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Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are bacterial defences that target bacteriophages and mobile genetic elements. How these defences evolve in novel host environments remains largely unknown. We studied the evolution of the CRISPR-Cas system in Mycoplasma gallisepticum (also named Mycoplasmoides gallisepticum ), a bacterial pathogen of poultry that jumped into a passerine host ~30 years ago. Over the decade following the host shift, all isolates displaying a functional CRISPR-Cas system were found not only to harbour completely new sets of spacers, but the DNA protospacer adjacent motif recognized by the main effector M. gallisepticum Cas9 (MgCas9) was also different. These changes in CRISPR-Cas diversity and specificity are consistent with a change in the community of phages and mobile elements infecting M. gallisepticum as it colonized the novel host. In the years following the host shift, we also detected a gradual rise in isolates displaying non-functional MgCas9. After 12 years, all circulating isolates harboured inactive forms only. This loss of CRISPR-Cas function comes at a time when the passerine host is known to have evolved widespread resistance, which in turn drove the evolution of increasing M. gallisepticum virulence through antagonistic coevolution. Such striking concordance in the rise of inactivated forms of CRISPR-Cas and the evolution of host resistance suggests that the inactivation of the CRISPR-Cas system was necessary for enabling adaptive bacterial responses to host-driven selection. We highlight the need to consider both host and pathogen selection pressures on bacteria for understanding the evolution of CRISPR-Cas systems and the key factors driving the emergence of a pathogenic bacterium in a novel host.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa , a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa . As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa . We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCE Pseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications., Competing Interests: The authors declare no conflict of interest.