Investigating Neisseria gonorrhoeae double-stranded bacteriophage

Abstract Neisseria gonorrhoeae is an obligate human pathogenic species. A few reports in the literature describe three or four genomic islands encoding potential double-stranded phage genomes, and only one manuscript reports the production of a double-stranded phage. However, there are issues with the rigor of that report. We have also published that sublethal hydrogen peroxide treatment induces the expression of some genes carried in these phage islands (Quillin, et al.-2018). Moreover, two saturating transposon screens failed to isolate transposon insertions in some phage island genes (Hu, et al.-2020, Remmele, et al.-2014). We have developed a CRISPR interference (CRISPRi) system for N. gonorrhoeae (Geslewitz, et al.-2024) and have used it to show that repression of two of the three phage-island encoded LexA orthologs is lethal. Down- regulation of one ortholog by CRISPRi results in the production of phage particles as assayed by transmission electron microscopy. We will determine whether one or more phage islands can produce phage, explore whether the two essential LexA orthologs are regulated similarly to the canonical LexA, and determine whether these phage(s) can infect Neisseria strains or species.