| Due to their life cycles, phages have the potential to be used as an alternative to antibiotics to
counter antibacterial resistance. Phage therapy is not yet widespread, partly due to bacterial
defense systems, such as CRISPR. To counter CRISPR, phages have developed point mutations
within their genomes. This study aims to identify and assess the mutation variations of the
protospacer regions within the phage genome. In vitro and computational analysis assessed natural
selection in protospacer-containing genes within cluster members of mesophilic and thermophilic
phages. The purification of thermophilic phages was unsuccessful, partly due to the specificity of
the isolated phages to host receptors. Analysis of protospacer-containing genes in mesophilic
phage genomes revealed widespread purifying selection consistent with strong functional
constraints on phage proteins, as most genes showed dN/dS < 1. However, positive selection
(dN/dS > 1) was observed, indicating that the phage would favor mutations in these regions, used
to evade the CRISPR-Cas system.
In addition to identifying a suitable host for phage isolation, the microbial diversity analysis
provided insight into the role of temperature in shaping microbial communities. 16S rRNA gene
sequencing generated 7,618 Amplicon Sequence Variants (ASVs). No significant difference in
alpha diversity indices among temperature groups (p > 0.05) was observed. Beta diversity analysis
(PERMANOVA, R² = 0.229, p = 0.001) indicated that temperature significantly influenced
community structure. Bipartite network plots showed that most ASVs were site-specific,
suggesting thermal specialization, while a smaller subset had broader temperature ranges and
occurred in multiple sites.
Keywords: CRISPR, protospacer-containing genes, dN/dS, purifying selection, ASV, microbial
community |