Dealing with the evolutionary downside of CRISPR immunity: bacteria and beneficial plasmids

Abstract

The immune systems that protect organisms from infectious agents invariably have a cost for the host. In bacteria and archaea CRISPR-Cas loci can serve as adaptive immune systems that protect these microbes from infectiously transmitted DNAs. When those DNAs are borne by lytic viruses (phages), this protection can provide a considerable advantage. CRISPR-Cas immunity can also prevent cells from acquiring plasmids and free DNA bearing genes that increase their fitness. Here, we use a combination of experiments and mathematical-computer simulation models to explore this downside of CRISPR-Cas immunity and its implications for the maintenance of CRISPR-Cas loci in microbial populations. We analyzed the conjugational transfer of the staphylococcal plasmid pG0400 into Staphylococcus epidermidis RP62a recipients that bear a CRISPR-Cas locus targeting this plasmid. Contrary to what is anticipated for lytic phages, which evade CRISPR by mutations in the target region, the evasion of CRISPR immunity by plasmids occurs at the level of the host through loss of functional CRISPR-Cas immunity. The results of our experiments and models indicate that more than 10(-4) of the cells in CRISPR-Cas positive populations are defective or deleted for the CRISPR-Cas region and thereby able to receive and carry the plasmid. Most intriguingly, the loss of CRISPR function even by large deletions can have little or no fitness cost in vitro. These theoretical and experimental results can account for the considerable variation in the existence, number and function of CRISPR-Cas loci within and between bacterial species. We postulate that as a consequence of the opposing positive and negative selection for immunity, CRISPR-Cas systems are in a continuous state of flux. They are lost when they bear immunity to laterally transferred beneficial genes, re-acquired by horizontal gene transfer, and ascend in environments where phage are a major source of mortality.

Figure 1. Different possibilities for the transfer of a beneficial plasmid into cells encoding CRISPR immunity against it.

S. epidermidis RP62a contains a CRISPR-Cas system with a spacer (pink) that produces crRNAs that match and target the nickase (nes) gene (also in pink) of staphylococcal conjugative plasmids, including pG0400. There are at least four different mechanisms that will allow the transfer of the plasmid in spite of CRISPR immunity: (i) mutation of the plasmid target (yellow), (ii) mutation or deletion of the anti-plasmid spacer, (iii) loss-of-function mutation of the cas genes required for immunity or partial or complete deletion of the CRISPR-Cas locus, or (iv) partial immunity that leads to tolerance of the plasmid.

Figure 2. Different mutations eliminate CRISPR immunity against conjugation in S. epidermidis.

(A) Summary of the different mutations found in this study and their proportions. (B) Distribution of mutations within the CRISPR-Cas locus. S. epidermidis RP62a harbors a CRISPR-Cas system containing four repeats (white boxes), three spacers (colored, numbered boxes) and nine cas/csm genes. Mutations found in CRISPR escapers include deletions in the repeat-spacer region (brackets), transposon insertions (red arrowheads; top, direct insertion; bottom, inverted) and single nucleotide deletions or substitutions (asterisks). Arrows indicate primers used to analyze transconjugants. (C) PCR analysis of the CRISPR array of transconjugants using primers L50/L6. Deletion of 1, 2 and 3 spacers observed in escapers R23, R10 and R2, respectively, is shown. M, DNA marker. wt, amplification using wild-type template DNA. (D) PCR analysis of the cas gene region of escapers using primers L23/L106. IS256 transposon insertions into csm5, csm6 and cas6 observed in escapers R60, B15 and R36, respectively, are shown. M, DNA marker. wt, amplification using wild-type template DNA.

Figure 3. CRISPR escapers accumulate deletions of the CRISPR/Cas region.

Schematic representation of the deletions on the S. epidermidis RP62a genome. Wild-type sequences are shown in green, deletions mediated by IS431 in pink, by IS256 in yellow, by Tn554 in orange, by recombination between SERP2353 and SERP2491 (96% identical at the nt level) or SERP2409 and SERP2493 (98% identical at the nt level) in light blue or brown, respectively, and by the excision of the SCCmec cassette in violet. Numbers represent genomic coordinates in kb.

Figure 4. Fitness of CRISPR inactivated and deleted transconjugants.

Pair-wise competition between transconjugants carrying different types of CRISPR-Cas mutations or deletions and wild-type S. epidermidis RP62a. The change in the relative frequency of plasmid-bearing cells (y-axis) is plotted against the number of transfers (one transfer per day, x-axis). In all cases the growth of wild-type cells was compared against: (A) control wild-type S. epidermidis RP62a (pG0400^mut), (B) R5, (C) R60, (D) R14, (E) R7, (F) B15, (G) B39. The black line indicates the average change in relative frequency (the values for each of three independent experiments are shown as a red triangle, blue circle and green rhombus). (H) Predicted changes in frequency for different selection coefficients, s. These are calculated from the equation, dq/dt  =  –q(1 – q)s, where q is the relative frequency of the plasmid bearing cells and s is the selection coefficient (s>0 indicates that the plasmid-bearing cells are at a disadvantage and s<0 that the plasmid-bearing cells have an advantage). We are assuming 1/100 dilutions or t = 6.64 generations in each transfer.

Figure 5. Simulation of plasmid competition with CRISPR-mediated immunity.

Changes in the densities of different populations over time are plotted. Standard parameters (defined in Supplementary Text 2) are: ν = 1.4, e = 5×10⁻⁷, k = 1, γ = 10⁻¹⁴, initial values, R = 2500, CP = 200, D1 = 100, CN = D1 = D2 = T1 = T2 = 0. (A) Same rate of CRISPR loss (µ) and plasmid escape mutations (ν), µ = ν = 10⁻⁷. (B) High rate of plasmid escape mutants, µ = 10⁻⁷, ν = 3×10⁻⁴. (C) High rate of CRISPR loss or deletion mutations, µ = 3×10⁻⁴, ν = 10⁻⁷.