Use of Synthetic Hybrid Strains To Determine the Role of Replicon 3 in Virulence of the Burkholderia cepacia Complex
Authors: Agnoli Kirsty , Freitag Roman , Gomes Margarida C. , Jenul Christian , Suppiger Angela , Mannweiler Olga , Frauenknecht Carmen , Janser Daniel , Vergunst Annette C. , Eberl Leo 
Published: April 2017

TheBurkholderia cepaciacomplex (Bcc) displays a wealth of metabolic diversity with great biotechnological potential, but the utilization of these bacteria is limited by their opportunistic pathogenicity to humans. The third replicon of the Bcc, megaplasmid pC3 (0.5 to 1.4 Mb, previously chromosome 3), is important for various phenotypes, including virulence, antifungal, and proteolytic activities and the utilization of certain substrates. Approximately half of plasmid pC3 is well conserved throughout sequenced Bcc members, while the other half is not. To better locate the regions responsible for the key phenotypes, pC3 mutant derivatives ofBurkholderia cenocepaciaH111 carrying large deletions (up to 0.58 Mb) were constructed with the aid of the FLP-FRT(FRT, flippase recognition target) recombination system fromSaccharomyces cerevisiae. The conserved region was shown to confer near-full virulence in bothCaenorhabditis elegansandGalleria mellonellainfection models. Antifungal activity was unexpectedly independent of the part of pC3 bearing a previously identified antifungal gene cluster, while proteolytic activity was dependent on the nonconserved part of pC3, which encodes the ZmpA protease. To investigate to what degree pC3-encoded functions are dependent on chromosomally encoded functions, we transferred pC3 fromBurkholderia cenocepaciaK56-2 andBurkholderia lata383 into other pC3-cured Bcc members. We found that although pC3 is highly important for virulence, it was the genetic background of the recipient that determined the pathogenicity level of the hybrid strain. Furthermore, we found that important phenotypes, such as antifungal activity, proteolytic activity, and some substrate utilization capabilities, can be transferred between Bcc members using pC3.

IMPORTANCETheBurkholderia cepaciacomplex (Bcc) is a group of closely related bacteria with great biotechnological potential. Some strains produce potent antifungal compounds and can promote plant growth or degrade environmental pollutants. However, their agricultural potential is limited by their opportunistic pathogenicity, particularly for cystic fibrosis patients. Despite much study, their virulence remains poorly understood. The third replicon, pC3, which is present in all Bcc isolates and is important for pathogenicity, stress resistance, and the production of antifungal compounds, has recently been reclassified from a chromosome to a megaplasmid. In this study, we identified regions on pC3 important for virulence and antifungal activity and investigated the role of the chromosomal background for the function of pC3 by exchanging the megaplasmid between different Bcc members. Our results may open a new avenue for the construction of antifungal but nonpathogenicBurkholderiahybrids. Such strains may have great potential as biocontrol strains for protecting fungus-borne diseases of plant crops.