Genetic Analysis of the CDI Pathway from Burkholderia pseudomallei 1026b

Abstract

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIII Bp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIII Bp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIII Bp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTII Bp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI+ inhibitor cells to the same extent as BTH_I0986+ cells, suggesting that LPS could function as a receptor for CdiAII Bp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIII Bp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species.

Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways

“…This latter result shows that the Burkholderia tRNase domain can be delivered into E. coli target cells, raising the possibility that E. coli lacks the pathway required for native CdiA-CT II Bp1026b import. We recently discovered that Burkholderia thailandensis ΔBTH_II0599 mutants are resistant to the CDI II Bp1026b system (26). BTH_II0599 encodes a member of the major facilitator superfamily (MFS), which are integral membrane transporters of small metabolites and antibiotics (27).…”

Contact-Dependent Growth Inhibition (CDI) and CdiB/CdiA Two-Partner Secretion Proteins

“…However, there are two examples where CDI acts across species. Enterobacter cloacae ATCC 13047 can inhibit E. coli K-12 derivatives using its CDI system 26 , and the CDI II Bp1026b system from B. pseudomallei 1026b inhibits closely related Burkholderia thailandensis E264 28; 66 . The receptors for these latter CdiA proteins have not been identified, but there is some evidence that lipopolysaccharide (LPS) may serve as a receptor for CdiA II Bp1026b .…”

“…The receptors for these latter CdiA proteins have not been identified, but there is some evidence that lipopolysaccharide (LPS) may serve as a receptor for CdiA II Bp1026b . Disruption of the BTH_I0986 locus in B. thailandensis target bacteria confers resistance to CDI II Bp1026b 66 . The BTH_I0986 gene encodes a predicted LPS glycosyltransferase, and ΔBTH_I0986 mutants have altered LPS and show decreased binding to inhibitor cells that express CdiA II Bp1026 66 .…”

“…Disruption of the BTH_I0986 locus in B. thailandensis target bacteria confers resistance to CDI II Bp1026b 66 . The BTH_I0986 gene encodes a predicted LPS glycosyltransferase, and ΔBTH_I0986 mutants have altered LPS and show decreased binding to inhibitor cells that express CdiA II Bp1026 66 . Thus, it seems likely that CdiA effectors exploit several different cell-surface receptors to identify susceptible target cells.…”

Can't you hear me knocking: contact-dependent competition and cooperation in bacteria

“…It is presently unclear whether CdiA EC536 also uses OmpC/OmpF for toxin translocation. However, because CdiA proteins exploit a variety of cell-surface receptors, including lipopolysaccharide ((32) and unpublished data), it appears that porins are not an obligate part of the CDI toxin translocation pathway.…”