Bacterial alginate regulators and phage homologs repress CRISPR–Cas immunity

Abstract

CRISPR–Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection1. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases2. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR–Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB–AlgB), previously characterized in the regulation of Pseudomonas aeruginosa alginate biosynthesis3,4, as a regulator of the expression and activity of the P. aeruginosa Type I-F CRISPR–Cas system. Downstream of KinB–AlgB, activators of alginate production AlgU (a σE orthologue) and AlgR repress CRISPR–Cas activity during planktonic and surface-associated growth5. AmrZ, another alginate regulator6, is triggered to repress CRISPR–Cas immunity upon surface association. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme and carry hijacked homologs of AmrZ that repress CRISPR–Cas expression and activity. This suggests that while CRISPR–Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

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Fig. 1: A forward genetic screen identified a role for an alginate-activating pathway in the repression of CRISPR–Cas immunity.
Fig. 2: The KinB–AlgB pathway modulates Cas3 and Csy protein and RNA levels.
Fig. 3: AmrZ is a surface-activated repressor of CRISPR–Cas immunity.
Fig. 4: Phage-derived AmrZ homologs control CRISPR–Cas immunity.

Data availability

Source data and statistics used to generate figures are provided with the paper. Additional data supporting the findings of this paper are available from the corresponding author on request.

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Acknowledgements

We thank D. Hung’s lab for providing ∆kinB,algB,algR and ∆algU mutants and G. O’Toole’s lab for providing the csy3::lacZ PA14 strain. We thank K. Trotta (S. Chou’s lab, UCSF) for advice in the development of fluorescent assays and A. Santiago-Frangos (B. Wiedenheft’s lab, MSU) for advice and consultation. J.B.-D.’s lab was supported by the UCSF Program for Breakthrough in Biomedical Research (funded in part by the Sandler Foundation), the Innovative Genomics Institute, a National Institutes of Health Office of the Director Early Independence Award (no. DP5-OD021344) and grant no. R01GM127489.

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Authors

Contributions

J.B.-D., A.L.B. and B.C. formulated the study design and plans. A.L.B. performed CRISPR–Cas activity and expression profiling and conducted bioinformatics analyses. B.C. conducted the genetic screen and constructed and characterized bacterial mutants. S.G. constructed sfCherry reporter strains. T.S. conducted CRISPRi assays. V.E. assisted in establishing reporter assays. J.B.-D. and A.L.B wrote the manuscript.

Corresponding author

Correspondence to Joseph Bondy-Denomy.

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Competing interests

J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics and a scientific advisory board member and cofounder of Acrigen Biosciences.

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Extended data

Extended Data Fig. 1 Mapped insertions from transposon mutagenesis screen.

All independent transposon insertions identified and mapped by visual screening with increased or decreased csy3::lacZ β-galactosidase activity. β-galactosidase activity is expressed as a percentage of the unmutagenized parent strain, and measurements were taken at a single timepoint after 8 h of growth in liquid culture. The insertion location in the PA14 genome is shown, along with the measured level of β-galactosidase enzyme at the 8 hour timepoint. These measurements were not determined (N/D) for strains with a growth defect.

Extended Data Fig. 2 Characterization of kinB::Tn mutants.

a. A streak plate on X-gal plates, showing strains involved in this study and isolated transposon (Tn) insertions. csy3::lacZ is a derivative of WT PA14, and is the unmutagenized parent of kinB::Tn 1-3. b. β-galactosidase measurements of strains grown in liquid culture for the indicuated time. Measurements for the unmutagenized (csy3::lacZ) parent strain and three isolated kinB transposon mutants (kinB::Tn1-3) are shown, as well as a control PA14 culture with no lacZ insertion. c. Phage titration on lawns of the kinB::Tn1 mutant transformed with empty vector or kinB. d. Spot titration of phages JBD26 (CR2_sp17, sp20-targeted, possessing acrIF4), JBD25 (CR1_sp1 targeted) on kinB::Tn mutants and ∆CRISPR-Cas. These experiments have been replicated at least 2 times with consistent results.

Extended Data Fig. 3 Double knockouts of pathway members.

a, b. Efficiency of immunity measurements for indicated mutants relative to WT. a. Double knockouts show ∆kinB combined with algB, algU, algR, or amrZ. EOI measurements are shown as the mean of 3 biological replicates, ± S.D. Mutants show increased EOI against DMS3macrIF4 relative to WT (∆kinB∆algB, P = 3.8 x 10-2, ∆kinB∆algU, P = 5.9 x 10-3, ∆kinB∆algR, P = 1.5 x 10-2, ∆kinB∆amrZ, P= 3.2 x 10-3) Two-tailed unpaired Student’s T-test was used to calculate P value, *p < 0.05, **p < 0.01. b. Indicated knockouts were combined with csy3::lacZ, EOI shown as the mean of two biological replicates.. These experiments have been replicated at least 2 times with consistent results. Source data

Extended Data Fig. 4 AmrZ activity in liquid growth.

a. qRT-PCR measurements of transcript levels of csy3 (light grey) and cas3 (dark grey) normalized to the housekeeping gene rpsL after 8 h of growth in liquid culture. Measurements are represented as the mean of 3 technical replicates. b. Measurement of the fluorescence levels of Csy1-sfCherry (light grey) or Cas3-sfCherry (dark grey) reporter strains after 10 h of growth in liquid culture. Fluorescence measurements are represented as the mean of 3 biological replicates +/- SD. Cas3-sfCherry (P = 0.26) and Csy1-sfCherry levels (P = 0.35) in ∆amrZ did not differ significantly from WT. Two-tailed unpaired Student’s T-test was used to calculate P value, ns = not significant c. csy3::lacZ β-galactosidase activity from PA14 WT csy3::lacZ transformed with either empty vector (EV) or a plasmid overexpressing AmrZ (+AmrZ). β-galactosidase reporter activity was measured after 8 h in liquid growth and is represented as the mean of 3 technical replicates. Experiment was replicated two times with consistent results. Source data

Extended Data Fig. 5 Mobile AmrZ homologs.

AmrZ homologs listed by the genome that encodes them, the accession number, and the mobile genetic element type.

Extended Data Fig. 6 AmrZ copy number analysis of two Pseudomonas aeruginosa strains.

AmrZ copy number analysis of two different strains of Pseudomonas aeruginosa. AmrZ homologs listed by accession number and their genomic coordinates. Phaster49 was used to identify the prophages encoding mobile AmrZ copies.

Extended Data Fig. 7 Cas and Csy RNA and protein levels across growth conditions.

a. Log2 of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) shown for each I-F cas gene in PA14 in the indicated growth condition43. b. Log2 of protein levels for each of the I-F Cas proteins in PA14 in the indicated growth condition44. Source data

Supplementary Information

Reporting Summary

Supplementary Table 1

Strains, plasmids and primers.

Source data

Source Data Fig. 1

Statistical source data.

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Source Data Extended Data Fig. 3

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Source Data Extended Data Fig. 7

Statistical source data.

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Borges, A.L., Castro, B., Govindarajan, S. et al. Bacterial alginate regulators and phage homologs repress CRISPR–Cas immunity. Nat Microbiol 5, 679–687 (2020). https://doi.org/10.1038/s41564-020-0691-3

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