Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans
Data files
Apr 24, 2024 version files 6.44 MB
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Benzetal2024_Dryad.xlsx
6.43 MB
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README.md
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Abstract
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.
https://doi.org/10.5061/dryad.8pk0p2nvp (NOT YET AVAILABLE)
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