Figure 1.
Ellipses - bacteria; Pentagons - phage; red and blue rectangles - acquired spacers; red and blue circles, regions of the phage genome corresponding to the acquired spacers (protospacers); stars, mutated protospacers generating CEMs. WT, Wild type bacteria and phage; BIMX and CEMX, bacteria with spacers for acquired resistance to WT phage and the first-order CRISPR Escape mutants (CEMX), respectively; BIMX2 and CEMX2, bacteria with spacers for acquired resistance to CEMX and the second-order CRISPR–escape mutants, respectively. Panel A: Infection and phage replication relationships. Solid black lines, phage adsorption and replication; broken red lines - phage adsorption and loss. Panel B: Changes in state. BIMX are produced by WT phage infecting WT cells and BIMX2 are produced by CEMX infecting BIMX. CEMX are produced by P0 infecting and replicating on B0 and CEMX2 are produced by CEMX infecting and replicating on BIMX.
Figure 2.
Short-term bacterial and phage growth dynamics in the absence of immunity: change in the density of WT bacteria and WT phage.
The broken lines are the densities predicted by the above model with the following parameters: v = 1.4 hr−1 per hour, e = 5×10−7 µg per cell, R (initial resource concentration) = 350 µg/ml, δ = 8×10−8 ml per cell per phage per hour, β = 80 phage particles per infected cell and, λ = 0.4 hours. At 24 hours, the estimated densities of bacteria and phage in these cultures were, respectively, 5×108 cells per ml for the control and 3.2×105 CFU and 6.2×107 PFU for the bacteria and phage in the mixed culture.
Figure 3.
Graphical representation of spacers across the two CRISPR loci for S. thermophilus BIMs.
Repeats are not included; only spacers are represented. Each spacer is represented by a combination of one select character in a particular color, on a particular background color, as previously described [36]. The color combination allows unique representation of a particular spacer. Similar color schemes (combination of character color and background color) represent identical spacers, whereas different color combinations represent distinguishable spacers.
Figure 4.
Nucleotide sequences in wild-type and mutant phages that correspond to the newly acquired spacers by the S. thermophilus BIM strains.
Each mutant is highlighted in red and yellow, bold and underlined.
Figure 5.
Changes in optical density of cultures with S. thermophilus and the corresponding phages to which these WT cells or BIMs are sensitive.
The first transfer was initiated with 40 µl of an overnight culture (for approx. 2×106 cells per ml) and phage (approx. 2×106 per ml) in 4 ml LM17Ca medium. The second transfer was initiated by adding 40 µl of the 24-hour first-pass cultures to 4 ml fresh LM17Ca. The heavy black lines with square tick marks indicate phage-free WT controls. The heavy red lines with diamond tick marks are the arithmetic mean ODs for the cultures with bacteria and phage. Panel A: WT S. thermophilus and WT phage (light blue) and nine first-order BIMs and their corresponding CEMs. Panel B: five second-order BIMs and their corresponding CEMs.
Figure 6.
Changes in optical density of cultures with S. thermophilus and high initial densities of phage (approx. 5×106/ml) to which the BIMs were resistant.
The first transfers (left) were initiated with 40 µl of overnight bacteria and phage in 4 ml LM17Ca medium. The second transfers (right) were initiated by adding 40 µl of the 24-hour first-pass cultures to 4 ml fresh LM17Ca. Panel A: WT phage with WT cells (medium-weight blue line) and 10 first-order BIMs. Panel B: five second-order BIMs and their corresponding first-order CEM phages. The heavy black lines with square tick marks are phage-free WT controls and the heavy red lines with diamond tick marks are the arithmetic mean densities of all the cultures with phage.
Figure 7.
Changes in optical density of cultures with S. thermophilus and low initial densities of WT phage.
The first transfer was initiated with 40 µl of an overnight culture of bacteria (approx. 2×106 cells per ml) and WT phage (approx. 2×104 ml) in 4 ml LM17Ca medium. The second transfer was initiated by adding 40 µl of the 24-hour first-pass cultures to 4 ml fresh LM17Ca. Panel A: WT phage with WT cells (medium-weight blue line) and 10 first-order BIMs. Panel B: WT phage with five second-order BIMs. The heavy black lines with square tick marks are phage-free WT controls. The heavy red lines with diamond tick marks are the arithmetic mean densities of all BIM cultures with phage.
Figure 8.
Changes in optical density of cultures with S. thermophilus first- and second-order BIMs and WT cells with low initial densities of WT phage.
The first transfer was initiated with 40 µl of an overnight culture of each BIM (approx. 2×106 per ml) and WT cells (approx. 2×106 per ml) and low densities (approx. 2×104 per ml) of WT phage in 4 ml LM17Ca medium. The second transfer was initiated by adding 40 µl of the 24-hour first-pass cultures to 4 ml fresh LM17Ca. Panel A: WT phage with WT cells and 10 first-order BIMs. Panel B: low densities of WT phage (approx. 2×104 per ml) with WT cells and five second-order BIMs. The heavy black lines with square tick marks are the phage-free controls (WT cells in (A) and BIM22 in (B)). The heavy red lines with diamond tick marks are the arithmetic mean densities of all the BIM cultures with phage.
Figure 9.
Changes in optical and viable cell density of S. thermophilus in cell-free lysates of wild-type phage.
BIM22 and SMQ-301 in a full concentration phage lysate (1.5×108 pfu/ml) and an LM17Ca control (1/100 dilution). Con – LM17Ca control; Lys – cell-free phage lysate. Panel A: optical density. Panel B: estimated cell density.
Figure 10.
Simulation results of the extended model.
Changes in the densities of bacteria and phage in cultures inoculated with WT phage (P0), second-order BIMs (B2), and WT cells upon which the phage can replicate (B0). The first-order BIMs (B1) and first-order phage (P1) are respectively produced by spacers added to the CRISPR region and generated by mutation, with probabilities of 10−6 per cell or phage per hour. The variable R is the concentration of the limiting resource. The bacterial growth and phage infection parameters in these simulations are the same as those in Figure 2. Save for (A) the parameters for the production and action of LY and persistence are as follows: vL = 10−6, KL = 106, η = 1.4, g = h = 0.01. The initial concentration of the resource at the start of a transfer is 350 µg/ml. The cultures were initiated with 2×106 B0 and B2 cells and 2×106 P0 phage. Panel A: No LY produced (vL = 0). Panel B: The first transfer of the culture. Panel C: The second transfer. Panel D: sequential transfers (approx. 2×104).
Figure 11.
Serial transfer experiment initiated with WT phage and WT cells.
Panel A: Optical densities at the end of successive passages. Panel B: estimated viable cell densities at the end of successive transfer. Panel C: estimated free phage densities at the end of successive transfers.