Involvement of a Plasmid-Encoded Type IV Secretion System in the Plant Tissue Watersoaking Phenotype of Burkholderia cenocepacia

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).

Role of phages in the pathogenesis of Burkholderia, or ‘Where are the toxin genes in Burkholderia phages?’

“…There are commonalities between the mechanisms of pathogenesis for the Bcc and B. pseudomallei: they are able to elaborate an exopolysaccharide that is required for full virulence; they possess one or more type III secretion systems (TTSS); and they are able to adhere to and invade host cells and persist in the intracellular compartment [6,7]. Two type IV secretion systems have been identified in B. cenocepacia belonging to the epidemic ET12 clonal lineage, with the plasmid encoded one related to elaboration of a plant watersoaking phenotype and the chromosomal one bearing homology to the VirB/D4 of Agrobacterium tumifaciens [15]. In plant pathogenic B. cepacia a plasmid encoded pectate hydrolase is a virulence factor necessary for maceration of onion tissue [16].…”

Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

“…Six of the eight branched-chain amino acid metabolism genes, including the porin implicated in inflammation during infection (55) (BxeC0438), are highly conserved between the two species. The LB400 megaplasmid also has a cluster of type IV secretion genes immediately adjacent to the latter amino acid gene cluster, which shares considerable sequence similarity with a B. cenocepacia type IV secretion system island encoded Ϸ46 kb downstream of the Cenocepacia island on the J2315 second chromosome (54). These corresponding LB400 megaplasmid regions do not possess the hallmarks of genomic islands, suggesting that the megaplasmid may represent an ancestral replicon from which the B. cenocepacia laterally acquired the Cenocepacia and type IV secretion islands.…”

“…We found potential virulence-related functions only present on islands 9, 11, and 4 (type IV secretion and adhesion functions; Table 4), suggesting that laterally acquired DNA has been retained by LB400 by selection in the natural environment rather than during primary infection. A plasmid-encoded type IV secretion system in B. cenocepacia has been implicated in the secretion of plant cytotoxic proteins (54); however, neither of the LB400 systems shares significant sequence similarity with the latter plant pathogenic gene cluster.…”

Development of a Species-Specific fur Gene-Based Method for Identification of the Burkholderia cepacia Complex

“…First described by Burkholder (6) as the causative agent of "sour skin" disease of onion bulbs, the BCC is now considered to be an important group of bacterial pathogens of plants, animals, and humans (4,11,13). This is especially true for people with cystic fibrosis (CF), where BCC infection can significantly contribute to the deterioration of pulmonary function.…”