Distribution of Genetic Determinants Associated with CRISPR-Cas Systems and Resistance to Antibiotics in the Genomes of Archaea and Bacteria

Abstract

The CRISPR-Cas system represents an adaptive immune mechanism found across diverse Archaea and Bacteria, allowing them to defend against invading genetic elements such as viruses and plasmids. Despite its broad distribution, the prevalence and complexity of CRISPR-Cas systems differ significantly between these domains. This study aimed to characterize and compare the genomic distribution, structural features, and functional implications of CRISPR-Cas systems and associated antibiotic resistance genes in 30 archaeal and 30 bacterial genomes. Through bioinformatic analyses of CRISPR arrays, cas gene architectures, direct repeats (DRs), and thermodynamic properties, we observed that Archaea exhibit a higher number and greater complexity of CRISPR loci, with more diverse cas gene subtypes exclusively of Class 1. Bacteria, in contrast, showed fewer CRISPR loci, comprising a mix of Class 1 and Class 2 systems, with Class 1 representing the majority (~75%) of the detected systems. Notably, Bacteria lacking CRISPR-Cas systems displayed a higher prevalence of antibiotic resistance genes, suggesting a possible inverse correlation between the presence of these immune systems and the acquisition of such genes. Phylogenetic and thermodynamic analyses further highlighted domain-specific adaptations and conservation patterns. These findings support the hypothesis that CRISPR-Cas systems play a dual role: first, as a defense mechanism preventing the integration of foreign genetic material-reflected in the higher complexity and diversity of CRISPR loci in Archaea-and second, as a regulator of horizontal gene transfer, evidenced by the lower frequency of antibiotic resistance genes in organisms with active CRISPR-Cas systems. Together, these results underscore the evolutionary and functional diversification of CRISPR-Cas systems in response to environmental and selective pressures.

Keywords: Archaea and Bacteria; CRISPR-Cas; horizontal gene transfer; resistance to antibiotics.

Figure 1

Phylogenetic tree of CAS1 proteins in archaeal genomes. The UPGMA tree of the CAS1 protein was generated using the MUSCLE algorithm in MEGA v12. Representative CAS1 proteins from all identified subtypes were selected. Evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 15.499 is shown (below the branches). The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The analytical procedure encompassed 34 amino acid sequences. The pairwise deletion option was applied to all ambiguous positions for each sequence pair, resulting in a final data set comprising 596 positions.

Figure 2

Phylogenetic tree of CAS1 proteins in bacterial genomes. The UPGMA tree of the CAS1 protein was generated using the MUSCLE algorithm in MEGA v12. Representative CAS1 proteins from all identified subtypes were selected. Evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 11.959 is shown (below the branches). The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The analytical procedure encompassed 31 amino acid sequences. The pairwise deletion option was applied to all ambiguous positions for each sequence pair, resulting in a final data set comprising 759 positions.

Figure 3

Comparison of CAS1 proteins in archaeal and bacterial genomes. (A) Percentage identity of CAS1 proteins in the archaeal genomes analyzed. (B) Percentage identity of CAS1 proteins in the bacterial genomes analyzed. (C) Percentage identity of CAS1 proteins in the analyzed archaeal and bacterial genomes. The cumulative curves show the fraction of CAS1 protein sequences with a percentage identity equal to or lower than the value indicated on the X-axis. The red line represents the cumulative distribution of sequence identities.

Figure 4

Conservation of direct repeats (DRs). The sequence logo was created using WebLogo 3.7.4. (A) Conservation of DRs in archaeal genomes. (B) Conservation of DRs in bacterial genomes. (C) Conservation of DRs in archaeal and bacterial genomes. Error bars are shown in gray.

Figure 5

Correlation between the length (bp) of the CRISPR arrays and the minimum free energy (MFE) of assembly of the CRISPR loci found in Archaea and Bacteria. A very strong positive correlation was observed (R² = 0.87).