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.
Highlights
Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition used by some Gram-negative species to inhibit the growth of neighboring bacteria [1,2,3]
The results presented here show that at least three genes, BTH_I0359, BTH_I0986 and BTH_II0599, are required for B. thailandensis cells to be fully inhibited by the CDIIIBp1026b system
We identified each gene in two independent selection experiments, suggesting that they represent the major non-essential genes required for the CDIIIBp1026b pathway
Summary
Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition used by some Gram-negative species to inhibit the growth of neighboring bacteria [1,2,3]. CdiA proteins are very large (180–650 kDa depending on the species) and are predicted to form long β-helical filaments that extend from the surface of inhibitor cells [2,5]. During CDI, CdiA binds to specific receptors on susceptible bacteria and delivers a toxin domain derived from its C-terminal region (CdiA-CT). CdiA-CT sequence diversity suggests a variety of toxin activities, and most characterized CDI toxins are nucleases with different cleavage specificities for DNA, tRNA or rRNA [1,7,8,9]. CdiA-CTEC93 from Escherichia coli EC93 appears to form pores in target-cell membranes [10], and sequence analysis suggests that other CDI toxins may have RNA deaminase and protease/peptidase activities [11]. CDI+ bacteria protect themselves from auto-inhibition by producing CdiI immunity proteins, which bind to CdiA-CT toxins and neutralize their activities
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