Original Mechanistic Investigation of CRISPR-Cas System Diversity and Activity in Environmental Bacteria from the Benue Trough: Implications for Phage-Bacteria Coevolution

CRISPR-Cas systems are adaptive immune mechanisms in bacteria and archaea that capture spacer sequences from invading mobile genetic elements, providing a molecular record of phage-bacteria coevolutionary arms races. Environmental microbiomes in geologically distinctive habitats may harbor novel CRISPR-Cas diversity not captured in existing databases. This dissertation presents an original mechanistic investigation of CRISPR-Cas system diversity, spacer acquisition dynamics, and anti-phage activity in environmental bacteria isolated from the Benue Trough geothermal springs and alluvial floodplain soils, North Central Nigeria. A culture-dependent and metagenomic dual approach was employed. Environmental DNA from 12 sampling sites was sequenced on Illumina HiSeq, and CRISPR arrays were identified using CRISPRCasFinder and minCED tools. Cas protein phylogenetic analysis was conducted alongside spacer protospacer origin tracing against viral and plasmid databases. Experimental validation of spacer acquisition was demonstrated in select culturable isolates challenged with environmental phage preparations. Thirty-seven distinct CRISPR-Cas loci were identified across 18 bacterial genera, with Type I-E, Type III-A, and a potentially novel Type IV-like system identified from assembled metagenome contigs. Spacer origin analysis mapped protospacers against 142 distinct phage genomes, 41 of which had no prior GenBank representation. The novel Type IV-like system lacked predicted nuclease activity in silico, suggesting a possible regulatory or anti-viral sensor function. These findings expand the known catalog of CRISPR-Cas diversity and provide the first systematic phage-bacteria coevolution data from the Benue Trough ecosystem. Keywords: CRISPR-Cas, phage-bacteria coevolution, environmental metagenomics, novel Cas systems, Benue Trough.