DOI: 10.5061/dryad.8pk0p2nvp
title: Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans

description: Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module,
 feature a DinG helicase instead of a nuclease, and form ribonucleoprotein
 complexes. Type IV-A3 systems are carried by conjugative plasmids that
 often harbor antibiotic resistance genes. Their CRISPR array contents
 suggest a role in inter-plasmid conflicts, but this function remains
 unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system
 co-opts the type I-E adaptation machinery from its host, Klebsiella
 pneumoniae, to update its CRISPR array. Furthermore, we reveal that robust
 interference of conjugative plasmids and phages is elicited through CRISPR
 RNA-dependent transcriptional repression. By silencing plasmid core
 functions, type IV-A3 impacts the horizontal transfer and stability of
 targeted plasmids, supporting its role in plasmid competition. Our
 findings shed light on the mechanisms and ecological function of type
 IV-A3 systems and demonstrate their practical efficacy for countering
 antibiotic resistance in clinically relevant strains.
description: # Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring
 spacers in trans - SOURCE DATA [https://doi.org/10.5061/dryad.8pk0p2nvp
 (**NOT YET AVAILABLE**)](https://doi.org/10.5061/dryad.8pk0p2nvp) ##
 Description of the data and file structure About the project: All data
 generated in this study are publicly available as of the date of
 publication, DOIs and accession numbers are listed in key resource table.
 Standardised data types (sequencing datasets) are available from
 **Bioproject PRJNA1066244**, all other source data are deposited here.
 Analysed plasmid sequences are freely available at RefSeq (state March
 2021). All source data of this publication is collected in one excel
 spreadsheet. Each tab contains data for a particular figure/ data
 analysis. Data within each spreadsheet is formatted for analysis in R. The
 different tabs correspond to figures in the published manuscript and are:
 ### **Fig1B: Csf2 (Cas7) protein sequences for alignments to build
 phylogenetic tree.** ### **Fig1E: p1530 transfer rates** Recipient - is
 the recipient species used as conjugation partner (_1/2/3/4 indicate
 replicate number) D.t, R.t, T.t, D.0, R.0, and T.0 - are donor/ recipient/
 transconjugant population densities at time point t and 0. psi.D, psi.T,
 and psi.R - are growth rates of donor/ transconjugant/recipient
 populations. t - is the duration of the mating assay/ measurement time of
 transconjugants, transfer_rates.estimate - is the estimated transfer rate
 transfer_rates.method - the preferred method selected in the R package
 conjugator. rates_transformed - log10 transformed transfer rates ###
 **Fig3B\_FigS6D** and **Fig3D\_FigS6E: Plasmid interference incoming/
 outgoing** Gene - is the targeted gene with the targeted strand indicated
 (coding vs. template) R_T_per_mL, D_T_per_mL, T_per_mL - are
 recipient+transconjugant / donor+transconjugant/ transconjugant population
 densities Fraction - Indicates transconjugant fraction
 (T_per_mL/R_T_per_mL) ### **Fig3C: Plasmid interference FACS** Gene - is
 the targeted gene with the targeted strand indicated (coding vs. template)
 Events - is the number of recorded bacterial events. Signal - specifies
 which fluorescent signal was recorded under events. Note that the reported
 fraction was calculated by dividing mCherry_GFP by mCherry_total ###
 **Fig3E\_FigS6F: Plasmid interference maintenance** Day - is the time
 point of measurement Gene - is the targeted gene with the targeted strand
 indicated (coding vs. template) LB_per_mL, Tet_per_mL - are total and
 pkjk5-carrying population densities Note: when TET_per_mL = 0 the
 pkjk5-carrying population density was below the detection limit and we
 used the detection limit of 100 CFU/ mL to perform statistical analyses
 ### **Fig3G: Phage interference** Gene - is the targeted gene with the
 targeted strand indicated (coding vs. template) PFU_mL - is plaque forming
 units (PFU)/ mL ### **Fig4C: In vivo transcriptional repression**
 IVA3_variant - is the IV-A3 CRISPR-Cas variant: WT = wild-type, mut =
 catalytically inactive DinG, D = ΔdinG knockout mutant Target -
 differentiates whether the target sequence is located in the gene or the
 promoter Strand_Target - indicates the specific target location (coding or
 template strand, position p/1/2/3) as illustrated in Fig 4B OD600 -
 Measured OD600 values at T= 15h mCherry - fluorescence intensity
 (Excitation 582 nm, Emission 620 nm) at T= 15h mCherry_relativeTo_OD -
 mCherry signal normalized with the corresponding OD600 measure at T= 15h
 ### **Fig4E: ABres reversal plasmid** Treatment - is T for targeting
 treatment and nt_control for the non-targeting control treatment
 TOTAL_permL, ESBL_permL - are densities for the total population and the
 resistant subpopulation ### **Fig4F: ABres reversal chromosome** Treatment
 - is WT for K. pneumoniae 808330 (no targeting), SHV_KO for ΔSVH mutant K.
 pneumoniae 808330(no targeting), T for WT under IV_A3 targeting,
 nt_control for WT with nt_control MIC - estimated minimal inhibitory
 concentration Specific - is exact if the MIC could be evaluated directly
 and higher if the MIC is higher than the highest antibiotic concentration
 used. ### **FigS3D: Size exclusion chromatography** IVA3_variant - is the
 IV-A3 CRISPR-Cas variant: WT = wild-type, D = ΔdinG knockout mutant NM -
 is the wavelength at which absorbance was measured in nm Trace - is the
 measure of absorbance (mAU) Elution_volume_mL - Elution volume in mL ###
 **FigS6G: Growth curves** Time - is the time point of measurement in hours
 Gene - is the targeted gene with the targeted strand indicated (coding vs.
 template) OD600 - is the blanked OD value ### **FigS8C: TXTL**
 IVA3_variant - is the IV-A3 CRISPR-Cas variant: WT = wild-type, mut =
 catalytically inactive DinG, D = ΔdinG knockout mutant crRNA - is the
 location at which degfp is targeted (on nt_control) RFU - relative
 fluorescence units Block - is experimental block: This experiment was
 performed in two independent experimental blocks. ### **FigS8D-E: BLI**
 IVA3_variant - is the IV-A3 CRISPR-Cas variant: WT = wild-type, D = ΔdinG
 knockout mutant Time - is the time point of measurement in seconds
 IV-A3_dilution - Dilution of IV-A3 complex in nM Data - BLI data Fit -
 fittings