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CRISPR-Cas systems are considered as barriers to horizontal gene transfer (HGT). However, the influence of such systems on HGT within species is unclear. Also, little is known about the impact of CRISPR-Cas systems on bacterial evolution at the population level. Here, using Bacillus cereussensu lato as model, we investigate the interplay between CRISPR-Cas systems and HGT at the population scale. We found that only a small fraction of the strains have CRISPR-Cas systems (13.9% of 1871), and most of such systems are defective based on their gene content analysis. Comparative genomic analysis revealed that the CRISPR-Cas systems are barriers to HGT within this group, since strains harboring active systems contain less mobile genetic elements (MGEs), have lower fraction of unique genes and also display limited environmental distributions than strains without active CRISPR-Cas systems. The introduction of a functional CRISPR-Cas system into a strain lacking the system resulted in reduced adaptability to various stresses and decreased pathogenicity of the transformant strain, indicating that B. cereusgroup strains could benefit from inactivating such systems. Our work provides a large-scale case to support that the CRISPR-Cas systems are barriers to HGT within species, and that in the B. cereusgroup the inactivation of CRISPR-Cas systems correlated with acquisition of MGEs that could result in better adaptation to diverse environments.

CRISPR-Cas systems are considered as barriers to horizontal gene transfer (HGT). However, the influence of such systems on HGT within species is unclear. Also, little is known about the impact of CRISPR-Cas systems on bacterial evolution at the population level. Here, using Bacillus cereussensu lato as model, we investigate the interplay between CRISPR-Cas systems and HGT at the population scale. We found that only a small fraction of the strains have CRISPR-Cas systems (13.9% of 1871), and most of such systems are defective based on their gene content analysis. Comparative genomic analysis revealed that the CRISPR-Cas systems are barriers to HGT within this group, since strains harboring active systems contain less mobile genetic elements (MGEs), have lower fraction of unique genes and also display limited environmental distributions than strains without active CRISPR-Cas systems. The introduction of a functional CRISPR-Cas system into a strain lacking the system resulted in reduced adaptability to various stresses and decreased pathogenicity of the transformant strain, indicating that B. cereusgroup strains could benefit from inactivating such systems. Our work provides a large-scale case to support that the CRISPR-Cas systems are barriers to HGT within species, and that in the B. cereusgroup the inactivation of CRISPR-Cas systems correlated with acquisition of MGEs that could result in better adaptation to diverse environments.

ABSTRACTUnderstanding the genetic basis of host shifts is a key genomic question for pathogen and parasite biology. The Bacillus cereusgroup, which encompasses Bacillus thuringiensisand Bacillus anthracis, contains pathogens that can infect insects, nematodes, and vertebrates. Since the target range of the essential virulence factors (Cry toxins) and many isolates is well known, this group presents a powerful system for investigating how pathogens can diversify and adapt to phylogenetically distant hosts. Specialization to exploit insects occurs at the level of the major clade and is associated with substantial changes in the core genome, and host switching between insect orders has occurred repeatedly within subclades. The transfer of plasmids with linked crygenes may account for much of the adaptation to particular insect orders, and network analysis implies that host specialization has produced strong associations between key toxin genes with similar targets. Analysis of the distribution of plasmid minireplicons shows that plasmids with orf156and orf157, which carry genes encoding toxins against Lepidoptera or Diptera, were contained only by B. thuringiensisin the specialized insect clade (clade 2), indicating that tight genome/plasmid associations have been important in adaptation to invertebrate hosts. Moreover, the accumulation of multiple virulence factors on transposable elements suggests that cotransfer of diverse virulence factors is advantageous in terms of expanding the insecticidal spectrum, overcoming insect resistance, or through gains in pathogenicity via synergistic interactions between toxins.IMPORTANCEPopulation genomics have provided many new insights into the formation, evolution, and dynamics of bacterial pathogens of humans and other higher animals, but these pathogens usually have very narrow host ranges. As a pathogen of insects and nematodes, Bacillus thuringiensis, which produces toxins showing toxicity to many orders of insects and other invertebrates, can be used as a model to study the evolution of pathogens with wide host ranges. Phylogenomic analysis revealed that host specialization and switching occur at the level of the major clade and subclade, respectively. A toxin gene co-occurrence network indicates that multiple toxins with similar targets were accumulated by the same cell in the whole species. This accumulation may be one of the strategies that B. thuringiensishas used to fight against host resistance. This kind of formation and evolution of pathogens represents a different path used against multiple invertebrate hosts from that used against higher animals.