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item Engledow, Amanda
item Medrano, Enrique - Gino
item Mahenthiralingam, Eshwar
item Lipuma, John
item Gonzalez, Carlos

Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/1/2004
Publication Date: 9/1/2004
Citation: Engledow, A.S., Medrano, E.G., Mahenthiralingam, E., LiPuma, J.J., Gonzalez, C.F. 2004. Involvement of a plasmid-encoded type IV secretion system in the plant tissue watersoaking phenotype of Burkholderia cenocepacia. Journal of Bacteriology. 186(18):6015-6024.

Interpretive Summary: The bacterium Burkholderia cenocepacia can cause a disease called soft rot in onions, and it can also cause chronic lung infections in patients with cystic fibrosis. Information concerning the infection processes used by this bacterium is limited. A B. cenocepacia strain originally isolated from an infected lung of a patient with cystic fibrosis was found to be capable of producing disease on onion. The mechanisms employed by the bacterium to infect the onion tissue were examined. We found that toxic chemicals were transported into the plant cells. The transport apparatus involved in B. cenocepacia onion rotting is analogous to transfer systems known to be used by other plant and human organisms that cause diseases where they inject toxins into their hosts. It is possible that the system used to infect onions also may be used to infect the lungs of persons with cystic fibrosis. This research contributes to our knowledge of how B. cenocepacia may infect humans, and may assist in providing a target for vaccines.

Technical Abstract: Burkholderia cenocepacia strain K56-2, a representative of the Burkholderia cepacia complex, is part of the epidemic and clinically problematic ET12 lineage. The strain produced plant tissue watersoaking (ptw) on onion tissue, which is a plant disease-associated trait. Using plasposon mutagenesis, mutants in the ptw phenotype were generated. The translated sequence of a disrupted gene (ptwD4) from a ptw-negative mutant showed homology to VirD4-like proteins. Analysis of the region proximal to the transfer gene homolog identified a gene cluster located on the 92-kb resident plasmid that showed homology to type IV secretion systems. The role of ptwD4, ptwC, ptwB4, and ptwB10 in the expression of ptw activity was determined by conducting site-directed mutagenesis. The ptw phenotype was not expressed by K56-2 derivatives with a disruption in ptwD4, ptwB4, or ptwB10 but was observed in a derivative with a disruption in ptwC. Complementation of ptw-negative K56-2 derivatives in trans resulted in complete restoration of the ptw phenotype. In addition, analysis of culture supernatants revealed that the putative ptw effector(s) was a secreted, heat-stable protein(s) that caused plasmolysis of plant protoplasts. A second chromosomally encoded type IV secretion system with complete homology to the VirB-VirD system was identified in K56-2. Site-directed mutagenesis of key secretory genes in the VirB-VirD system did not affect expression of the ptw phenotype. Our findings indicate that in strain K56-2, the plasmid-encoded Ptw type IV secretion system is responsible for the secretion of a plant cytotoxic protein(s).