Relaxed specificity of BcpB transporters mediates interactions between Burkholderia cepacia complex contact-dependent growth inhibition systems.

Belonging to the two-partner secretion family of proteins, contact-dependent growth inhibition (CDI) systems mediate interbacterial antagonism among closely related Gram-negative bacteria. The toxic portion of a large surface protein, BcpA/CdiA, is delivered to the cytoplasm of neighboring cells where it inhibits growth. Translocation of the antibacterial polypeptide out of the producing cell requires an associated outer membrane transporter, BcpB/CdiB. Some bacteria, including many Burkholderia species, encode multiple distinct CDI systems, but whether there is interaction between these systems is largely unknown. Using Burkholderia cepacia complex species as a model, here we show that related BcpB transporters exhibit considerable secretion flexibility and can secrete both cognate and non-cognate BcpA substrates. We also identified an additional unique Burkholderia dolosa CDI system capable of mediating interbacterial competition and demonstrated that its BcpB transporter has similar relaxed substrate specificity. Our results showed that two BcpB transporters (BcpB-2 and BcpB-3) were able to secrete all four of the B. dolosa BcpA toxins, while one transporter (BcpB-1) appeared unable to secrete even its cognate BcpA substrate under the tested conditions. This flexibility provided a competitive advantage, as strains lacking the full repertoire of BcpB proteins had decreased CDI activity. Similar results were obtained in Burkholderia multivorans, suggesting that secretion flexibility may be a conserved feature of Burkholderia CDI systems. Together these findings suggest that the interaction between distinct CDI systems enhances the efficiency of bacterial antagonism. IMPORTANCE The Burkholderia cepacia complex (Bcc) is a group of related opportunistic bacterial pathogens that occupy a diverse range of ecological niches and exacerbate disease in patients with underlying conditions. Contact-dependent growth inhibition (CDI) system proteins, produced by Gram-negative bacteria, contain antagonistic properties that allow for intoxication of closely related neighboring bacteria via a secreted protein, BcpA. Multiple unique CDI systems can be found in the same bacterial strain, and here we show that these distinct systems interact in several Bcc species. Our findings suggest that the interaction between CDI system proteins is important for interbacterial toxicity. Understanding the mechanism of interplay between CDI systems provides further insight into the complexity of bacterial antagonism. Moreover, since many bacterial species are predicted to encode multiple CDI systems, this study suggests that interactions between these distinct systems likely contribute to the overall competitive fitness of these species.

Recipient Cell Factors Influence Interbacterial Competition Mediated by Two Distinct Burkholderia dolosa Contact-Dependent Growth Inhibition Systems.

Contact-dependent growth inhibition (CDI) systems mediate interbacterial antagonism between Gram-negative bacteria by delivering the toxic portion of a large surface protein (termed BcpA in Burkholderia species) to the cytoplasm of neighboring bacteria. Translocation of the antibacterial polypeptide into recipient cells requires specific recipient outer and inner membrane proteins, but the identity of these factors outside several model organisms is unknown. To identify genes involved in CDI susceptibility in the Burkholderia cepacia complex member Burkholderia dolosa, a transposon mutagenesis selection approach was used to enrich for mutants resistant to BcpA-1 or BcpA-2. Subsequent analysis showed that candidate regulatory genes contributed modestly to recipient cell susceptibility to B. dolosa CDI. However, most candidate deletion mutants did not show the same phenotypes as the corresponding transposon mutants. Whole-genome resequencing revealed that these transposon mutants also contained unique mutations within a three gene locus (wabO, BDAG_01006, and BDAG_01005) encoding predicted lipopolysaccharide (LPS) biosynthesis enzymes. B. dolosa wabO, BDAG_01006, or BDAG_01005 mutants were resistant to CDI and produced LPS with altered core oligosaccharide and O-antigen. Although BcpA-1 and BcpA-2 are dissimilar and expected to utilize different outer membrane receptors, intoxication by both proteins was similarly impacted by LPS changes. Together, these findings suggest that alterations in cellular regulation may indirectly impact the efficiency of CDI-mediated competition and demonstrate that LPS is required for intoxication by two distinct B. dolosa BcpA proteins. IMPORTANCEContact-dependent growth inhibition (CDI) system proteins, produced by many Gram-negative bacteria, are narrow spectrum antimicrobials that inhibit the growth of closely related neighboring bacteria. Here, we use the opportunistic pathogen Burkholderia dolosa to identify genes required for intoxication by two distinct CDI system proteins. Our findings suggest that B. dolosa recipient cells targeted by CDI systems are only intoxicated if they produce full-length lipopolysaccharide. Understanding the mechanisms underlying antagonistic interbacterial interactions may contribute to future therapeutic development.