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Examines the effect of elevated temperature on virulence, VirB protein accumulation and T-pilus formation in Agrobacterium tumefaciens and A. vitis strains. Denaturation of protein complexes; Transfer of virulence factors to the membrane-bound components of transporters; Correlation between protein content and bacterial virulence.

Analyzes the role of VirB6 in the stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens. Reduction in the cellular levels of VirB5 and VirB3 in the absence of VirB6; VirB5 as a component of T pilus formation of plants; Regulation of T pilus formation through direct interaction between VirB6 and VirB5.

Examines the association of VirB5 with the T pilus of Agrobacterium tumefaciens. Stabilization of VirB5 by Vir proteins; Role of bacterial pili in cell-cell interaction; VirB2 as major component of the T pilus; Confirmation on T pilus high-molecular-weight structure; Mediation of assembly of the T pilus by interactions between VirB proteins.

Investigates the function of TraC in conjugative plasmid transfer. Molecular basis of extracellular complementation; Analysis of Escherichia coli carrying pKM101; Purification of soluble TraC from the periplasma of Escherichia coli; Constitution in TraC cross-linking of a biochemical assay for assembly of plasmid transfer complex.

Type IV secretion systems mediate conjugative plasmid transfer as well as the translocation of virulence factors from various gram-negative pathogens to eukaryotic host cells. The translocation apparatus consists of 9 to 12 components, and the components from different organisms are believed to have similar functions. However, orthologs to proteins of the prototypical type IV system, VirB of Agrobacterium tumefaciens, typically share only 15 to 30% identical amino acids, and functional complementation between components of different type IV secretion systems has not been achieved. We here report a heterologous complementation in the case of A. tumefaciens virB1 defects with its orthologs from Brucella suis (VirB1s) and the IncN plasmid pKM101 (TraL). In contrast, expression of the genes encoding the VirB1 orthologs from the IncF plasmid (open reading frame 169) and from the Helicobacter pylori cag pathogenicity island (HP0523) did not complement VirB1 functions. The complementation of VirB1 activity was assessed by T-pilus formation, by tumor formation on wounded plants, by IncQ plasmid transfer, and by IncQ plasmid recipient assay. Replacement of the key active-site Glu residue by Ala abolished the complementation by VirB1 from B. suis and by TraL, demonstrating that heterologous complementation requires an intact lytic transglycosylase active site. In contrast, the VirB1 active-site mutant from A. tumefaciens retained considerable residual activity in various activity assays, implying that this protein exerts additional effects during the type IV secretion process. [ABSTRACT FROM AUTHOR]