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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 ofAgrobacterium 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 heteroiogous complementation in the case ofA. 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.

Results show that VirB and VirD4 protein levels in Agrobacterium bacterial species are reduced subsequent to a temperature shift from 20 C to 28 C or 37 C. Data reveal substantial differences in the virulence protein abundance, which reflect the bacterial virulence at different temperatures.

Results show that in the absence of the VirB6 lipoprotein, VirB3 and VirB5 biosynthesis is reduced affecting T pilus formation and other genetic processes. Data further suggest that interaction of VirB6 with VirB5 regulates T pilus formation indicating to its stabilizing effect on VirB5.

Research has been conducted on the VirB proteins from Agrobacterium tumefaciens. The influence of Vir proteins on the stability of VirB1, VirB2 and VirB5 is analyzed.

The aim of this study is to demonstrate TraC function in the conjugative plasmid transfer. Biochemical analyses of Escherichia coli carrying pKM101 indicate that proteins interacting with other plasmid transfer components mediate TraC membrane attachment.