elliptic differential equations13; of motion for two-dimensional, turbulent, incompressible13; flows in Reynolds-averaged- form in general non-orthogonal13; coordinate system. The prediction procedure13; uses a standard K- f turbulence model embodied in13; a finite volume algorithm, originally developed by the13; first author at the Institute for hydromechanics, University13; Karlsruhe, Germany. The code is thoroughly modified13; to handle the boundary conditions at the cut line13; behind the aerofoil for the C-type grids used. Computations13; are carried out for NACA 0012 aerofoils at13; zero angle of attack and for a Korn-Garabadian Aerofoil13; at 5' angle of attack when asymmetric wake is developed. The prediction is validated against experimental results. Experimental validation confirms the prediction procedure to be reasonably accurate for surface pressure, skin-friction coefficient for attached boundary layer flows. In case of asymmetric wake, poor quantitative agreement between prediction and experiment may be attributed to the coarse grid, the upwind/central hybrid discretisation scheme and the inadequacy of Standard Wall Function in resolving the surface boundary layer specially at adverse pressure gradient

An investigation on the applicability of Cartesian grid approach to calculate flow over arbitrary terrain

465-472An investigation is made on the applicability of method of representing by Cartesian grid to calculate flow over natural terrain. Model hill geometry with different maximum slope angles is chosen and calculations by both Cartesian and Boundary-fitted coordinate representations are made. Computations are performed for laminar flow at different Reynolds numbers and turbulent flow conditions. Results in terms of mean velocity distribution, streamline, vorticity distribution and velocity vector close to the surface are analyzed and discussed. The study indicates that to calculate flow over undulating natural terrain, rectangular coordinate method of representing the geometry is able to capture most of the flow phenomena and it can be a viable alternative