Kind discrimination and competitive exclusion mediated by contact-dependent growth inhibition systems shape biofilm community structure

Abstract

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.

Figure 1. BtE264 and Bt-Bp chimeric bcpAIOB loci diagram.

Gray indicates BtE264 DNA. Colors indicate variable DNA sequences encoding BcpA-CT, BcpI, and BcpO proteins from BtE264 and Bp1106a. Yellow shading indicates Bp1106a DNA that replaced BtE624 DNA.

Figure 2. Bt-Bp1106a-1 and Bt-Bp1106a-2 mediated interbacterial competition.

CDI-mediated competition between A) Bt-Bp1106a-1 or B) Bt-Bp1106a-2 (inhibitors) and BtΔbcpAIOB bacteria without immunity, with cognate immunity, or with heterologous immunity (targets). Samples of bacteria from the center (C) and edge (E) of colony biofilms taken after 24 hours were plated with antibiotics to determine the log competitive index (C.I. (Log)), blue data points. Red data points indicate only inhibitor bacteria were recovered, and the actual C.I. is therefore greater than or equal to the represented value.

Figure 3. Lack of Bt-Bp1106a-3 and Bt-Bp1106a-3b mediated interbacterial competition.

Samples of bacteria from the center (C) and edge (E) of colony biofilms taken after 24 hours were plated with antibiotics to determine the log competitive index (C.I. (Log)) (blue data points) for competitions between A) Bt-Bp1106a-3 or B) Bt-Bp1106a-3b (inhibitors) and BtΔbcpAIOB bacteria without immunity or with cognate immunity (targets), C) BtE264 (inhibitor) and Bt-Bp1106a-3 without immunity or with cognate immunity (targets), D) BtE264 (inhibitor) and Bt-Bp1106a-3b without immunity or with cognate immunity (targets). Red data points indicate only inhibitor bacteria were recovered, and the actual C.I. is therefore greater than or equal to the represented value.

Figure 4. Competition between CDI⁺ bacteria expressing different bcpA alleles.

The log competitive index (C.I. (Log)) is plotted for competitions between A) BtE264 and Bt-Bp1106a-1 and B) BtE264 and Bt-Bp1106a-2 at 24 hours in the center (C) and edge (E) of colony biofilms with starting ratios of 1∶1, 10∶1, and 1∶10. A positive C.I. (Log) indicates BtE264 bacteria outcompeted the Bt-Bp chimeric strains, and negative C.I. (Log) indicates the Bt-Bp chimeric strains outcompeted BtE264 bacteria. Blue data points indicate the C.I. (Log). Red data points indicate only the “winning” (outcompeting) strain was recovered, the actual C.I. is therefore greater than or equal to the represented value. C) and D) Microscopy of colony biofilms mixed with BtE264::rfp and Bt-Bp1106a-1::gfp or Bt-Bp1106a-2::gfp at the indicated ratios. Images were taken of the center and edge at 24 hours. Note that bacteria completely span the field of view for all images taken (edge images were taken, therefore, just inside the edge). Scale bar = 10 µm.

Figure 5. Dependence of bacterial numbers on CDI-mediated competition between opposing CDI⁺ bacteria.

A) The log competitive index (C.I. (Log)) is plotted for competitions between Bt-Bp1106a-1 and Bt-Bp1106a-2 bacteria at a 1∶1 starting ratio (with and without immunity as indicated) in the center (C) and edge (E) of colony biofilms. A positive C.I. (Log) indicates the inhibitor bacteria outcompeted the target bacteria, and a negative C.I. (Log) indicates the target bacteria outcompeted the inhibitor bacteria. B) The log competitive index (C.I. (Log)) is plotted for competitions between Bt-Bp1106a-1 and Bt-Bp1106a-2 bacteria at the indicated starting ratios. A positive C.I. (Log) indicates Bt-Bp1106a-1 bacteria outcompeted Bt-Bp1106a-2 bacteria. All samples were taken at 24 hours. Blue data points indicate the C.I. (Log). Red data points indicate only the “winning” (outcompeting) strain was recovered, the actual C.I. is therefore greater than or equal to the represented value.

Figure 6. Analysis of Bt-Bp chimeric strains early in biofilm development.

Confocal microscopy of early time points in biofilm development of BtE264, Bt-Bp1106a-1, Bt-Bp1106a-2, and BtΔbcpAIOB bacteria, each constitutively expressing gfp. A) and B) Images taken at 6 and 24 hours in the plane of focus at the bottom of the biofilm (i.e., of bacteria directly attached to the glass bottom biofilm chambers). White scale bar = 10 µm. C) Fluorescent and DIC merged images taken at 24 hours in a plane of focus approximately half way between the top and bottom of the biofilm. Arrows indicate pillar structures. Black scale bar = 32 µm.

Figure 7. Analysis of pillar structure formation throughout biofilm development.

CLSM and quantification of representative pillar structures in biofilms formed by BtE264, Bt-Bp1106a-1, Bt-Bp1106a-2, and BtΔbcpAIOB bacteria, each expressing gfp constitutively. A) Images in Ai) and Aii) represent examples of small and large, respectively, pillar structures formed by Bt-Bp chimeric strains, and represent typical pillar structures in both cases for BtE264 and BtΔbcpAIOB bacteria, at 24 hours post inoculation. Z stack image cross-sections are shown in the plane parallel to the glass coverslip (shown by large center image) at 6 µm above the substratum. Scale bar = 10 µm. For each of the strains in A), COMSTAT analysis of Z stacks collected after 24 hours of biofilm development were used to determine B) total biomass, C) average height, and D) maximum height of the biofilms. BtE264, purple bars; Bt-Bp1106a-1, orange bars; Bt-Bp1106a-2, red bars; and BtΔbcpAIOB, gray bars. Bars represent the mean of at least three independent experiments and error bars indicate the SEM. Significance was determined by two-tailed t-tests; * p<0.05, *** p<0.0001. E) and F) CLSM images of representative large pillar structures formed by the indicated strains at 48 and 72 hours, respectively, post inoculation. Z stack image cross-sections were taken at 6 µm (48 hours) or 10 µm (72 hours) above the substratum. Scale bar = 10 µm.

Figure 8. Pillar structure formation by strains at a higher inoculum.

Confocal microscopy of biofilms formed with the “high inoculum”. Fluorescent and DIC merged images taken at 24 hours in a plane of focus approximately half way between the top and bottom of biofilms formed by BtE264, Bt-Bp1106a-1, Bt-Bp1106a-2, and BtΔbcpAIOB bacteria, each constitutively expressing gfp. Black scale bar = 32 µm.

Figure 9. CDI system-mediated kind discrimination during polymicrobial biofilm formation of Bt-Bp1106a-1 and Bt-Bp1106a-2 bacteria.

Confocal microscopy and quantitative analysis of polymicrobial biofilms 72∶1 starting ratio with the following strains: I. Bt-Bp1106a-1 and Bt-Bp1106a-2, II. Bt-Bp1106a-1 and Bt-Bp1106a-2::bcpI_{Bp 1106a-1}, III. Bt-Bp1106a-1 and Bt-Bp1106a-2::bcpI_{Bt E264}, and IV. Bt-Bp1106a-1::bcpI_{Bp 1106a-2} and Bt-Bp1106a-2::bcpI_{Bp 1106a-1}. For A) and B), strains in red carry a constitutive rfp gene, strains in green carry a constitutive gfp gene. For COMSTAT analysis in C) and D), green bars correspond to gfp expressing strain. Bars represent the mean of at least three independent experiments and error bars indicate the SEM. Significance was determined by two-tailed t-tests; *** p<0.0006; ns, not significant, comparing each strain to Bt-Bp1106a-2 (roman numeral I). A) Images taken of the plane at the bottom of the biofilm (substratum). Scale bar = 10 µm. B) Fluorescent and DIC merged images taken in a plane of focus approximately half way between the top and bottom of the biofilms. Scale bar = 32 µm. C) COMSTAT analysis of a single plane at the bottom of the biofilm (substratum), as shown in A). D) COMSTAT analysis of the total biofilm (i.e., all planes), from experiments shown in B).

Figure 10. bcpAIOB allele-dependent competitive exclusion from an established community.

A) Biofilms were inoculated with the “established strain,” Bt-Bp1106a-1::bcpI_{Bt E264}, constitutively expressing rfp, incubated for 24 hours, washed, and then re-inoculated with the “invading strain,” I. BtE264 or II. BtE264::bcpI_{Bp 1106a-1}, constitutively expressing gfp, re-incubated for 48 hours, washed, and then imaged by confocal microscopy. Images shown are of fluorescent and DIC channels merged, taken in a plane of focus approximately half way between the top and bottom of the biofilms. Scale bar = 32 µm. B) CLSM of representative RPF⁺ pillar structures. Images are from the same experiment as in A). Z stack image cross-sections were taken at 10 µm above the substratum. Scale bar = 10 µm. C) Single strain biofilms formed by BtE264 and BtE264::bcpI_{Bp 1106a-1}, constitutively expressing gfp, 24 hours post inoculation. Images shown are of fluorescent and DIC channels merged, taken in a plane of focus approximately half way between the top and bottom of the biofilms. Scale bar = 32 µm.