The Brucella suis type IV secretion system assembles in the cell envelope of the heterologous host Agrobacterium tumefaciens and increases IncQ plasmid pLS1 recipient competence.
Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related alpha(2)-proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.
Carle
Anna
A
McMaster University, Department of Biology, 1280 Main Street West, Hamilton, Ontario LS8 4K1, Canada.
Höppner
Christoph
C
Ahmed Aly
Khaled
K
Yuan
Qing
Q
den Dulk-Ras
Amke
A
Vergunst
Annette
A
O'Callaghan
David
D
Baron
Christian
C
D016960:Agrobacterium tumefaciens / Q000166:cytology* / Q000276:immunology*; D041021:Brucella suis / Q000235:genetics / Q000276:immunology*; D002006:Brucellosis / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology*; D002462:Cell Membrane / Q000276:immunology; D005455:Fluorescent Antibody Technique; D009876:Operon; D010957:Plasmids / Q000235:genetics*