Nasty Viruses, Costly Plasmids, Population Dynamics, and the Conditions for Establishing and Maintaining CRISPR-Mediated Adaptive Immunity in Bacteria Bruce R. Levin* Department of Biology, Emory University, Atlanta, Georgia, United States of America Abstract Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses. Citation: Levin BR (2010) Nasty Viruses, Costly Plasmids, Population Dynamics, and the Conditions for Establishing and Maintaining CRISPR-Mediated Adaptive Immunity in Bacteria. PLoS Genet 6(10): e1001171. doi:10.1371/journal.pgen.1001171 Editor: David S. Guttman, University of Toronto, Canada Received June 2, 2010; Accepted September 21, 2010; Published October 28, 2010 Copyright: ß 2010 Bruce R. Levin. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by US National Institutes of Health GM091875 (BRL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: blevin@emory.edu Introduction For many species of bacteria, adaptive evolution is through the expression of chromosomal and extrachromosomal (plasmid- and prophage - borne) genes or clusters of genes (pathogenicity and nicer islands) acquired by horizontal gene transfer (HGT) from the same or even quite distant species [1,2]. Thus, on first consideration it may seem that bacteria and their accessory genetic elements would have mechanism to promote the acquisition, incorporation and expression of genes from without. And, indeed there are mechanisms like integrons [3–7] that appear to have that function. On the other side, DNA acquired from external sources may be deleterious. This is certainly the case when that DNA is borne on lytic bacteriophage, but also for plasmids that engender fitness costs [8,9] or chromosomal DNA from the wrong source [10,11]. To deal with these contingencies, it would seem that bacteria would have mechanisms to protect themselves against infection by deleterious foreign DNA [12]. And indeed there are systems like restriction-modification (restriction endonucleases) which appear to have that role [13,14]. The most recently discovered mechanism postulated to provide bacteria immunity to infectious genetic elements are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). For recent reviews see [15,16]. CRISPR is particularly intriguing because of its ubiquity, appearing in ,90% and ,40% of archaeal and eubacterial sequenced genomes, respectively, and because of the adaptive mechanism by which it provides immunity to infections by a virtually indefinite diversity of bacteriophage and plasmids. DNA from infecting phage and plasmids is incorporated into the CRISPR array. Through a yet to be fully elucidated mechanism, bacteria abort the replication of infecting phage [17] or the establishment of conjugative plasmids [18] bearing copies of the DNA incorporated into their CRISPR arrays, also see [19]. Further support for CRISPR being an adaptive immune system that is maintained because it protects bacteria from infection with phage comes from studies of the community ecology of bacteria and phage; DNA in the CRISPR regions of the bacteria from those communities corresponds to that in the co-existing phage [20–23]. For an intriguing perspective on CRISPR as a witness to the coevolutionary history of bacteria and phage, see [24]. PLoS Genetics | www.plosgenetics.org 1 October 2010 | Volume 6 | Issue 10 | e1001171