Identifying essential genes in using a highly-saturated transposon library.

The unique epibiotic-parasitic relationship between type strain TM7x, a member of the newly identified Candidate Phyla Radiation, now referred to as and its basibiont, strain XH001 (formerly , require more powerful genetic tools for deeper understanding of the genetic underpinnings that mediate their obligate relationship. Previous studies have mainly characterized the genomic landscape of XH001 during or post TM7x infection through comparative genomic or transcriptomic analyses followed by phenotypic analysis. Comprehensive genetic dissection of the pair is currently cumbersome due to the lack of robust genetic tools in TM7x.

High-Throughput Mutant Screening in via Transposon Sequencing.

Transposon mutagenesis greatly facilitates the study of gene function in microorganisms ranging from viruses to fungi. Traditionally, one would study individual transposon mutants with interesting phenotypes one mutant at a time. Here, we describe methods for the study of tens of thousands of transposon mutants in parallel in the bacterial pathogen using transposon-sequencing.

High-Throughput Mutant Screening via Transposon Sequencing.

Transposon mutagenesis has been the method of choice for genetic screens and selections in bacteria by virtue of the transposon being linked to the disrupted gene, simplifying its identification. Transposon sequencing (Tn-seq) is a high-throughput version of transposon mutant screening, in which massively parallel sequencing is used to simultaneously follow the fitness of all mutants in a complex library. In a single experiment, one can use Tn-seq to interrogate the contribution of all genes of a bacterium to fitness under a condition of interest.

Identification of Spacer and Protospacer Sequence Requirements in the Vibrio cholerae Type I-E CRISPR/Cas System.

The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of , the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5'-TT-3' protospacer-adjacent motif (PAM) for interference.

Expanding the repertoire of conservative site-specific recombination in Clostridioides difficile.

Recent genomic analysis of an epidemic ribotype 027 (RT027) Clostridioides difficile strain revealed the presence of several chromosomal site-specific invertible sites hypothesized to control the expression of adjacent genes in a bimodal on-off mode. This process, named phase variation, is thought to enhance phenotypic variability under homogeneous conditions ultimately increasing population fitness in unpredictable environmental fluctuations. The full extent of phase variation mediated by DNA-inversions in C.

Genome-wide detection of conservative site-specific recombination in bacteria.

The ability of clonal bacterial populations to generate genomic and phenotypic heterogeneity is thought to be of great importance for many commensal and pathogenic bacteria. One common mechanism contributing to diversity formation relies on the inversion of small genomic DNA segments in a process commonly referred to as conservative site-specific recombination. This phenomenon is known to occur in several bacterial lineages, however it remains notoriously difficult to identify due to the lack of conserved features.