Kind Discrimination and Competitive Exclusion Mediated by Contact-Dependent Growth Inhibition Systems Shape Biofilm Community Structure

<div><p>Contact-Dependent Growth Inhibition (CDI) is a phenomenon in which bacteria use the toxic C-terminus of a large exoprotein (called BcpA in <i>Burkholderia</i> 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 <i>Burkholderia</i>) and amongst the small immunity proteins (BcpI) that protect against CDI in an allele-specific manner. In addition to CDI, the BcpAIOB proteins of <i>Burkholderia thailandensis</i> 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 <i>B. thailandensis</i> 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.</p>