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

An Estimate of the Numbers and Density of Low-Energy Structures (or Decoys) in the Conformational Landscape of Proteins

The conformational energy landscape of a protein, as calculated by known potential energy functions, has several minima, and one of these corresponds to its native structure. It is however difficult to comprehensively estimate the actual numbers of low energy structures (or decoys), the relationships between them, and how the numbers scale with the size of the protein.We have developed an algorithm to rapidly and efficiently identify the low energy conformers of oligo peptides by using mutually orthogonal Latin squares to sample the potential energy hyper surface. Using this algorithm, and the ECEPP/3 potential function, we have made an exhaustive enumeration of the low-energy structures of peptides of different lengths, and have extrapolated these results to larger polypeptides.We show that the number of native-like structures for a polypeptide is, in general, an exponential function of its sequence length. The density of these structures in conformational space remains more or less constant and all the increase appears to come from an expansion in the volume of the space. These results are consistent with earlier reports that were based on other models and techniques

A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly

The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys-Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5Å resolution and of a two-domain (D2-D3) construct at 1.87Å resolution, show that each of the three Ig-like domains contains a single Lys-Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.open0

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

Factors Influencing <i>GUS </i>Expression in Cucumber (<i>Cucumis sativus </i>Linn.)

344-349The transformation efficiency of two strains of Agrobacterium tumefaciens, EHA 105 and LBA 4404 with three cucumber cultivars, viz. Endeavor, Green Long and Poinsett 76 have been investigated. Factors such as age of explants, co-cultivation period, efficiency of strains, acetosyringone treatment, use of Kanamycin and Basta as selection agents were assessed. GUS histochemical assay was performed to assess transformation efficiency. 5-day-old cotyledons and 3-day co-cultivation period for Endeavor and Green Long and 2-day co-cultivation period for Poinsett 76 were suitable for efficient transformation. EHA 105 was more virulent and the transformation efficiency was two to three folds higher than LBA 4404. Among the three cultivars, Endeavor was found to be more responsive to transformation as evidenced by GUS expression followed by Green Long and Poinsett 76. Acetosyringone (20μ M- 50μ M) enhanced the efficiency of transformation in all the three cultivars. Basta at 10 mg/l was more efficient selection agent than Kanamycin at 100 mg/l. Shoot regeneration occurred within 6 weeks after co-cultivation

Shoot regeneration from immature cotyledonary nodes in black gram [<i style="">Vigna</i> <i style="">mungo</i> (L.) Hepper]

551-555Eighteen-day-old immature cotyledonary node explants (18 d after anthesis) of black gram produced multiple shoots in MS salts+B5 vitamins containing medium in the presence of BA (1.0 mg/L), TDZ (0.1 mg/L) and AdS (15 mg/L). Maximum shoot proliferation (28 shoot/explant) occurred at the end of second subculture after 45 d. Periodic excision of regenerated shoots from explants increased shoot regeneration efficiency during subculture. The combination of TDZ and AdS with BA significantly increased shoot proliferation. Elongation and rooting were performed in GA3 (0.6 mg/L) and IBA (1.0 mg/L) containing media, respectively. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the field with a survival rate of 60%