Reconstituting the History of Cronobacter Evolution Driven by Differentiated CRISPR Activity

Cronobacter strains harboring the CRISPR-Cas system are important foodborne pathogens causing serious neonatal infections. However, the specific role of the CRISPR-Cas system in bacterial evolution remains relatively unexplored. In this study, we investigated the impact of the CRISPR-Cas system onCronobacter evolution and obtained 137 new whole-genomeCronobacter sequences by next-generation sequencing technology. Among the strains examined (n = 240), 90.6% (193/213) of prevalent speciesCronobacter sakazakii ,Cronobacter malonaticus , andCronobacter dublinensis strains had intact CRISPR-Cas systems. Two rare species,Cronobacter condimenti (n = 2) andCronobacter universalis (n = 6), lacked and preserved the CRISPR-Cas system at a low frequency (1/6), respectively. These results suggest that the presence of one CRISPR-Cas system is important for aCronobacter species to maintain genome homeostasis for survival. TheCronobacter ancestral strain is likely to have harbored both subtype I-E and I-F CRISPR-Cas systems; during the long evolutionary process, subtype I-E was retained while subtype I-F selectively degenerated inCronobacter species and was even lost by the majorCronobacter pathovars. Moreover, significantly higher CRISPR activity was observed in the plant-associated speciesC .dublinensis than in the virulence-related speciesC. sakazakii andC .malonaticus . Similar spacers of CRISPR arrays were rarely found among species, suggesting intensive change through adaptive acquisition and loss. Differentiated CRISPR activity appears to be the product of environmental selective pressure and might contribute to the bidirectional divergence and speciation ofCronobacter .IMPORTANCE This study reports the evolutionary history ofCronobacter under the selective pressure of the CRISPR-Cas system. One CRISPR-Cas system inCronobacter is important for maintaining genome homeostasis, whereas two types of systems may be redundant and not conducive to acquiring beneficial DNA for environmental adaptation and pathogenicity. Differentiated CRISPR activity has contributed to the bidirectional divergence and genetic diversity ofCronobacter . This perspective makes a significant contribution to the literature by providing new insights into CRISPR-Cas systems in general, while further expanding the roles of CRISPR beyond conferring adaptive immunity and demonstrating a link to adaptation and species divergence in a genus. Moreover, our study provides new insights into the balance between genome homeostasis and the uptake of beneficial DNA related to CRISPR-based activity in the evolution ofCronobacter .