Contact-Dependent Growth Inhibition (CDI) is a phenomenon in which bacteria use the toxic C-terminus of a large exoprotein (called BcpA in Burkholderia species) to inhibit the growth of neighboring bacteria upon cell-cell contact. CDI systems are present in a wide range of Gram-negative proteobacteria and a hallmark feature is polymorphism amongst the exoprotein C-termini (BcpA-CT in Burkholderia) and amongst the small immunity proteins (BcpI) that protect against CDI in an allele-specific manner. In addition to CDI, the BcpAIOB proteins of Burkholderia thailandensis mediate biofilm formation, and they do so independent of BcpA-mediated interbacterial competition, suggesting a cooperative role for CDI system proteins in this process. CDI has previously only been demonstrated between CDI+ and CDI- bacteria, leaving the roles of CDI system-mediated interbacterial competition and of CDI system diversity in nature unknown. We constructed B. thailandensis strains that differed only in the BcpA-CT and BcpI proteins they produced. When co-cultured on agar, these strains each participated in CDI and the outcome of the competition depended on both CDI system efficiency and relative bacterial numbers initially. Strains also participated in CDI during biofilm development, resulting in pillar structures that were composed of only a single BcpA-CT/BcpI type. Moreover, a strain producing BcpA-CT/BcpI proteins of one type was prevented from joining a pre-established biofilm community composed of bacteria producing BcpA-CT/BcpI proteins of a different type, unless it also produced the BcpI protein of the established strain. Bacteria can therefore use CDI systems for kind recognition and competitive exclusion of 'non-self' bacteria from a pre-established biofilm. Our data indicate that CDI systems function in both cooperative and competitive behaviors to build microbial communities that are composed of only bacteria that are related via their CDI system alleles.

Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram-negative bacteria, contact-dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C-terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell-contact. Termed the BcpA-CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC-type transporter, are required for entry of CDI system protein BcpA-2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA-CT that shared sequences within the predicted "translocation domain" of BcpA-2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK-utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA-2-mediated interbacterial antagonism, suggesting that BcpA-2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia-specific sequences at the GltK C-terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane.

Microbes have evolved many strategies to adapt to changes in environmental conditions and population structures, including cooperation and competition. One apparently competitive mechanism is contact dependent growth inhibition (CDI). Identified in Escherichia coli, CDI is mediated by Two-Partner Secretion (TPS) pathway proteins, CdiA and CdiB. Upon cell contact, the toxic C-terminus of the TpsA family member CdiA, called the CdiA-CT, inhibits the growth of CDI(-) bacteria. CDI(+) bacteria are protected from autoinhibition by an immunity protein, CdiI. Bioinformatic analyses indicate that CDI systems are widespread amongst α, β, and γ proteobacteria and that the CdiA-CTs and CdiI proteins are highly variable. CdiI proteins protect against CDI in an allele-specific manner. Here we identify predicted CDI system-encoding loci in species of Burkholderia, Ralstonia and Cupriavidus, named bcpAIOB, that are distinguished from previously-described CDI systems by gene order and the presence of a small ORF, bcpO, located 5' to the gene encoding the TpsB family member. A requirement for bcpO in function of BcpA (the TpsA family member) was demonstrated, indicating that bcpAIOB define a novel class of TPS system. Using fluorescence microscopy and flow cytometry, we show that these genes are expressed in a probabilistic manner during culture of Burkholderia thailandensis in liquid medium. The bcpAIOB genes and extracellular DNA were required for autoaggregation and adherence to an abiotic surface, suggesting that CDI is required for biofilm formation, an activity not previously attributed to CDI. By contrast to what has been observed in E. coli, the B. thailandensis bcpAIOB genes only mediated interbacterial competition on a solid surface. Competition occurred in a defined spatiotemporal manner and was abrogated by allele-specific immunity. Our data indicate that the bcpAIOB genes encode distinct classes of CDI and TPS systems that appear to function in sociomicrobiological community development.

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.