A Brucella suis mutant with a nonpolar deletion in the virB8 gene was attenuated in a macrophage infection model. Complementation with the B. suis VirB8 protein expressed from the virB promoter restored virulence. Expression of TraJ, a VirB8 homologue from plasmid pSB102, did not restore virulence; however, virulence was partially restored by a chimeric protein containing the N terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ.Type IV secretion systems (T4SS) are used by gram-negative bacteria to transport macromolecules across the bacterial envelope into a recipient cell (4, 6). Transported substrates include nucleoprotein complexes, transported during bacterial conjugation or during transfer of transferred DNA from Agrobacterium tumefaciens, as well as protein virulence factors translocated into eukaryotic host cells by pathogens.The T4SS is a complex multiprotein structure spanning the bacterial envelope. Most T4SS are composed of up to 11 individual protein building blocks plus, in many cases, a 12th "coupling protein," thought to bring the macromolecular substrates to the secretion system. Biochemical analysis of the A. tumefaciens VirB system, the T4SS paradigm, is beginning to show how the individual components assemble in the bacterial envelope and how the substrates are secreted (5). The VirB1 protein is a murein-digesting lytic transglycosylase that locally digests the peptidoglycan, allowing the assembly of the T4SS. The VirB3, VirB6, and VirB7 to VirB10 proteins are believed to form a channel-like structure spanning both bacterial membranes; the VirB4 and VirB11 proteins, with the VirD4 coupling protein, are ATPases associated with the cytoplasmic face of the inner membrane; and the VirB2 and VirB5 proteins form a pilus-like structure on the bacterial surface.The VirB8 protein has been shown to play a key role in the assembly of the T4SS machinery. Using microscopic, biochemical, and genetic approaches to study function in A. tumefaciens, it was discovered that VirB8 acts as a nucleation center that is required to recruit VirB9 and VirB10 into clusters in the outer membrane (10) and to localize VirB proteins at the cell pole (8). The amino terminus of VirB8, encompassing the first 67 amino acids, contains a short cytoplasmic tail followed by a single hydrophobic transmembrane domain. The carboxy-terminal moiety of the protein of 172 amino acids is believed to be entirely periplasmic. Recently, the three-dimensional structures of the periplasmic domain of VirB8 from both Brucella suis and A. tumefaciens have been determined (1, 18). Using site-directed mutagenesis to change selected residues of the B.suis VirB8 protein, we have shown that changes that inhibit VirB8 dimerization or that inhibit the interactions with VirB4 or VirB10 also affect T4SS assembly and virulence (12). Here, we describe the construction and characterization of the B. suis strain with a nonpolar deletion of virB8 used in that study and show, for the first time, complementation with a chimeric pr...
The origin and evolution of B chromosomes could be explained by the specific DNA sequence on them. But the specific sequences known were quite limited. To investigate maize B chromosome sqicific DNA sequeces, maize genomes with and without B chromosomes were analyzed by AFLP. Only 5 markers were found specific to genomes with B chromosomes among about 2000 AFLP markers. Southern hybridization and sequence analysis revealed that only the sequence of M8-2D was a B chromosome specific sequence. This sequence contained the telomeric repeat unit AGGGTTT conserved in plant chromosome telomeres. In addition, the sequence of M8-2D shared low homology to clones from maize chromosome 4 centromere as well. M8-2D were localized to B chromosome centromeric and telomeric regions.
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