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The Rcs stress response inversely controls surface and CRISPR–Cas adaptive immunity to discriminate plasmids and phages

Nature Microbiology volume 6, pages 162–172 (2021)Cite this article

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Abstract

Bacteria harbour multiple innate defences and adaptive CRISPR–Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR–Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR–Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR–Cas systems. Rcs-mediated repression of CRISPR–Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.

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Fig. 1: Overview of the type III-A csm promoter, fluorescent reporter plasmid and high-throughput SorTn-seq workflow.
Fig. 2: SorTn-seq reveals type III-A CRISPR–Cas regulators.
Fig. 3: Rcs pathway activation represses type III CRISPR–Cas activity.
Fig. 4: Induction of the Rcs pathway represses type I CRISPR–Cas activity and leads to decreased CRISPR adaptation and greater plasmid retention.
Fig. 5: Mutations inducing the Rcs pathway confer innate immunity against phage infection.
Fig. 6: Proposed model of phage defence strategy and effects on HGT on Rcs phosphorelay activation.

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Data availability

Additional data that support the findings of the present study are available from the corresponding author upon request. Sequencing data are available in the Sequence Read Archive under BioProject no. PRJNA601789. The annotated genome of Serratia sp. ATCC 39006—LacA is available through the NCBI (reference sequence NZ_CP025085.1) (https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP025085.1). Source data are provided with this paper.

Code availability

Custom R scripts (SorTnSeq.R and SorTnSeq_Statistics.R) and files required for data processing (serratia_master_features_table.xlsx) are available on GitHub (https://github.com/JacksonLab/SorTn-seq).

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Acknowledgements

This work was supported by the Marsden Fund from the Royal Society of New Zealand (to P.C.F. and P.P.G.) and the School of Biomedical Sciences Bequest Fund from the University of Otago (to P.C.F., S.A.J., J.E.U. and P.P.G.). L.M.S. and L.M.M. were supported by University of Otago Doctoral Scholarships. We thank the staff of the Otago Micro and Nano Imaging facility for assistance with electron microscopy, M. Wilson for help with cell sorting and A. Jeffs of the Otago Genomics Facility for assistance with sequencing. We thank H. Hampton, A. Rey Campa, M. Yevstigneyeva and G. Salmond for providing strains and plasmids, and members of the Fineran laboratory for helpful discussions.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

    Leah M. Smith, Simon A. Jackson, Lucia M. Malone, James E. Ussher & Peter C. Fineran

  2. Genetics Otago, University of Otago, Dunedin, New Zealand

    Simon A. Jackson, Paul P. Gardner & Peter C. Fineran

  3. Department of Biochemistry, University of Otago, Dunedin, New Zealand

    Paul P. Gardner

  4. Bio-Protection Research Centre, University of Otago, Dunedin, New Zealand

    Paul P. Gardner & Peter C. Fineran

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  1. Leah M. Smith
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Contributions

L.M.S. designed experiments, performed the SorTn-seq and the bioinformatic analyses, constructed plasmids and mutants, performed all reporter, interference, adaptation, phage adsorption and microscopy experiments, and prepared all the figures. S.A.J. designed experiments, performed bioinformatic analyses, constructed plasmids and provided supervision. L.M.M. generated strains and provided phages. J.E.U. provided supervision and input into the flow cytometry. P.P.G. assisted with bioinformatic analyses and supervision. P.C.F. conceptualized the study, designed experiments and supervised the project. L.M.S. and P.C.F. wrote the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to Peter C. Fineran.

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The authors declare no competing interests.

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Peer review information Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Classification of significantly enriched genes and model of csm regulation in Serratia.

a,b, Categories shown are Gene Ontology classifications93,94 from the a, low expression bin and b, high expression bin. Locus tags and Gene Ontology information is detailed in Supplementary Table 2 (NA = no Gene Ontology term assigned). c, Main regulatory pathways controlling type III-A CRISPR-Cas immunity in Serratia. Features identified through csm SorTn-seq are shown in dark blue. Dashed lines indicate that the mechanism of regulation (direct versus indirect) is unknown. Connections between pathways that have been established in other organisms but have not yet been demonstrated in Serratia are indicated by a question mark (QS = quorum sensing).

Extended Data Fig. 2 Levels of csm expression and Rcs pathway-related promoters.

a, Activity of the csm promoter was measured in strains containing a chromosomal LacZ reporter (Pcsm-lacZ) or no reporter controls (lacZ—controls) (n = 6 biologically independent samples). b, Expression of the csm promoter was partially restored in the igaA mutant background upon expression of igaA from a low copy number plasmid (pPF2143, RK2 origin; T5-lac promoter) versus control plasmid (pPF1622) (n = 5 biologically independent samples). MFI, median fluorescence intensity. c, Organization of a predicted capsular polysaccharide biosynthesis locus and predicted rcsA gene in Serratia. d, Expression levels of the rcsA promoter (PrcsA) is elevated in strains igaA and mdoG, which suggests positive autoregulation of rcsA upon Rcs-pathway induction (n = 5 biologically independent samples). e, Capsule locus expression, measured from a wza promoter fusion to eYFP (Pwza), was reduced in Rcs-activated strains igaA and mdoG. The decrease in capsule locus expression is consistent with TEM imaging which showed no evidence of capsule synthesis in the igaA mutant (n = 5 biologically independent samples). All bars shown are the mean and error bars represent the s.e.m. For panels d and e, two-sided t-tests were used to determine statistical significance. Detailed statistical testing can be found in the accompanying Source Data file. ****P < 0.0001; ***P < 0.001.

Source data

Extended Data Fig. 3 The Rcs pathway mediated changes in type I expression and interference.

a, Repression of type I-E cas3 expression requires rcsB in both strains mdoG and igaA, while rcsA contributes to repression only in strain igaA. The reduction of cas3 expression is independent of rprA (n = 5 biologically independent samples). MFI, median fluorescence intensity. b, Repression of type I-F cas1 expression requires both rcsB and rcsA in strain igaA and is independent of rprA (n = 5 biologically independent samples). c, CRISPR-Cas interference measured through conjugation efficiency of plasmids carrying protospacers matching endogenous CRISPR arrays (targeted) or control plasmids (untargeted). Type I-E CRISPR-Cas interference of the igaA strain is restored upon deletion of rcsB, and partially restored upon deletion of rcsA (I-E targeted). Type I-F interference levels of the igaA strain are restored upon deletion of rcsB or rcsA (I-F targeted). Conjugation efficiency of untargeted plasmids is similar amongst rcsA or rcsB deletions and isogenic parental strains (untargeted) (n = 3 biologically independent samples). All bars shown are the mean and error bars represent the s.e.m. For panels a and b, two-sided t-tests were used to determine statistical significance. Detailed statistical testing can be found in the accompanying Source Data file. ****P < 0.0001; **P < 0.01; ns, not significant.

Source data

Extended Data Fig. 4 Activation of the Rcs pathway results in attenuated phage infection and reduced swimming motility.

a, During infection on plates, strain igaA is resistant to all phages. Faint clearing of the mdoG lawn is visible at high titers for flagellatropic phages OT8, JS26 and PCH45 (-1; ~109 PFU/ml and -2; ~108 PFU/ml). Infection of mdoG by LC53 is reduced on plates compared to WT. b, Strain mdoG is resistant to infection by flagellatropic phages in liquid, while remaining susceptible to phage LC53 (n = 3 biologically independent samples; lines represent the mean + /- the standard deviation). MOI, multiplicity of infection. c, Reduced swimming in mdoG and null swimming in igaA mutants is consistent with activation of the Rcs pathway. Deletion of rcsB (ΔrcsB) restores swimming motility to wild-type levels in mdoG and igaA mutant backgrounds, while deletion of the rcsA homolog RS09790 (ΔrcsA) does not. The partial restoration of swimming in strain igaA upon rcsA deletion is consistent with partial increase in flhDC expression (Extended Data Fig. 6), as well as partial re-sensitization to flagellatropic phages. (Extended Data Fig. 5). Deletion of both rcsA and rcsB (ΔrcsAB), results in similar swimming motility as the rcsB deletion.

Source data

Extended Data Fig. 5 Phage resistance in strains igaA and mdoG is dependent on the Rcs pathway.

a, Deletion of rcsB restores susceptibility of strains igaA and mdoG to flagellatropic phage OT8. Deletion of rcsA partially sensitizes igaA to OT8, while mdoG remains largely resistant (n = 3 biologically independent samples). MOI, multiplicity of infection. These results support the partial role for rcsA in swimming motility (Extended Data Fig. 4) and flagella gene repression (Extended Data Fig. 6) observed in igaA but not mdoG. b, Deletion of either rcsB or rcsA restores susceptibility of strain igaA to phage LC53 resistant (n = 3 biologically independent samples). Lines in a and b represent the mean +/− the standard deviation.

Source data

Extended Data Fig. 6 Genetic organization and expression levels of Rcs-affected loci.

a, Expression of the flhDC operon (flagella master regulator) promoter (PflhDC) is reduced in Rcs-activated strains. In strain igaA, restoration of expression to WT levels occurs upon deletion rcsB and rcsD, while rcsA deletion partially restores expression. Repression of PflhDC is independent of rprA. In mdoG, flhDC expression is increased upon deletion of rcsB and rcsD but is unaffected by rcsA deletion (n = 5 biologically independent samples). MFI, median fluorescence intensity. b, Expression of the ompW promoter (PompW) is reduced in Rcs-activated strains. In strain igaA, an increase in expression is observed upon deletion rcsB, rcsD, or rcsA. In mdoG, the slight decrease in ompW expression does not alter phage LC53 infectivity in liquid but may contribute to an observed reduction in plaque formation on solid media (Extended Data Fig. 4). Repression of ompW is independent of rprA in the igaA mutant (n = 5 biologically independent samples). All bars indicate the mean and error bars represent the s.e.m. Two-sided t-tests were used to determine statistical significance. Detailed statistical testing can be found in the accompanying Source Data file. ****P < 0.0001; **P < 0.01; ns, not significant.

Source data

Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Figs. 1–2 and Supplementary Tables 1–6.

Reporting summary

Supplementary Data 1

Complete edgeR output of genes and intergenic regions tested during csm SorTn-seq analysis. Fold changes and associated P values are indicated for features in the high and low libraries, as compared against the depleted control.

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Unprocessed gels.

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Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 6

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Smith, L.M., Jackson, S.A., Malone, L.M. et al. The Rcs stress response inversely controls surface and CRISPR–Cas adaptive immunity to discriminate plasmids and phages. Nat Microbiol 6, 162–172 (2021). https://doi.org/10.1038/s41564-020-00822-7

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