Use of Synthetic Hybrid Strains To Determine the Role of Replicon 3 in Virulence of the Burkholderia cepacia Complex

Abstract

The Burkholderia cepacia complex (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 of Burkholderia cenocepacia H111 carrying large deletions (up to 0.58 Mb) were constructed with the aid of the FLP-FRT (FRT, flippase recognition target) recombination system from Saccharomyces cerevisiae. The conserved region was shown to confer near-full virulence in both Caenorhabditis elegans and Galleria mellonella infection 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 from Burkholderia cenocepacia K56-2 and Burkholderia lata 383 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. IMPORTANCEThe Burkholderia cepacia complex (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 nonpathogenic Burkholderia hybrids. Such strains may have great potential as biocontrol strains for protecting fungus-borne diseases of plant crops.

The afc antifungal activity cluster, which is under tight regulatory control of ShvR, is essential for transition from intracellular persistence of Burkholderia cenocepacia to acute pro-inflammatory infection

“…mellonella and rats [35,36]. In zebrafish, we previously described that this strain can show substantial variation between experiments [37], with mortality rates between 20 and 60% at 4 days post fertilisation (dpf), but always less virulent than K56-2 (Fig 9). We also found that H111 does not show afc -dependent antifungal activity as seen for K56-2 [37].…”

Trade, Diplomacy, and Warfare: The Quest for Elite Rhizobia Inoculant Strains

“…These properties can, in theory, permit the construction of “hybrid” strains by combining accessory replicons that harbor gene functions related to competition, symbiotic efficiency, and possibly tolerance to harsh soil conditions, from different donor strains in a single genome. This would be similar to the approach recently employed by Agnoli et al (2017) with Burkholderia cepacia complex species. Additionally, the large pangenomes may harbor many genes related to the important “social” phenotypes referred in the previous paragraphs.…”

An investigation ofBurkholderiacepacia complex methylomesviaSMRT sequencing and mutant analysis

“…https://doi.org/10.1101/2020.03.09.983379 doi: bioRxiv preprint Given that the accepted main purpose of RM systems is to limit the intrusion of foreign DNA into the genome [34], we tested the RM null mutant for efficiency of replicon introduction by conjugation. As previously mentioned, the third replicon of the Bcc, pC3, can be moved between certain Bcc members, after the integration of an origin for conjugal transfer into pC3 [7]. Transfer efficiency was determined using B. cenocepacia K56-2 as the donor, and either the original RM null mutant (NullΔpC3) or H111Δc3 [5] as recipient.…”

“…Galleria mellonella pathogenicity assays were carried out as described previously [7], using larvae purchased from BioSystems Technology, UK. Assays were performed in triplicate and 10 larvae were used per strain and control.…”

“…Upon discovery of the spontaneous loss of pC3 that occurred during the construction of NullpC3 -, pC3 was moved back into the strain, as described in [7], to give NullpC3 + . Finally, an unmarked RM null mutant strain was constructed by repeating the deletion of the Type II methylase gene (I35_4914) on C2 of QM, with care taken to select a pC3-containing clone.…”