Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR–cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host–phage interactions in a model CRISPR–cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR–escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10−6), our population studies indicate that there is more to the dynamics of phage–host interactions and the establishment of a BIM–CEM arms race than predicted from existing assumptions about phage infection and CRISPR–cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of these results for the ecology and (co)evolution of bacteria and phage.
Highlights
The experimental demonstrations, in 2007 and 2008, that the Clustered Regularly Interspaced Short Palindromic Repeats that abound in the genomes of the vast majority of archaea and nearly half of bacteria can serve as part of an adaptive immune system (CRISPR–cas) that protects these prokaryotes against lytic phage [1] and conjugative plasmids [2] raised many intriguing questions and stimulated a great deal of research
The evidence that the CRISPR regions of the genomes of archaea and bacteria play a role in the ecology andevolution of these microbes and their viruses is overwhelming: (i) the spacers of these prokaryotes are homologous to the DNA of viruses in their communities; (ii) experimentally, the acquisition and incorporation of spacers of viral DNA can protect these organisms from subsequent infection by these viruses; (iii) experimentally, viruses evade this immunity by mutation in homologous protospacers or protospacer-adjacent motifs (PAMs)
We use mathematical models, experiments with Streptococcus thermophilus and the phage 2972, and DNA sequence analyses to explore the contribution of CRISPR– cas immunity to the ecology andevolution of bacteria and their viruses
Summary
The experimental demonstrations, in 2007 and 2008, that the Clustered Regularly Interspaced Short Palindromic Repeats that abound in the genomes of the vast majority of archaea and nearly half of bacteria can serve as part of an adaptive immune system (CRISPR–cas) that protects these prokaryotes against lytic phage [1] and conjugative plasmids [2] raised many intriguing questions and stimulated a great deal of research. Long before the immunological role of CRISPR in these microorganisms was recognized, differences in the number of these palindromic repeats had been used as markers for studies of the genetic (molecular) epidemiology and forensics of pathogenic bacteria [13,14,15,16]. Clusters of genes, borne on plasmids, phage, or chromosomal genes, and acquired by horizontal transfer, code for the virulence
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