CRISPRi-mediated repression of three cI repressors induces the expression of three related Neisseria gonorrhoeae bacteriophages.

Abstract

The Neisseria gonorrhoeae FA1090 isolate encodes nine prophage islands (Ngoɸ1-9). Ngoɸ1-3 contain genes consistent with a Siphoviridae-dsDNA bacteriophage (phage). Saturating transposon-sequencing screens using two different N. gonorrhoeae isolates predicted that multiple prophage genes were essential, including three putative transcriptional repressors: ngo0479 (present in Ngoɸ1), ngo1116 (present in Ngoɸ2), and ngo1630 (present in Ngoɸ3). All three genes display homology to the Lambda phage cI, a regulator important for maintaining the lysogenic state and inhibiting lytic induction, but these proteins are not close paralogs. Using a Neisseria lactamica-derived Type I-C CRISPR-interference system, we show that these cI orthologs are essential, as the knockdown of each gene results in bacterial death. We determined that the repression of the three cI orthologs resulted in the significant induction of phage gene expression. Finally, we detected Siphoviridae-like phage particles released from N. gonorrhoeae following repression of ngo0479, ngo1116, or ngo1630. We hypothesize that these cI orthologs are critical for preventing phage lytic infection and cell death and allow N. gonorrhoeae to benefit from the carriage and expression of prophage genes.IMPORTANCEBacteriophage, or phage, are bacteria-infecting viruses and are the most abundant natural entities in the world. Here, we report that Neisseria gonorrhoeae's three most complete double-stranded DNA prophage islands each encode essential and related transcriptional repressors. CRISPRi-mediated repression of these transcriptional repressors leads to a significant increase in prophage gene expression and phage induction. This study marks an important initial step in studying the interaction between N. gonorrhoeae and its resident phage.