Cell culture and cloning
A. baylyi strain ADP1 was obtained from the American Type Culture Collection (ATCC #33305). Cells were propagated in standard LB media at 30 or 37 °C. DNA constructs were prepared using Gibson Assembly or Golden Gate cloning methods to generate linear DNA, which was used to transfom A. baylyi directly, by incubating 20-100 μl cells plus DNA for 30 minutes to a few hours. Clones were picked from selective plates, genomic DNA was purified using a genomic DNA miniprep kit, regions of interest were amplified by PCR, and the transformants were verified by Sanger sequencing. For PrecA-GFP, clones were screened for low-level GFP fluorescence by placing the plate on a blue transilluminator, rather than antibiotic selection. Multiplex CRISPR arrays were assembled using a method described previously 19.
Three genomic loci were used in the experiments described here. One was the natural CRISPR array located between ACIAD2484 and ACIAD2500. A second was the gene remnant ACIAD2826, which was replaced as a neutral locus for insertions, denoted ntrl1. A third was a neutral locus used previously, replacing ACIAD2186, 2187, and part of 2185, into which the ectopic CRISPR arrays were inserted40. For ectopic CRISPR arrays, the inserts included 684 bp upstream of the first repeat from the endogenous array. For PrecA-GFP, the insert included 214 bp of promoter from upstream of the recA gene, as described previously23.
A. baylyi was grown overnight in LB broth at 30°C with shaking. Just before experiments, cultures were washed and resuspended in fresh LB. 20 μl of cells were mixed with 40 ng of purified DNA and incubated at 30°C with shaking for 2 hours. Samples were then serially 10-fold diluted and spotted on LB agar plates with and without selective antibiotics (35 μg/ml kanamycin or spectinomycin), with 3 technical replicates for each dilution level. Plates were incubated at 30°C overnight, and colonies were counted at the lowest dilution level that still had clearly separated colonies. Counts for the three technical replicate spots were averaged. Each experiment was repeated at least 3 times, on at least 2 separate days.
For pulse-chase experiments, cells were growth overnight at 30°C, washed and diluted 1/13x into fresh LB, and re-grown for 3 hours at 37°C. The cells were then washed into fresh LB again, divided into 20 μl aliquots, mixed with 40 ng DNA, and incubated at 37°C. After 30 minutes, DNase and DNase buffer were added, and incubation continued for another 15 minutes at 37°C to digest remaining donor DNA. The cultures were then diluted into 600 μl fresh LB, and incubated at 37°C, with aliquots taken for serial dilution and spotting after 15 minutes and 3-4 hours.
Relative fitness was calculated using the equation , where A is cells transformed by DNA containing a given target, B is cells transformed by control DNA without a protospacer, and the subscripts indicate time points. Killing rates were calculated as , where γ is the growth rate of transformants with non-targeted control DNA without a protospacer, and n is the number of transformants at each time point.
Serial dilutions produce data best compared and plotted on a log scale, so data were plotted and compared after taking base 10 logarithms. Hypothesis testing was performed in Matlab using single-tailed t-tests. Where multiple samples were tested at once, the standard significance threshold of p<.05 was divided by the number of samples (Bonferroni correction for multiple hypothesis testing). Where data points were below detection, they were manually set to the limit of detection as the most conservative way to include them in the statistical analysis.
Visualizing DNA damage
The construct PrecA-GFP was prepared containing 214 bp of the A. baylyi recA promoter fused to GFP23, with approximately 1 kb homology arms surrounding a vgrG operon, and transformed into A. baylyi. This is a Type VI secretion system toxin-antitoxin operon, so in the absence of cells with the toxin, this insertion is neutral. In addition, cells contained a spectinomycin resistance gene in another neutral locus. Donor DNA for visualization experiments consisted of 3 kb covering that spectinomycin resistance gene and surrounding DNA, with a 32 bp protospacer inserted before the spectinomycin gene. Thus, the donor DNA had a 32 bp insert embedded within 3 kb of total homology to recipient cells. Cells were grown overnight in LB with shaking at 30 °C, washed, diluted 1:20 into fresh LB, and mixed 1:1 with donor DNA. Agar pads were prepared by pouring molten LB containing 2% wt/vol agar into 35 mm glass-bottom dishes, allowed to solidify, scooped out with a spatula, and placed upside-down. Spots of 0.5 μl of the cell-DNA mixtures were deposited onto the raised pads created from the glass window cutout and allowed to dry, after which the pad was placed back into the glass-bottom dish. Each experimental run contained 6 spots, including a no-DNA control. Three experiments were run for each CRISPR target and two for each plasmid. An additional 5 spots were imaged for protospacer 1 to obtain higher statistical power. A total of 9 no-DNA control spots were imaged, one for each experimental run. Experiments using plasmid DNA also included integrating DNA spots as positive controls. Time-lapse movies were obtained on a Nikon TE microscope using a 20x objective, with a 30-minute time step. Time points for analysis were selected at the point when growth significantly slowed, generally after 36 hours. GFP images were analyzed using the following procedure:
- Preprocessing: Images were preprocessed by dividing by a blank background image, which was itself first smoothed with a Gaussian filter with a radius of 5 pixels.
- Excluding empty areas: Regions with no cells were outlined manually, refined using the Image Segmenter app in Matlab, and excluded from further analysis.
- Background autofluorescence: To compensate for increased background fluorescence in the centers of microcolonies, an image-specific background was constructed. First, a Gaussian curve was constructed using the mean and variance of the GFP pixel histogram. Pixels greater than the 99th percentile of that curve were suppressed down to the 99th percentile level. The resulting image was smoothed with a Gaussian filter of radius 20 pixels to create an autofluorescence background. This was subtracted from the pre-processed GFP image, yielding the final background-corrected image.
- Histogram normalization: A histogram of GFP pixel intensity was constructed, and a new Gaussian curve was fit to the lower 60% of pixels, representing an expected distribution in the absence of GFP expression. The histogram was shifted laterally to be centered at 0, its width was normalized to σ = 1, and its height was normalized such that the total area under the curve was 1, to allow visual histogram overlays (Supplemental Fig. S5).
- Bright pixels: Bright pixels were defined as those greater than the 99.5 percentile of the aligned Gaussian.
Article TitleCRISPR-Cas Inhibits Natural Transformation Through Altruistic Group Defense and Self-Sacrifice
CRISPR-Cas systems present an evolutionary tradeoff: does defense against phages and other parasitic DNA also prevent cells from acquiring potentially helpful new genes? Genomic analyses of this conundrum have arrived at often contradictory conclusions. Meanwhile, experimental studies have focused mainly on phages, conjugation, or artificial transformation, but less work has examined natural competence, a major driver of evolution and antibiotic resistance. Here, we use Acinetobacter baylyi, which combines high natural competence with a functional CRISPR-Cas system, to experimentally probe the interactions between CRISPR-Cas and natural competence. In these bacteria, the endogenous CRISPR array largely allows natural transformation by targeted DNA. However, CRISPR-Cas then kills the newly autoimmune cells in a form of programmed cell death. CRISPR-Cas often allows self-targeting cells to form colonies, albeit with fitness costs. Thus CRISPR-Cas appears to block natural transformation in a process more akin to altruistic group defense than an individual immune system.