DOI: 10.5061/dryad.2ngf1vj1w
title: Data from: CRISPRi-mediated repression of three cI repressors induces the expression of three related <em>Neisseria gonorrhoeae</em> bacteriophages

description: he 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.
 Bacteriophage, 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.
description: # Data from: CRISPRi-mediated repression of three cI repressors induces
 the expression of three related *Neisseria gonorrhoeae* bacteriophages ##
 Description of the data and file structure These files comprise the raw
 data for the paper “CRISPRi-mediated repression of three cI repressors
 induces the expression of three related *Neisseria gonorrhoeae*
 bacteriophages” published in the *Journal of Bacteriology* in May 2025. We
 have submitted our raw CFU/mL Liquid Death curve counts for each
 CRISPRi-carrying *N. gonorrhoeae* strain with a *ngo0479*, *ngo1116*, or
 *ngo1630*-targeting spacer treated with or without IPTG
 (CRISPRi-NGO0479_Liquid_Death_Curve.xlsx,
 CRISPRi-NGO1116_Liquid_Death_Curve.xlsx,
 CRISPRi-NGO1630_Liquid_Death_Curve.xlsx), raw RNA-sequencing data from the
 CRISPRi-NGO0479, CRIPSRI-NGO1116, and CRISPRi-NGO1630 strains following
 bacterial liquid growth, supplemented with or without IPTG (Raw
 CRISPRi_RNA-sequencing_Raw_data.xlsx), folder containing raw Transmission
 Electron Microscopy images labeled according to the figure each image
 corresponds with in the paper (TEM_Raw_Images.zip), measurements of phage
 heads released following growth with or without IPTG for the
 CRISPRi-NGO0479, CRISPRi-NGO1116, and CRISPRi-NGO1630 strains
 (Phage_Head_Measurements.xlsx), measurements of phage tails released
 following growth with IPTG for the CRISPRi-NGO0479, CRISPRi-NGO1116, and
 CRISPRi-NGO1630 strains (Phage_Tail_Measurements-1.xlsx). A copy of the
 README is included in RTF format (README.rtf). ###
 CRISPRi-NGO0479_Liquid_Death_Curve.xlsx,
 CRISPRi-NGO1116_Liquid_Death_Curve.xlsx,
 CRISPRi-NGO1630_Liquid_Death_Curve.xlsx * These are 3 separate files, each
 file representing the strain which it is named after * Hours = How long
 the bacteria were grown in liquid culture before isolating samples to
 perform CFU/mL dilutions and calculations * 1 mM or 0 mM IPTG = The
 concentration of IPTG added to the liquid culture at the start of the
 experiment * Data represent 3 biological replicates with 9 technical
 replicates per condition and time point * CFU/mL = data represented as
 “Colony Forming Units per milliliter” ### Raw
 CRISPRi_RNA-sequencing_Raw_Data.xlsx * Tabs * NGO0479 0 mM vs 1 mM IPTG =
 *N. gonorrhoeae* CRISPRi-NGO0479 strain grown with and without IPTG; RNA
 was isolated and sequenced. This tab shows the RNA transcript abundance
 for every gene in the bacteria * NGO0479 Significant Genes = this tab
 contains the transcript abundance for the genes that displayed significant
 differences in RNA transcript abundance between 0 and 1 mM IPTG * NGO1116
 0 mM vs 1 mM IPTG = *N. gonorrhoeae* CRISPRi-NGO1116 strain grown with and
 without IPTG; RNA was isolated and sequenced. This tab shows the RNA
 transcript abundance for every gene in the bacteria * NGO1116 Significant
 Genes = this tab contains the transcript abundance for the genes that
 displayed significant differences in RNA transcript abundance between 0
 and 1 mM IPTG * NGO1630 0 mM vs 1 mM IPTG = *N. gonorrhoeae*
 CRISPRi-NGO1630 strain grown with and without IPTG; RNA was isolated and
 sequenced. This tab shows the RNA transcript abundance for every gene in
 the bacteria * NGO1630 Significant Genes = this tab contains the
 transcript abundance for the genes that displayed significant differences
 in RNA transcript abundance between 0 and 1 mM IPTG * Columns: * Locustag
 = refers to the locus in which the gene corresponds in the *N.
 gonorrhoeae* N-1-60 strain * Gene = 4-letter name for gene; if the gene
 did not have a name, “N/A” is written * Feature Type = feature in the
 genome; CDS = coding sequencee * Pvalue = P-value of the difference in
 transcript abundance * Benjamini_Hochberg_Adjusted_PValue =
 Benjamini-Hochberg P-value Adjustment * (0479, 1116, or 1630 1mM_2 hr) =
 RNA transcript abundance for each strain following growth in liquid media
 supplemented with 1 mM IPTG for 2 hours * (0479, 1116, or 1630 0mM_2 hr) =
 RNA transcript abundance for each strain following growth in liquid media
 supplemented with 0 mM IPTG for 2 hours ### TEM_Raw_Images.zip * This
 folder contains the RAW TEM (Transmission Electron Microscopy) tif images
 used for the paper. The name of each file corresponds to the figure in
 which the image was used for the paper. The number value indicates which
 image in the Figure row it corresponds to (from left to right, 1 to 3 [if
 a row contains 3 images] or 1 to 2 [if a row contains 2 images]) * In each
 image, the bottom of the image is a scale * “nm” = nanometer ###
 Phage_Head_Measurements.xlsx * This file contains the head diameter
 measurements of phage — or non-phage vesicles — isolated from the
 CRISPRi-NGO0479, CRISPRi-NGO1116, and CRISPRi-NGO1630 strains grown with
 or without IPTG * Measurements were acquired by analyzing TEM images with
 ImageJ * Each cell represents a single phage head or vesicle * All values
 are nanometer (“nm”) measurements * Tabs: * 1 mM IPTG CRISPRi-*ngo0479*
 (nm) = phage particle head measurements (in nanometers) from
 CRISPRi-NGO0479 strain grown with 1 mM IPTG * 0 mM IPTG CRISPRi-*ngo0479*
 (nm) = vesicle measurements (in nanometers) from CRISPRi-NGO0479 strain
 grown with 0 mM IPTG * 1 mM IPTG CRISPRi-*ngo1116* (nm) = phage particle
 head measurements (in nanometers) from CRISPRi-NGO1116 strain grown with 1
 mM IPTG * 0 mM IPTG CRISPRi-*ngo1116* (nm) = vesicle measurements (in
 nanometers) from CRISPRi-NGO1116 strain grown with 0 mM IPTG *
 1 mM IPTG CRISPRi-*ngo1630* (nm) = phage particle head measurements (in
 nanometers) from CRISPRi-ngo1630 strain grown with 1 mM IPTG *
 0 mM IPTG CRISPRi-*ngo1630* (nm) = vesicle measurements (in nanometers)
 from CRISPRi-NGO1630 strain grown with 0 mM IPTG ###
 Phage_Tail_Measurements-1.xlsx * This file contains the tail lengths
 measurements of phage isolated from the CRISPRi-NGO0479, CRISPRi-NGO1116,
 and CRISPRi-NGO1630 strains grown with IPTG * All measurements are in
 nanometers (“nm”); each cell represents a single tail * Measurements were
 made through ImageJ analysis of TEM images * Tabs: * Cummulative = the
 lengths of all tails measured from the CRISPRi-NGO0479, CRISPRi-NGO1116,
 and CRISPRi-NGO1630 strains, combined in a single list * CRISPRi-NGO0479
 +IPTG (nm) = the lengths of tails measured from the CRISPRi-NGO0479 strain
 following growth with 1 mM IPTG; nm = nanometers * CRISPRi-NGO1116 +IPTG
 (nm) = the lengths of tails measured from the CRISPRi-NGO1116 strain
 following growth with 1 mM IPTG; nm = nanometers * CRISPRi-NGO1630 +IPTG
 (nm) = the lengths of tails measured from the CRISPRi-NGO1630 strain
 following growth with 1 mM IPTG; nm = nanometers