Three-coordinate organometallic complexes are rare, especially with the prototypical methyl ligand. Using a hindered, rigid bidentate ligand (L), it is possible to create 12-electron methyliron(II) and 13-electron methylcobalt(II) complexes. These complexes are thermally stable, and 1 H NMR spectra suggest that the low coordination number is maintained in solution. Attempts to create the 14-electron LNiCH3 led instead to the three-coordinate nickel(I) complex LNi(THF). Single crystals of LMCH3 are isomorphous with the new three-coordinate chloride complexes LNiCl and LCoCl. Along with the recently reported LFeCl (Smith, J. M.; Lachicotte, R. J.; Holland, P. L. Chem. Commun. 2001, 1542), these are the only examples of threecoordinate iron(II), cobalt(II), and nickel(II) complexes with terminal chloride ligands, enabling the systematic evaluation of the effect of coordination number and metal identity on M-Cl bond lengths. Electronic structure calculations predict the ground states of the trigonal complexes.
This paper is the first detailed description of the development and use of new genetic tools specifically for the safe manipulation of highly pathogenic Francisella tularensis subsp. tularensis. Most of these tools are also demonstrated to work with other F. tularensis subspecies. Kanamycin and hygromycin resistance determinants that function as genetic markers in F. tularensis subsp. tularensis strain Schu and sets of episomal shuttle vectors that are either unstable or stably maintained in the absence of selection were developed. In addition, the hyg gene, expressed from the F. tularensis groESL promoter, was successfully used as a marker for transposon mutagenesis. This work also includes the development of sacB-based suicide plasmids expressing kanamycin resistance that can be used for electroporation-mediated allelic exchange of unmarked mutations in Schu and the F. tularensis live vaccine strain (LVS). Using these plasmids, the two predicted b-lactamase genes, blaA and blaB, in Schu and LVS were deleted. Only the DblaB1 mutants had increased susceptibility to ampicillin, and this phenotype was complemented by a plasmid expressing blaB + . The results suggest that the b-lactam antibiotic resistance phenotype of Schu and LVS is likely due to only one of the two b-lactamase genes present and that ampicillin resistance can be used as an additional selectable marker in b-lactamase deletion mutants. The collection of tools presented in this report will be helpful for the genetic analyses of F. tularensis subsp. tularensis pathogenesis.
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