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.
Table 1.
Distribution of bcpAIOB alleles.
Table 2.
Summary of CDI results.
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::bcpIBp1106a-1, III. Bt-Bp1106a-1 and Bt-Bp1106a-2::bcpIBtE264, and IV. Bt-Bp1106a-1::bcpIBp1106a-2 and Bt-Bp1106a-2::bcpIBp1106a-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::bcpIBtE264, constitutively expressing rfp, incubated for 24 hours, washed, and then re-inoculated with the “invading strain,” I. BtE264 or II. BtE264::bcpIBp1106a-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::bcpIBp1106a-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.
Table 3.
Relevant strain list.